Diabetic foot ulcers (DFUs) present with different grades of ischemia and infection and are associated with high mortality and disability rates with little effective treatment. We used Fu Subcutaneous Needling (FSN) to treat 2 cases with DFUs and achieved satisfactory results.Two cases of DFUs showed poor recovery after conventional wound care treatment, and case 2 was confronted with the risk of amputation.Two patients with history of diabetes were diagnosed with DFUs, presenting with lower leg and foot ulcers.Case 1 received 6 sessions of FSN treatment in 8 days, and case 2 received 10 sessions of FSN treatment in 14 days.Case 1 completely healed from a 1 × 0.5-cm blister and a 0.5 × 0.5-cm ulcer of the right lower leg 14 days after the first FSN treatment. The ulcer area of the left foot in case 2 decreased from 6 × 7 cm to 4 × 3.5 × 0.2 cm. Three months of follow-up revealed full wound closure.FSN is effective for healing with DFUs, and it may be used as an adjunctive healing strategy for DFUs patients when conventional treatments such as infection, glycemic control, and local ulcer care are not available. DFUs are among the main causes of disability and death in diabetic patients. With the incidence of diabetes gradually increasing worldwide, the World Health Organization reported that approximately 422 million people have diabetes, 15 to 25% of whom will undergo DFUs.–4 According to statistics, 40 to 60% of non traumatic amputations of the lower limbs are performed in diabetic patients worldwide. The high incidence of disability and morbidity due to DFUs has become an important public health problem. Glycemic control, wound care, and prompt revascularization are general treatments, although with poor healing rates. Adjunctive therapies for DFUs such as acupuncture, massage, and acupoint injection have shown positive effects.Diabetic foot ulcers (DFUs) are common and one of the most serious complications of diabetes mellitus, with varying grades of ischemia and infection. FSN is used to stimulate the subcutaneous layer, which is connected to the muscle layer to improve blood circulation, and is applied for painful musculoskeletal problems with positive effects.,10 Herein we report 2 cases of DFUs that were effectively treated with FSN.Fu's subcutaneous needling (FSN) has been an innovative acupuncture therapy since 1996 .A 71-year-old man with a history of diabetes for over 6 years was admitted to the Foshan Hospital of Traditional Chinese Medicine for aggravating swelling, pain, and increased exudate of the right ankle dorsum on May 28, 2021. The patient had a 22-year-long history of hypertension, which was well controlled by losartan and nifedipine tablets. Although repaglinide  and metformin  were administered before admission, the patient had poor glycemic control. The patient presented with swelling, decreased skin pliability, and coldness of his right lower leg for 2 years, which aggravated a week prior to hospitalization. His right foot became swollen with pain and a blister grew on the surface of the skin  within the muscle belly were palpated and felt tight and less elastic by the practitioner’s fingers. In these 2 cases, MTrPs were found in the ipsilateral tibialis anterior and gastrocnemius muscles. The FSN needle was then inserted at an angle of 15 to 25° from the skin into the subcutaneous layer around the belly of the muscles. The swaying movement was performed at a frequency of approximately 100 times per minute for 2 minutes. Meanwhile, the reperfusion approach was performed with muscle movement to cause contraction against the physician’s resistance for approximately 8 seconds. For example, ankle flexion is a type of contraction of the tibialis anterior , rapid-acting insulin , and metformin hydrochloride tablets  to control glucose. Moreover, irrigation with 3% boric acid solution, debridement, and dressing treatment were applied daily for wound care. On the 5th day of admission, the blister ruptured and a pus cavity formed, with necrotic tissue inside Fig. B. HoweveThe tissue around the ulcers regained a ruddy appearance after the first FSN treatment Fig. D, the ne9/L; neutrophils: 7.93 × 109/L; red blood cell count: 5.81 × 1012/L; albumin-creatinine ratio: 2.8 mg/mmol; microalbumin: 29.2 mg/L; glucose: 7.89 mmol/L; fructosamine: 2.18 L. Urine rountine test: glucose: 3+; uric bravery former: 1+; leukocyte esterase 2+. A highly resistant strain of Methicillin-resistant Staphylococcus aureus was detected by bacterial culture of pus secretions. Magnetic resonance imaging revealed swelling of the surrounding subcutaneous soft tissue. A diagnosis of diabetic foot with mild infection was made .A 48-year-old man was admitted to the same hospital as case 1 with the complaining of 2 deteriorative purulent ulcers on his left foot for 2 months. He also had parethesia, such as coldness and numbness in both lower legs. The patient had a 7-year-long history of diabetes. Physical examination revealed a 6 × 7-cm blister on the plantar aspect of the forefoot and a 2 × 2 × 0.5-cm abscess on the lateral aspect Fig. A. PhysicS. aureus . The patient was administered levofloxacin  for 14 days. Providencia stuartii, Pseudomonas Aeruginosa and Myroides were detected in pus and wound secretion examination after 14 days, and piperacillin-tazobactam  was used as the most sensitive antibiotic for another 14 days. Besides, continuous insulin aspart was used in insulin pump (1.4 U/h in 7 am–7 pm and 0.7 U/h in 7 pm–7 am) to control glucose, and 3% boric acid solution irrigation and dressing treatment were used to treat wound infection locally every day. Based on the condition that bone and tendon exposure appeared from the bottom of the left big toe after debridement (Fig. Drug sensitivity test showed that levofloxacin was the most effective antibiotic against Methicillin-resistant In search of a better treatment, FSN treatment was applied once a day from the 11th day of admission. The detailed steps were performed in a manner similar to case 1. The excretion decreased, and the necrotic tissue of the ulcers decreased from 25 to 20% Fig. C. In the Impaired microvascular reactivity diminishes the blood supply to ulcerated areas, and insufficient vascularization, tissue hypoxia, and a high probability of infection contribute to delayed wound healing. For these 2 cases, case 2, especially poor glycemic control and inadequate microcirculation, led to severe DFUs. However, conventional treatments such as infection, glycemic control, and local ulcer care failed to promote ulcers healing in these 2 cases, suggesting that a sufficient supply of blood and oxygen is essential for accelerating tissue healing.Diabetic foot lesions develop following injury and are usually accompanied by peripheral neuropathy and arterial diseases. MTrPs are hard, palpable nodules in a taut band of skeletal muscle, which were palpated in the tibialis anterior and gastrocnemius in the 2 cases, revealing that muscles related to ischemic areas appear to be in tightened condition. FSN is a therapy that inactivates MTrPs to relieve muscle spasticity and dilate the blood vessels, which are compressed by tightened muscles to promote local blood circulation. Moreover, with muscle movement of contraction of relaxation in FSN treatment, the combination of vasodilation and increase in tissue blood flow may potentially accelerate the production of collagen fibers and granulation tissue and facilitate ulcer healing.Ischemia is the underlying cause of chronic muscle spasm.This case report provides promising evidence that FSN is an adjunctive strategy to treat DFUs because of its unique features: needling points are remote from the ulcerative area and are therefore much less likely to increase the risk of infection; although the FSN needle does not reach the muscle layer, it can act on muscles by performing only in the subcutaneous layer, which surrounds and separates the muscles.These were 2 successful cases of accelerated DFUs healing caused by FSN. Further randomized controlled trials are needed to prove evidence for FSN to be an adjunctive therapy and to help millions of people suffering from DFUs.In addition to conventional treatments, including infection, glycemic control, and local ulcer care, FSN can effectively inactivate MTrPs to relieve tightened muscles and promote local blood circulation, which may accelerate the production of collagen fibers and granulation tissue, facilitate ulcer healing, and reduce the risk of amputation.We are grateful to the patients for their participation in the study and allowing us to publish these case reports. We thank Dr Alexander B. Mearns, Private Clinic, Tain, Ross-Shire, Scotland for his excellent technical assistance.Conceptualization: Fei Qi, Huiyi Huang.Investigation: Fei Qi.Methodology: Fei Qi, Huiyi Huang, Yanyan Cai.Project administration: Fei Qi, Huiyi Huang, Zhonghua Fu.Supervision: Huiyi Huang, Zhonghua Fu.Validation: Fei Qi.Visualization: Fei Qi, Huiyi HuangWriting – original draft: Fei Qi, Yanyan Cai.Writing – review & editing: Huiyi Huang, Zhonghua Fu.


Giardia duodenalis is a common zoonotic intestinal parasitic protozoan and sheep are among its hosts; however, limited information is available on sheep kept in large-scale housing. The Hu sheep is a first-class protected local livestock breed in China. In this study, we investigated the seasonal dynamics of G. duodenalis infection in Hu sheep and the environmental contamination of large-scale sheep farms. We collected 474 fecal samples and 312 environmental samples from Hu sheep on a large-scale sheep farm in Henan, China. The prevalence of G. duodenalis was determined by nested PCR targeting the β‑giardin (bg) gene. The assemblages and multilocus genotypes (MLGs) were investigated based on analyses of three genetic loci, i.e. bg, glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi). To detect mixed infections of different assemblages, assemblage A/E-specific PCRs were performed to amplify the tpi gene. The prevalence of G. duodenalis infection in sheep was 17.9% (81/474) and the positivity rate in environmental samples was 0.96% (3/312). Genetic analysis revealed the presence of two assemblages (assemblages A and E), with assemblage E being detected in both fecal and environmental samples, and assemblage A detected only in fecal samples. A total of 23 MLGs were obtained in fecal and environmental samples, all of which belonged to assemblage E. These results indicate the seasonal dynamics of G. duodenalis infection in sheep and environmental contamination on large-scale housing sheep farms and provide an important reference for the prevention and control of G. duodenalis on large-scale housing sheep farms. Giardia duodenalis is a typical intestinal parasitic protozoan that is most often spread through polluted food and water. Most animals and humans are susceptible to infection by this parasite, which hinders the reproductive ability of adult animals and the growth of young animals. Giardiasis, which is usually asymptomatic but can cause symptoms such as bloating, diarrhea, nausea, vomiting, and indigestion, affects more than 28.5 million people in China each year and is the second most common cause of infectious diarrhea in humans after viruses ..KT922248KJ027408 as a reference, six bg locus sequences were identified, three of subtype AI and three with two single-nucleotide polymorphisms, named A1. Similarly, the GenBank sequence FJ560569 was utilized as a reference, and the 15 sequences at the tpi locus were used to classify the parasites into three subtypes, including two known subtypes (A4 and A6) and a novel subtype A8 . The sequences obtained from the 3 environmental samples formed 3 MLGs . Other samples were mixed infections of assemblages A and E . Phylogeodenalis .Ruzhou City, Henan Province, has a warm temperate semi-humid continental monsoon climate with four distinct seasons: spring , summer , autumn  and winter .G. duodenalis infections in Hu sheep and environmental contamination of large-scale housing sheep farms in China. The results showed that the incidence of G. duodenalis in Hu sheep was 17.09% (81/474), and in environmental samples, it was 0.96% (3/312). The prevalence of G. duodenalis in Hu sheep was higher than the previously reported prevalence of G. duodenalis infections in sheep in Henan Province (1.82–5.64%) [This paper reports, for the first time, the seasonal dynamics of 2–5.64%) ; howeverGiardia cysts can survive for months in high-humidity, low-temperature, low-sunlight-level, and low-salinity environments [G. duodenalis infection rates are highest in spring (30.37%). Furthermore, the relatively cold winter temperatures, as well as the dry air in Ruzhou, may be the reason why the infection rate is lower in winter (19.23%) than spring. It is possible that the summer is not suitable for G. duodenalis transmission, resulting in lower infection rates (9.43%), and the influence of the summer results in lower infection rates in the autumn (7.75%).ronments , 11. RuzG. duodenalis infection rates and age [G. duodenalis were the highest in weaning lambs , which may be because weaning is a period of transition of feeding methods, and the lambs’ immunity is low because of stress. Moreover, the parasite prevalence in lactating lambs  was lower than that in fattening lambs , probably because the lactating lambs were fed by fewer infected lactating ewes, reducing the probability of cross-infection. It was also found that the infection rate of late-gestation ewes was lower than that of early-gestation ewes and non-pregnant ewes. This phenomenon may be attributable to pregnant ewes being fed a more varied diet than their non-pregnant counterparts. Furthermore, pregnant ewes are typically better cared for and housed in more comfortable, quiet, and secure enclosures. Because of interactions among these factors, pregnant ewes may have had higher immunity and disease resistance, resulting in lower infection rates than non-pregnant ewes.A literature search shows that most reports describe a negative correlation between  and age , 32, 36,G. duodenalis was present in the environment. Because of the high density of livestock on large-scale sheep farms, G. duodenalis in the environment is likely to be consumed by sheep, allowing the transmission cycle to continue [In this study, 312 environmental samples were collected. Although only three positive samples were found in the summer , the results suggest that continue , 12.G. duodenalis in sheep, while assemblages A and B have also been occasionally reported [bg, gdh, and tpi, assemblage E was still the dominant cluster of G. duodenalis in Hu sheep, followed by assemblage A, while assemblage B was not found. A total of 18 samples were detected with mixed infections of assemblages A and E using specific primers for the tpi locus of these assemblages. Because commonly used primers for the tpi locus were unable to detect two or more assemblages in the same sample in the past, it can be inferred that the identification of assemblage A and mixed infections in previous investigations were relatively low because of the specificity of the primers [G. duodenalis assemblage, and the risk of sheep acting as potential G. duodenalis conservator hosts have been simultaneously underestimated.Available studies suggest that assemblage E is the dominant assemblage of reported . The res primers . The pubgdh and tpi) of 84 bg-positive samples were analyzed with high resolution via the multilocus genotyping tool. In the fecal samples from Hu sheep, seven assemblage E subtypes were identified using the gdh locus, and four assemblage E subtypes as well as three assemblages A subtypes were identified using the tpi locus. In the environmental samples, three assemblage E subtypes were identified using the gdh locus, and two assemblage E subtypes were identified using the tpi locus  and the rate of G. duodenalis in the environment was 0.96% (3/312), indicating that the prevalence of the protozoa in Hu sheep is high and it can be transmitted through the environment. Large-scale farms with a high density of sheep should take measures to avoid horizontal transmission of G. duodenalis. In addition, mixed infections of assemblages A and E in Hu sheep were more severe than reported by previous studies, suggesting that the presence of assemblage A has been underestimated. Therefore, the significance of G. duodenalis in sheep for public health may need to be re-examined.This dynamic survey of


However, little information is available on the HTLV-1 seroconversion rate in pregnant mothers and the impact of new HTLV-1 infection on mothers and babies during the perinatal period.Human T-cell leukemia virus type-1 (HTLV-1) is transmitted vertically from an infected mother to her child From the database of a prefecture-wide antenatal adult T-cell leukemia prevention program in Nagasaki, Japan, we extracted data on 57,323 pregnant women who were screened for anti-HTLV-1 antibody during 2011–2018. Data on the 16,863 subjects whose HTLV-1 proviral load (PVL) was measured more than twice were included in our analyses.rrm = 0.25) with no perinatal problems.In total, 133 (0.79%) pregnant women were HTLV-1-positive during their first pregnancy and nine (0.05%) seroconverted before or during subsequent pregnancies (between pregnancies). The median PVL  was significantly lower in the seroconverted mothers (0.10%) than in the initially seropositive mothers (0.15%). A repeated measures correlation analysis for the individual PVLs of the HTLV-1-positive pregnant women showed that PVL increased with parity number (The HTLV-1 seroconversion rate between pregnancies was 0.05%, and their HTLV-1 PVL increased annually but no perinatal problems were noted. Globallee modes : verticatfeeding , horizonercourse , and iatnsfusion .via prolonged breastfeeding by the infected mother was presented in 1985 by researchers from Nagasaki University, located in southwest Japan  HTLst Japan ; and (3)armosets . Soon afarmosets  through As previously reported, APP Nagasaki led to a marked reduction in mother-to-child HTLV-1 transmission rates from 20.5% before the APP began to 2.4% after its implementation . Other iRecently, researchers from APP Nagasaki reported that the HTLV-1 proviral load (PVL), induced regulatory T-cell population, and soluble interleukin-2 receptor (sIL-2R) levels of HTLV-1-infected pregnant mothers remained stable during pregnancy but became elevated after delivery . HoweverThe present study therefore aimed to assess the HTLV-1 seroconversion status in multiparous mothers who participated in APP Nagasaki, the individual changes of the HTLV-1 PVL between pregnancies in HTLV-1-positive mothers, and the impact of HTLV-1 positivity on pregnancy outcomes.From its initiation in July 1987 to April 2018, the APP Nagasaki database included information on ~330,000 pregnant mothers, from almost all areas of Nagasaki Prefecture, who were tested for HTLV-1 infection . The datAll of the negative screening test results from the clinical laboratory companies were sent back to the respective obstetric gynecologists, for them to inform the respective pregnant mothers. From 2011 onward, in the case of a positive or suspected positive screening test result, the residual blood sample was forwarded to the APP Nagasaki office at Nagasaki University Hospital where a confirmatory test was performed by western blotting and the HTLV-1 PVL was quantified by a real-time polymerase chain reaction (RT-PCR) assay in the Department of Laboratory Medicine. The final test results were reported to the respective obstetric gynecologists, who then informed the respective mothers by 35 weeks of gestation . The obsIn APP Nagasaki, a quantitative RT-PCR assay was routinely performed from 2011 onward to quantify the HTLV-1 PVL in peripheral blood mononuclear cells (PBMCs) from anti-HTLV-1 antibody-positive pregnant mothers and those with indeterminate western blotting results. The details of the method used to quantify the HTLV-1 PVL have been previously published , and theWithin 1 week of delivery, each of the HTLV-1-positive mothers who participated in APP Nagasaki was required to answer questions about their birthplace, pregnancy history, the feeding method chosen by their own mother  during their infancy, and the feeding method that they chose for their own baby. Their obstetric gynecologist was also required to answer questions regarding pregnancy/delivery complications, the health condition of the baby, and the medical history of the pregnant mother .The dataset used for analysis in this study was extracted from the APP Nagasaki database after obtaining approval from the Ethics Committee of the Institutional Review Board of Nagasaki University Hospital, Nagasaki, Japan . The APP Nagasaki was initiated in 1987, and modern techniques to measure the HTLV-1 PVL using an RT-PCR assay have been applied to participating mothers only since 2011 . Therefohttp://www.openepi.com, Atlanta, GA, United States; https://CRAN.R-project.org). To analyze changes over time in the HTLV-1 PVL by age and by parity of the HTLV-1-positive mothers, we conducted repeated measures correlation (rmcorr) analyses using the R package version 0.3.0 . All statistical tests were performed at the two-tailed 5% level of significance.We analyzed the available data on pregnant mothers who were tested for HTLV-1 infection at least twice during pregnancy. The demographic data were summarized with standard descriptive statistics, such as frequencies (percentages) for categorical variables, and median (min–max) or median  values for continuous variables. The pregnant mother age at enrollment in the APP was treated as a continuous variable or categorized into four groups . The method of feeding that the pregnant mothers themselves received from their own mothers was categorized into three groups: long-term breastfeeding (6 months or longer), short-term breastfeeding (shorter than 6 months), and formula feeding alone. The mothers were also categorized into subgroups based on their history of gravidity (the number of pregnancies), parity (the number of live births), and divorce. Continuous variables were compared using Wilcoxon rank-sum tests. Categorical variables were compared using the Chi-squared test or Fisher’s exact test, as appropriate. All of the basic statistical analyses were performed using OpenEpi Statistical Software version 3.01  of the 16,730 pregnant mothers who were found to be negative for HTLV-1 during the initial screening subsequently seroconverted to HTLV-1 positive . Thus, tn = 133) and those of mothers who were HTLV-1 seronegative at study entry but later seroconverted to anti-HTLV-1 antibody-positive (n = 9). The median age at first entry to APP Nagasaki was 30 years old  in mothers of known HTLV-1-seropositive status and 31 years old  in HTLV-1-seroconverted mothers. Although there was no statistically significant difference in the age distribution between the two groups, approximately half of those already known to be HTLV-1-seropositive were in their late 20 s at their first pregnancy, whereas approximately half of the HTLV-1-seroconverted mothers were in their 30 s at their first pregnancy. There was also no significant difference in the proportions of mothers with a family history of ATL, a divorce history, or particular screening test values or in the proportion of western blot-positive or -indeterminate individuals between the two groups.p < 0.024). Regarding whether the mothers had known about the HTLV-1 infection status of their husband or sexual partner, only two pregnant mothers reported HTLV-1 infection in their partner; the rest of the study participants did not know their partner’s HTLV-1 infection status.In contrast, there were significant differences between the two groups regarding the feeding method that the participating mothers themselves received from their mothers , gravidity history, parity history, and HTLV-1 PVL. In brief, compared with the group of those already known to be HTLV-1-positive before pregnancy, the group of mothers that underwent HTLV-1 seroconversion between pregnancies had lower HTLV-1 PVL  the proportion of those who underwent seroconversion from anti-HTLV-1 antibody negative to positive among pregnant mothers was 0.054%, with a seroconversion incidence rate of 17.5 per 100,000 person-years during the period 2011–2018, (2) HTLV-1 infection had no adverse effect on pregnancy outcomes in either the HTLV-1-infected or HTLV-1-seroconverted mothers; however, (3) the HTLV-1 PVL increased with increased parity number, and this showed a statistically significant positive correlation in both the HTLV-1-infected and the HTLV-1-seroconverted pregnant mothers.The estimated incidence rate  of HTLV-1 seroconversion among pregnant mothers aged 17–41 years in this analysis was higher than that for female blood donors aged 16–49 years  in Kyushu-Okinawa, where HTLV-1 is endemic , despiteRegarding the PVL in HTLV-1-positive pregnant mothers, we previously reported that the PVL was the highest during the postpartum period  as compared with that during pregnancy terms , which suggests that HTLV-1-infected women of reproductive age are continuously exposed to HTLV-1 horizontally from their HTLV-1-infected partners. Therefore, further careful follow-up is needed for both HTLV-1-positive pregnant mothers and their babies.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the Institutional Review Board of Nagasaki University Hospital, Nagasaki, Japan . The patients/participants provided their written informed consent to participate in this study.NK and NF analysis the data of HTLV-1 test among pregnant women. MI and KM organized the study protocol. NK, MI, and KM wrote the manuscript. YH, SM, NF, and KM collected the data of HTLV-1 tests among pregnant women. HM collected the data regarding Mother to Child transmission of HTLV-1. KY and KM performed the HTLV-1 screening test of pregnant women in Nagasaki. All authors contributed to the article and approved the submitted version.This work was partially supported by the Research Program  on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) awarded to KM. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of AMED.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Its activity, as a master tuner of many mechanisms underlying the aging process, for example in the skin, is still presumptive, however its crucial position in the complex cross-talk between mitochondria and the process of cell survival, should suggest that l-lactate may be not a simple waste product but a fine regulator of the aging/survival machinery, probably via mito-hormesis. Actually, emerging evidence is highlighting that ROS are crucial in the signaling of skin health, including mechanisms underlying wound repair, renewal and aging. The ROS, including superoxide anion, hydrogen peroxide, and nitric oxide, play both beneficial and detrimental roles depending upon their levels and cellular microenvironment. Physiological ROS levels are essential for cutaneous health and the wound repair process. Aberrant redox signaling activity drives chronic skin disease in elderly. On the contrary, impaired redox modulation, due to enhanced ROS generation and/or reduced levels of antioxidant defense, suppresses wound healing via promoting lymphatic/vascular endothelial cell apoptosis and death. This review tries to elucidate this issue. A possible reason can be hypothesized by observing the reduction in GLUT-1 receptors alongside with the aging process  pathway was reported  , which act as transcription factors, modulate the genetic expression of several enzymes generating uridine monophosphate, alongside with LDHA, therefore controlling l-lactate production  to glycolysis via HIF-1α and stabilizes the activity of the same hypoxic factor HIF-1α, probably in order to support the signaling function of ROS in human fibroblasts  and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) with l-lactate in the regulation of a glycemic-induced senescent phenotype should be particularly interesting. In type 2 diabetic patients, with a chronical hyperglycemic state,  the role of l-lactate in bioenergetics and mitochondria biology; (b) the role of l-lactate in stem cell commitment in the skin and mesenchymal differentiation. Both functions are intertwined with ROS biology.Two different of fundamental routes are to be put in the spotlight to further elucidate the involvement of l-lactate in the mitochondria-ROS signaling, and therefore in the aging process, skin differentiation should be affected by l-lactate turnover and metabolism. Actually, l-lactate participates in the complex milieu of skin cellular components by interacting with the interplay stromal cells-immune cells, for example by switching off the pro-inflammatory immune response and promoting tissue renewal and repair  and its major cofactor, peroxisome proliferator-activator receptor gamma coactivator 1-beta (PGC1-β), are also involved   production  and Jagged-1 (Jag-1)  and the mitochondrial uncoupling protein 3 (UCP3) gene expression , reported that 4-hydroxynonenal (4-HNE), an α-β unsaturated hydroxy-alchenal from lipidic peroxidation, dampens the age-related increase in ROS, due to the increased state IV of oxidative respiration from mitochondria by the UCP3 overexpression , which regulates mitochondria biogenesis and mitophagy, i.e., mitochondrial and peroxisome fission, regulates also endoplasmic reticulum-generated droplets in the adipose tissue, so correlating the correct lipid storage in adipocytes with the mitochondrial survival  biosynthetic pathway may affect l-lactate regulation, as the CoQ10 deficiency, which may be caused by a homozygous stop variant in COQ9 c.730C > T, pArg244*, should lead to neonatal lactic acidosis, general development delay and intractable seizures , is fundamental to comprehend skin aging, particularly for photoaging, and the involvement of mitochondria biology  enhancement in intracellular proline pool and collagen synthesis , which is regulated by the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3k)/Akt, and protein kinase C (PKC) signaling pathways (Feng et al. l-lactate as a signaling molecule might provide biomedical research with heuristically valid insights, particularly about the role of this metabolite in lowering senescence in the skin and to elucidate further the mechanisms underlying skin aging.Aging in the skin is a major process where the activity of l-lactate is a major signaling molecule able to interact with mitochondria biogenesis and turnover and tune the ability of cells to respond to stressors;l-lactate works as a signaling molecule involve mechanisms known as “mito-hormesis”The fundamental way by which l-lactate plays fundamental roles in signaling of brain development related with exercise and in immune regulation;Despite its nature of apparently useless byproduct, Its role in skin rejuvenation and aging control can be supported by the survival machinery led by mitochondrial biogenesis.In this review we have fundamentally highlighted that:l-lactate should be included in the wider role of small molecules working as signaling compounds, able to finely regulate cell survival, cycle and development. In this sense, the importance to deepen the activity and the biological meaning of this glycolysis byproduct is paramount.The role of


Results reveal that all HHP-treatments produced the highest (p < 0.05) log-reduction of molds (log 1.85 to 3.72), and yeast (log 3.19), in comparison with PEF-treatments. Increments in pH, acidity, and TSS values attributed to compounds’ decompartmentalization were found. PME activity was partially inactivated by HHP-treatment at 600 MPa/10 min (22% of inactivation) and PEF-treatment at 200 Hz/168.4 kJ/L (19% of inactivation). Increment in vitamin C and TPC was also observed. The highest increment in TPC (79% of increment) and vitamin C (77% of increment) was observed with PEF at 200 Hz/168.4 kJ/L. The putative effect of HHP and PEF on microbial safety, enzyme inactivation, and phytochemical retention is also discussed in detail. In conclusion, HHP and PEF improve phytochemical compounds’ content, microbial safety, and quality of black/red raspberry juice.Black and red raspberries are fruits with a high phenolic and vitamin C content but are highly susceptible to deterioration. The effect of high hydrostatic pressure (HHP 400–600 MPa/CUT-10 min) and pulsed electric fields  processes on black/red raspberry juice was studied. The effect on the inactivation of microorganisms and pectin methylesterase (PME) activity, physicochemical parameters , and water activity ( Rubus occidentalis L.) and red raspberry (Rubus idaeus L.) are two fruits, belonging to the Rosacea family, whose name is derived from the Anglo-Latin term raspise which means “a sweet rose-colored wine” [Black raspberry , and PME activity, as well as on the phytochemical parameters and vitamin C and phenolic compounds content.L-ascorbic acid (AA), methanol, citrus pectin , ammonium acetate, gallic acid (GA), and Folin–Ciocalteu’s phenol reagent were purchased from Sigma-Aldrich . Sodium carbonate, sodium chloride, and sodium phosphate dibasic were purchased from J.T. Baker, . Potassium chloride and sodium hydroxide were purchased from Fermont . Water was provided by a Milli-Q water purification system .Acetic acid (99.9%), Rubus ulmifolius L.) and red raspberry (Rubus idaeus L.) fruits from Organic Driscoll’s, México, in the mature stage for consumption were purchased in a local supermarket in Monterrey, Mexico. The fruits were carefully selected to avoid deteriorated berries and they were then washed with tap water. Then, 3.3 kg of each berry (1:1 w/w) was mixed using an industrial blender  for 1 min until obtaining a homogenous sample. Concentrated juice was diluted at 70% by adding distilled water. The final juice has 7.5 °Brix and the pH was adjusted to 3.25 with distilled water and citric acid, respectively [Black raspberry (ectively . After twa) was determined with an AquaLab  at room temperature (25 °C) [The pH of the black raspberry/red raspberry juice was determined by the official method AOAC 981.12.) using a potentiometer , and the TSS was expressed as °Brix using a refractometer , water activity  and vacuum-sealed  for HHP treatments in an AVURE 2 L HHP unit  using water as a pressurizing medium at room temperature (25 °C) as an initial condition (20 °C). Samples were treated at 400, 500, and 600 MPa during the come-up time (CUT), 2, 5, and 10 min. Each treatment was carried out by duplicate and untreated samples were used as a control. Due to adiabatic heating during the holding time, the average process temperatures were 27, 29, and 31 °C at 400, 500, and 600 MPa, respectively [The juice was treated in a continuous-flow, system ELCRACK-HVP 5 . The PEF system delivers square-wave pulses in bipolar mode and is composed of two cells that have two stainless steel electrodes separated by a gap of 7 mm at a flow rate of 60 L/h. The juice samples were treated at frequencies of 100, 200, and 500 Hz, with a pulse width of 25 μs, a pulse number of 100, an electric field strength from 11.3 to 23.3 kV/cm, and specific energy from 19.7 to 168.4 kJ/L. About 100 mL of the sample was collected at the outlet of the equipment, placed in polyethylene bags, and stored at 4 °C until analysis. Each treatment was carried out by duplicate and untreated samples were used as a control. Immediately after processing, the juice was analyzed for microbial and enzyme inactivation, and phytochemical content.Molds and the yeast population of the black/red raspberry juice were determined in the untreated juice and the treated juice immediately after processing by plate count agar according to Official Methods of Analysis ,28. Aftesample) was mixed with 20 mL of a solution consisting of 1% citrus pectin and 0.1 M NaCl previously maintained at 44 °C on a hot plate  to have a final temperature of 30 °C after mixing with the juice. The mixture was maintained at constant agitation (1200 rpm) at 30 °C  and then the pH ) was immediately adjusted to approximately 7.0 with a 2N NaOH solution and readjusted at pH 7.7 with a 0.5 N NaOH solution. An excess of 30 µL (VNaOH) of 0.5 N NaOH (NNaOH) was added, and the time (t) required to return to pH 7.7 was registered. The PME activity (At) at each treatment condition was calculated with Equation (1) and the PME residual activity (RAt) was expressed as a percentage of the untreated sample with Equation (2).PME activity was measured according to the methodology indicated by Escobedo-Avellaneda et al. . Briefly3PO4 43%: bipyridyl 4%: FeCl3 3%; in a percentage content of 33%, 26.7%, 26.7%, and 13.3%, respectively were added to all assay tubes. After 1 h incubation at 37 °C, 200 μL of the sample was transferred to a Costar clear 96-flat bottom well microplate , and the absorbance was measured at 525 nm (maximum absorption point confirmed by wavelength scan 400 to 650 nm) in a microplate reader . A five-point calibration curve (R2 = 0.9948) in a 0.1–1.0 mM range of AA was used to obtain vitamin C concentrations as mg AA/100 g sample on a wet basis. Then, three replicates with triplicates analyses were performed (n = 9) [The vitamin C content was determined according to that described by Escobedo-Avellaneda et al.  with som (n = 9) .v/v). The mixture was vortexed at 3000 rpm for 1 min  and placed in a water bath at 90 °C for 2 h  shaking in a vortex every 30 min. After that, the sample was centrifuged at 5000 g/4 °C for 10 min. The supernatant was recovered and made up to 5 mL with methanol. For the phenolic analysis, 50 µL of the methanol extract was mixed with 650 µL of distilled water and 50 µL of the Folin–Ciocalteau reagent. After 5 min at room temperature (25 °C), 250 µL sodium carbonate 0.5 M was added. After 2 h incubation at 37 °C, 200 μL sample was transferred to a Costar clear 96-flat bottom well microplate , and the absorbance was measured at 765 nm in a microplate reader . A six-point calibration curve (R2 = 0.9966) in a 50–300 ppm range of GA was used to obtain phenolic concentrations as mg GAE/100 g sample on a wet basis. Following this, three replicates with triplicates analyses were performed (n = 9) [Total phenolic compounds were measured according to the method described by Escobedo-Avellaneda et al. . Phenoli® 16.1.1 Statistical Software for Windows, USA).Results were reported as the mean and standard deviation of three independent samples (triplicate). The analyses of variance (ANOVA) were performed at each treatment and a Tukey test at a 95% CI was carried out to identify significant differences between mean values . The application of 400 MPa under CUT condition led to less than 1.85-log reduction, while, at the same pressure (400 MPa) but with an increment in time of 2 min achieved a 3.72-log reduction of molds inhibition. This suggests that HHP-treatment of 400 MPa required at least 2 min to produce a microbial inactivation, while higher pressures such as 500–600 MPa require at least CUT conditions to produce 3.72-log reduction.Results showed that the initial microbial population of molds and yeasts was 5.30 × 10log reduction) at all conditions tested. Similar inactivation results have been reported in different juice drinks [Regarding the effect of the HHP-treatments on yeast inactivation, it was observed that it was completely inactivated  and yeasts (1.55 × 103 UFC/mL) during the PEF treatments was less reduced in comparison with HHP treatments a values of the black/red raspberry juice subject to HHP and PEF treatments are shown in The results of the pH, titratable acidity (% citric acid), total soluble solids (°Brix), and water activity (p < 0.05) after the treatments of HHP and PEF. In the HHP-treated juice, the pH value was in the range of 3.39 to 3.45, while in the PEF-treated juice the pH value was from 3.36 to 3.38. Suggesting that HHP-treatments produce a higher effect on the pH value in comparison with PEF-treatments  pH value (3.38 ± 0.01) was obtained in the PEF-treatments of 100 Hz/19.7 kJ/L, 500 Hz/113.8 kJ/L, and 100 Hz/79.7 kJ/L, while the lowest pH value (3.36 ± 0.01) was in the treatment of 200 Hz/42.9 kJ/L. This suggests that frequency plays an important role in the change in pH value, i.e., the higher increment in the pH value was observed at the highest frequency (500 Hz) and the lowest frequency (100 Hz); while the lowest increment in pH value was observed at the frequency of 200 Hz. The pH value of the HHP and PEF-treated black/red raspberry juice and untreated juices classifies them as acidic products, as reported by Aganovic et al. [In the black/red raspberry juice treated with HHP, the highest pH value was observed in the HHP-treatment of 500 MPa/10 min (3.45 ± 0.01), followed by 400 MPa/10 min (3.43 ± 0.01) and 600 MPa/10 min (3.41 ± 0.01). The lowest pH value was observed in the HHP-treatment of 400 MPa/2 min (3.36 ± 0.01). These results suggest that the change in pH value was dependent on the pressure and time of the treatment. At the pressure of 400 and 500 MPa, the pH increases with the increment in the treatment time, thus, a higher pH value is observed during the treatment time of 10 min rather than 2 min; while at the pressure of 600 MPa the contrary case is observed, the higher pH value is observed in the CUT, rather than 10 min of treatment. For the PEF-treated juices, the highest  for all HHP (0.8%) and PEF (0.9%) treated juice obtained (Titratable acidity (TA) measures the total acid concentration and salts contained in a fruit . In thisobtained . These rp < 0.05) was observed. All HHP-treated samples presented 8.0 ± 0.06 °Brix and all the PEF-treated juices showed 8.5 ± 0.06 °Brix. This increment was attributed to the release of compounds from the cell which dissolve in the aqueous phase and become part of the fruit juice, incrementing the TSS [In fruits, sweetness  is one of the desirable characteristics of commercially grown and a lack of it has been associated with low total soluble solids  content and pH value ,43. In t the TSS . Just as the TSS . Thus, t the TSS .wa) measures the availability of free water in a food system that is responsible for any biochemical reactions [wa can determine a food’s shelf stability [wa value in the controls and the HHP and PEF-treated juice of black/red raspberries were in the range of 0.97–0.98, and these values were statistically equal (p > 0.05), indicating that HHP and PEF treatment do not produce an effect in wa value. Generally, foods with an wa value < 0.6 are microbiologically more stable [wa value in the juice could be favorable to microbial growth, and any biochemical reactions responsible for juice deterioration.Water activity . Considering all of the physicochemical parameter results of the black/red raspberries juice, it was observed that HHP and PEF treatments produce an increment in the pH value, %TA, and °Brix, while the p < 0.05) from 6 (400 MPa/10 min) to 14% (400 MPa/CUT). In contrast, the treatment at 500 and 600 MPa caused a reduction from 7 to 22% in the PME activity (p < 0.05). The treatment at 500 MPa caused a reduction in PME activity (p < 0.05) at times of 10 min (13%), 5 min (9%), 2 min (7%), and CUT (7%). For samples subjected to 600 MPa, the same behavior was observed; however, a higher PME activity reduction in comparison with 500 MPa was observed. In the HHP at 600 MPa, the higher reduction (p < 0.05) was obtained after 10 min (22%), followed by 5 min (17%), 2 min (12%), and CUT (12%). These results reveal that among the HHP-treatments, the highest inhibition (p < 0.05) was observed with the HHP-treatment of 600 MPa/10 min (22% of reduction). Regarding PEF treatments, the higher inhibition was observed at 200 Hz/168.4 kJ/L (19%), followed by 100 Hz/79.7 kJ/L (13%), and 500 Hz/113.8 kJ/L (13%); while the lowest inhibition was obtained at 100 Hz/19.7 kJ/L (7%) and 200 Hz/42.9 kJ/L 9%). Overall results, the HHP and PEF treatments decrease the PME activity. This reduction was higher with the HHP-treatment at 600 MPa/10 min (22%), followed by PEF at 200 Hz/168.4 kJ/L (19%).In this work, the residual PME activity of the black/red raspberry juice untreated (controls) and treated under different conditions of HHP and PEF treatments was determined A. TreatmAccording to the PME activity, for HHP-treatments two effects were observed, an increment and a reduction of the PME activity, while for PEF only decrements in the PME activity were observed. The increment of the PME activity during the HHP treatment at 400 MPa  would be related to the enzyme liberation of the structure cell after the HHP-treatment. Once the enzyme and its substrate are decompartmentalized, they interact, and the enzyme reaction takes place leading to increased enzyme activity in the juice treated. This increment is also attributed to the reversible configuration of the enzyme after HHP-treatment, leading to structural changes that favor enzyme-substrate interaction  or the rL-ascorbic acid), therefore, the effect of the HHP and PEF treatments on the vitamin C in the black/red raspberry juice was determined (p < 0.05) in comparison with the control. The PEF treatments presented a concentration of 0.564–0.609 mg AAE/100 g of the sample, indicating an increase of 64–77%. In the HHP-treatments of 400 MPa, the CUT caused an increment of 67%, and in the treatment time of 2, 5, and 10 min, the increment was 56, 55, and 50%, respectively. The HHP-treatments of 500 and 600 MPa present the same behavior, the higher increment was in the CUT, followed by 2, 5, and 10 min. However, 500 and 600 MPa presented a lower increment (p < 0.05) in comparison with the treatment of 400 MPa. In the PEF treatments, vitamin C content also presents an increment (p < 0.05). In the treatment of 100 Hz/19.7 kJ/L, 200 Hz/42.9 kJ/L, and 500 Hz//113.8 kJ/L an increment of 64 and 65%, was obtained, while in the treatments of 100 Hz/79.7 kJ/L and 200 Hz/168.4 kJ/L, increments of 67 and 77%, respectively, were observed. The higher vitamin C content observed could be due to the capacity of HHP and PEF treatments to release the compound from compartments containing high concentrations of vitamin C, such as the cytosol and vacuole, producing an increment in the content [Berries are fruits with high content of vitamin C  in the juice was determined C. The TPThe increase in the phenolic compounds’ content by the HHP and PEF treatments can improve the intensity of color in the final beverage, as well as the nutritional value and antioxidant activity, attributed to the phenolic compounds’ bio-accessibility and bioavailability. In berries, the main phenolic compounds include flavonoids, anthocyanins , flavonols , flavanols (catechins and epicatechin). As well as phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids) and hydrolyzable tannins, such as ellagitannins. Therefore, the increment in TPC would be related to the increment of these components, which are mainly responsible for the health benefits of the consumption of berries.wa values), retention of vitamin C and phenolic compounds, and the enzyme inactivation of the PME activity of the black/red raspberry juice was proposed . The PME catalyzed the formation of insoluble calcium pectate gels which react with calcium ions (Ca2+) present in the juices, and then precipitate pulling the cloud with them causing juices clarification [2+  cross-linking of demethylated pectin was activated by the HHP treatment at 400 MPa but inhibited by the HHP-treatments at 500–600 MPa, and the PEF treatments at 500 Hz/113.8 kJ/L, 100 Hz/79.7 kJ/L, and 200 Hz/168.4 kJ/L. This suggests that the effect on PME activity depended on the pressure and electrochemical effect applied. Due to this, the HHP and PEF produce changes in the structure and conformation of enzymes, resulting in the modification of their enzyme catalytic activities [α-helix, β-sheets, etc.), tertiary , and quaternary (number and arrangement of protein subunits) structures of the protein [Results also reveal that HHP and PEF treatments favor other biochemical reactions that lead to activation or inhibition of the PME activity  log-reduction of molds (log 1.85 to 3.72) and yeast (log 3.19), in comparison with PEF treatments. Important differences regarding the quality values of the pH, %AT, and TSS (◦Brix), attributed to the compounds’ decompartmentalization and release by HHP and PEF treatments were found. An increment in the TPC and vitamin C in the HHP and PEF treated juice was also observed. Both phytochemical compounds are associated with a fresh flavor; therefore, HHP and PEF increased the quality of the black/red raspberry juice. The optimum condition for the highest increment in TPC (79%) and vitamin C (77%) was the PEF treatment with 200 Hz and 168.4 kJ/L. At this PEF condition, the inactivation of the PME activity of 19% and microbial inactivation of molds (2.12-log reduction) and yeast (3.19-log reduction) were obtained. Conversely, the optimum HHP condition for the highest increment in vitamin C was 400 MPA-CUT, while, for microbial safety, it was 400 MPA−2 min (3.72-log reduction of molds and 3.19-log reduction of yeast), and for PME activity inactivation (22% of inactivation) the optimum HHP-condition was 600/10 min. Overall, the effect on the black/red raspberry juice was dependent on the HHP and PEF treatment applied, as well as, on the microorganisms and phytochemical compounds. In addition, a putative effect of HHP and PEF treatments on microbial safety, enzyme inactivation, and phytochemicals retention was also disused in detail. Further studies with more treatment conditions should be applied to reach higher enzyme inactivation. This study showed the effect of the HHP and PEF processes on black/red raspberry juice. Both HHP and PEF are two non-thermal technologies that can be applied for microorganism inactivation and PME activity on fruit juice. Especially all HHP-treatments achieved the highest (


Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. are important vector-borne parasites of humans and animals. CNM is a relatively recently discovered pathogen of humans. Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide. The present study aimed to determine the prevalence of CNM and Hepatozoon spp. in three species of Microtus and to assess the occurrence of vertical transmission in naturally-infected voles. Molecular techniques were used to detect pathogen DNA in blood and tissue samples of captured voles and their offspring. The prevalence of CNM in the vole community ranged 24–47% depending on Microtus species. The DNA of CNM was detected in 21% of pups from three litters of six infected Microtus dams  and in 3/45 embryos (6.6%) from two litters of eight CNM-infected pregnant females. We detected Hepatozoon infection in 14% of M. arvalis and 9% of M. oeconomus voles. Hepatozoon sp. DNA was detected in 48.7% of pups from seven litters  and in two embryos (14.3%) obtained from one M. arvalis litter. The high prevalence of CNM infections in the Microtus spp. community may be a result of a relatively high rate of vertical transmission among naturally infected voles. Vertical transmission was also demonstrated for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens. Candidatus Neoehrlichia mikurensis (CNM) is a relatively recently discovered tick-borne pathogen from the family Anaplasmataceae3, one of the aetiological agents of so called ‘tick-borne fever’5. Neoehrlichiosis affects mainly immunocompromised individuals and has been diagnosed also in dogs6. At least eight species of rodents  have been recognised as reservoir hosts of CNM in Europe13, in addition to Rattus norvegicus, the latter species in the first report of the competence of rodents as reservoir for these bacteria1. In Central Europe, the main vector of CNM is Ixodes ricinus with reported prevalence ranging between 0.1–24.3%14. In Poland, CNM has been detected in I. ricinus ticks from different habitats including city parks/forests and natural forests with generally low prevalence (0.3–2.9%)16. Furthermore, CNM has been identified in five immunocompetent asymptomatic foresters from North-Eastern Poland17. However, data on the reservoir hosts of CNM in the region of Poland is still fragmentary18.8, there is also evidence for efficient vertical transmission of CNM in different species of rodents from Germany10.In addition to confirmed transmission by ticksHepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide26. As Hepatozoon does not affect livestock or humans, the systematics and transmission routes of these parasites are not well recognised, with many novel species/genotypes identified in rodent hosts still waiting for complete valid descriptions26. Only two main species parasitising dogs, Hepatozoon americanum and Hepatzoozon canis, are well studied22. Canine hepatozoonosis caused by H. canis is a common infection in dogs, originally reported from the Mediterranean area of Europe, and more recently also from Central Europe. The first cases of H. canis infection in Central Europe were recently recorded in dogs in Hungary27, Ukraine28, the Czech Republic29, Poland , and Germany30. Imported H. canis cases were also recently diagnosed in the United Kingdom31.Apicomplexan protists of the genus Hepatozoon spp. are vector-borne parasites, transmitted by the ingestion of different arthropods, including fleas (for rodent species) and ticks, for example the brown dog tick Rhipicephalus sanguineus for H. canis34. Other routes of transmission are also suspected, including intake of infected prey (i.e. infected rodents hunted by snakes24) and vertical transmission. Vertical transmission has been reported for H. canis in dogs in Japan35. The high prevalence of H. canis in free-living carnivores in Central Europe, in absence of the tick vector, R. sanguineus, has led to the conclusion of a possibly high efficiency of transplacental H. canis transmission in red foxes, grey wolves, and golden jackals39. In Poland, high prevalence of Hepatozoon spp. has been recorded in red foxes, but also in common woodland rodents, i.e. bank voles (Myodes (Clethrionomys) glareolus)40. Hepatozoon infection was detected also by microscopy in our previous study in common voles, Microtus arvalis41.Hepatozoon spp. in three species of voles, based on molecular typing of parasites and 2) to assess the occurrence of vertical transmission of these two vector-borne pathogens in naturally-infected voles.The present study aimed 1) to determine the prevalence of CNM and To investigate the occurrence of transplacental transmission, two field-based experiments were carried out. In the first year, we determined the presence of pathogens in embryos dissected from naturally infected females, since this should completely eliminate the possibility of vector-borne transmission to offspring.43.In the second year, to eliminate any possibility of contamination of offspring with maternal blood, we sampled pups obtained from captured, pregnant female voles that were ectoparasite freeMicrotus arvalis, root vole Microtus oeconomus, and field vole Microtus agrestis) were trapped in different microhabitats extending up gentle hills (greatest elevation 5 m) from two small mid-field ponds. The local terrain provides a sufficient difference in height for a gradation in physical conditions and vegetation: from marshland submerged during rainy periods that are a suitable habitat for the root vole, M. oeconomus, to a dryer grassland habitat preferred by M. arvalis. Individuals of M. agrestis were trapped mostly in the intermediate zones. Voles were trapped using mixed bait comprising fruit (apple), vegetables (carrot and/or cucumber), and grain. Two traps were set every 10 m along transects at dusk for five consecutive nights, and were checked each morning. Unoccupied traps were then closed after the morning inspection to prevent animals entering during daytime, when excessive heat from exposure of traps to direct sunlight might have affected animals detrimentally, and were re-baited and re-set on the following afternoon. Traps were closed also during periods of rainfall. All the captured animals were transported in their traps to the laboratory for inspection.Voles were live-trapped in the summers of 2013 and 2014, in long-term abandoned fields near Urwitałt (field station of the University of Warsaw), in the Mazury Lake District of North-Eastern Poland . Three species of voles  based on body weight and nose-to-anus length together with reproductive condition 42. Two thin blood smears were prepared from blood samples taken by the cardiac puncture of each animal trapped in 2013; additionally, 200 42. Identification of the Microtus species was performed as described previously43. Foetuses were isolated from the uteri, washed in sterile water, and frozen at a temperature of − 20°C42.In 2013, necropsies were carried out following terminal isoflurane  anaesthesia42, during which all ectoparasites were removed. A blood sample for blood smears and PCR examination was taken from the tail tip of each animal. Males, non-pregnant females, and juvenile voles were then released near to their trapping points. Females suspected of being pregnant were transferred to the animal house to be kept in vector-free conditions. Each female was placed in an individual clean sterile cage provided with sawdust, nest material, food , and water ad libitum, where they were kept until parturition and then with their pups. No ectoparasites were noted on these captive voles at any time after initial caging. Pups were kept together with their dams for one month. In the third week of life, the pups were weighed and blood samples were collected from their tail tips. Pups and dams were then released at the trap lines near to where the dams had been originally caught43.In the summer of 2014, all voles were live-processed under temporary anaesthesia as described in Tołkacz et al.43.Embryos were isolated from uteri and individually autopsied following two washes in sterile water, to minimise contamination with maternal blood. We necropsied 111 embryos from 20 litters  or Hemacolor  staining kits, according to the manufacturers' instructionsHepatozoon spp. under oil immersion (× 1000 magnification). A sufficient number of fields of vision were examined to enable up to 50 leukocytes to be inspected (no fewer than 200 fields of vision).Smears from all captured animals and pups were examined for Hepatozoon spp. DNA was used for the identification of infections in all trapped voles , embryos and pups. The primers and thermal profiles used in this study have been described previously45. The PCR amplification of the 470 bp fragment of the 16S rRNA with species-specific primers enabled the detection of CNM1. PCR amplification and sequencing of the 914 bp fragment of the heat-shock protein gene (groEL gene)45 were used for the detection and species identification of CNM. As positive controls, we used the genomic DNA of CNM extracted from the tick I. ricinus15.Specific amplification of CNM and Hepatozoon spp. were performed by PCR amplification and sequencing of the 660 bp gene fragment of the 18S rRNA, as described previously44. The DNA of Hepatozoon erhardovae from a bank vole19 was used as a positive control. Negative controls, consisting of sterile water, were included in each set of PCRs.The detection and genotyping of PCR products were subjected to electrophoresis on a 1.5% agarose gel, and stained with Midori Green stain . Samples that tested positive on two consecutive occasions were considered to be positive. Selected positive products from the PCR reactions were subsequently sequenced .Hepatozoon-positive PCR products derived from 18 trapped voles, 16 products obtained from pups, and two products obtained from embryos were sequenced from both directions .Thirty six 16S rRNA gene and 33 for groEL gene) from trapped voles and their offspring were sequenced .Eighty three CNM-positive PCR products (50 for https://www.megasoftware.net/). The evolutionary model was chosen according to the data and bootstrapped over 1000 randomly generated sample trees. The Maximum Likelihood method was used for tree-construction. Phylogenetic analyses encompassed the sequences obtained in the current study and sequences of Hepatozoon sp. and CNM deposited in the GenBank database46.All the sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) v. 11.0 open access software  maximum likelihood techniques based on log-linear analysis of contingency tables (in SPSS vs 21) was applied. The results are presented as percentages with 95% confidence limits in parentheses (CL), calculated with bespoke software based on the tables of Rohlf and Sokal (1995), by courtesy of F.S. Gilbert and J. M. Behnke from the University of Nottingham, UK. For analysis of the prevalence of infections in wild-caught voles, we fitted prevalence of infection as a binary factor with host species , host sex , host age , and year  used as factors43. Subsequent analyses were carried out for each host species separately.The statistical approach adopted has been documented comprehensively in our earlier publicationsHepatozoon and CNM in females/dams may facilitate congenital transmission to their embryos/pups, we fitted models with CNM infection of female/dam and embryo/pup as an additional factor . For each level of analysis in turn, beginning with the most complex model, involving all possible main effects and interactions, those combinations not contributing significantly to the explanation of variation in the data were eliminated stepwise, beginning with the highest-level interaction, as applied in our earlier papers43. A minimum sufficient model was then obtained, for which the likelihood ratio of χ2 was not significant, indicating that the model was sufficient in explaining the data. The success of vertical transmission to each litter, calculated as the fraction of positive pups/litter, was correlated with litter size using the Spearman rank correlation test (SPSS v. 21)43.For analysis of the prevalence in pups, we implemented pup survival as a binary factor . In order to test the hypothesis that co-infection of 43. All animal care in the current study was conducted in accordance with ARRIVE  guidelines 2.051.All of the procedures were conducted with the approval of the First Warsaw Local Ethics Committee for Animal Experimentation in Poland  according to the principles governing experimental conditions and care of laboratory animals required by the European Union and the Polish Law on Animal ProtectionM. arvalis; 76 root voles, M. oeconomus and 17 field voles, M. agrestis. Prevalence of Hepatozoon sp. infection, based on PCR results by year of study, host species, and sex is provided in Table Hepatozoon was found in M. arvalis (13.7% [95% CL: 9.1–19.8%]) and 9.2% [95% CL: 4.1–18.7%] of M. oeconomus tested positive, but no Hepatozoon infections were detected in M. agrestis . Differences in prevalence of Hepatozoon between the two years of the study, between males and females . Gamonts of Hepatozoon sp. were not observed in any of the inspected blood smears.In total, 217 voles of three species were trapped and sampled: 124 common voles, Hepatozoon-positive M. oeconomus female) were too small to enable isolation . Prevalence was 30% (95% CL: 16.3–48.3%) in pregnant M. arvalis and 20% (95% CL: 3.7–55.4%) in M. oeconomus females , Table Hepatozoon DNA in 97 embryos of the 17 Hepatozoon-negative females , however, none of the seven dams tested positive for Hepatozoon.The DNA of Hepatozoon sp. DNA was detected in 48.7% (95% CL: 33.9–63.2%) of pups , in comparison to 48% (16/33 [95%CL: 33.9–63.2%]) of positive pups from six M. arvalis dams , our Hepatozoon sequences clustered within a large clade composed by many Hepatozoon genotypes associated with rodents and reptiles from different parts of the world  and felids (Hepatozoon felis). The topology of the tree supported the closest similarity of Hepatozoon sp. from Microtus spp. to Hepatozoon erhardovae, originating from bank voles (M. glareolus) from the same location in NE Poland21. However, sequences from Microtus voles differed slightly from both main conserved genotypes of H. erhardovae in bank voles across Europe52 and formed a separate branch. Sequences of Hepatozoon obtained in a mother and in the offspring were identical. Representative sequence have been deposited in GenBank under accession number ON994872.In the phylogenetic tree inferred from the rld Fig. . This clMicrotus voles in the community. The highest prevalence of CNM was detected in M. agrestis (47.1% [95% CL: 25.3–71.3%]) and the lowest in M. oeconomus (23.7% [95% CL: 14.7–35.4%]) but the difference in prevalence between the three host species was not significant (NS).Prevalence of CNM infection by year of study, host species and sex is provided in Table χ2 = 9.05, df = 1, P < 0.05). There was a significant interaction of year of study and host age and prevalence of CNM . In 2013 prevalence declined gradually with increasing host age but in 2014, a year with generally low prevalence, this pattern was reversed  , could not be evaluated because of the early stage of pregnancy, with embryos too small to enable reliable isolation of fetal tissues.The DNA of CNM was detected in 12 euthanised pregnant females. Infection in four litter  of pups . There was also no significant difference in the percentage of congenital infections between male and female pups born to infected dams: 16% (3/19) of males and 29% (4/14) of females were CNM-positive.All the pups born to CNM infected dams survived in comparison to two litters of non-infected dams that died after delivery . There was almost no diversity among the obtained sequences displaying 99.8–100% identity to CNM ‘MgUR’ isolate (KJ561570) derived from a bank vole, M. glareolus, from the same area in our earlier study . A representative groEL sequence has been deposited in GenBank under accession number OP158204 . There was no diversity among the obtained sequences displaying 100% identity to CNM WAW5 isolate (KJ123754) derived from a asymptomatic patient in Warsaw17, but also to isolate Omsk-41_Micagr (MN736126) derived from M. agrestis in Syberia . A representative 16S rRNA sequence has been deposited in GenBank under accession number (OQ152532).Fifty sequences of the CNM Hepatozoon and CNM were detected in four dams . The vertical transmission of both Hepatozoon and CNM had occurred in three out of those litters. Another two dams (2014/25 and 2014/112) were infected with CNM but not with Hepatozoon and in this case congenital CNM infection was not detected in pups. Vertical transmission of Hepatozoon occurred in three litters of dams infected only with Hepatozoon but not with CNM .Co-infections of Hepatozoon infection in a dam was associated with Hepatozoon infection in pups . CNM infection in a dam was associated with CNM infection in pups . Hepatozoon infection in a dam was not associated with congenital infection of CNM, while CNM infection in a dam was not associated with congenital infection of Hepatozoon in pups (NS).In a minimal sufficient model obtained from this analysis, only Hepatozoon in a litter (percentage of the litter with Hepatozoon) with the success of vertical transmission of CNM in the litter (percentage of litter with CNM), for offspring of co-infected females/dams (n = 4) but no correlation was evident (NS).Furthermore, focusing on the infection status of offspring, we correlated the success of vertical transmission of Microtus vole community inhabiting a rural area in North-Eastern Poland. Moreover, we have provided further evidence that this high prevalence is likely to have been maintained by a significant rate of congenital infections . Our study is among the first to assess the prevalence of Hepatozoon sp. and to determine the genetic identity of this pathogen in a Microtus spp. community, providing support also for the possibility of vertical transmission of Hepatozoon among vole species.In the present study, we have reported on the relatively high prevalence of infection with the zoonotic bacterium CNM in a sympatric Microtus voles. Although CNM has been previously reported in M. arvalis and M. agrestis53, this is the first report of CNM in M. oeconomus. Thus, we have expanded the list of rodent species serving as reservoirs of these zoonotic bacteria. Our study has confirmed that rodents are the main reservoir hosts for CNM because no CNM infections have been detected previously in insectivores53 and other Neoehrlichia species have been found only in carnivores54.We have identified CNM in three species of Microtus species. This is a moderate rate of prevalence, similar values having been reported in at least seven papers for the most commonly studied rodent species in several countries in Europe56. In one study, much lower values  were reported7 and in two studies in Germany prevalence (> 55%) was found to be slightly higher compared to that in our work12. Hence, there is a some disparity in prevalence values for the most commonly studied species, ranging 0.3–33% for A. agrarius57, 1.7–65% for A. flavicollis57, 1.1–58% for M. glareolus57, and 11–33% for A. sylvaticus56.The prevalence of CNM in our vole community ranged 24–47% depending on Microtus spp. have been less well studied than mice and bank voles, with reported CNM prevalences of 5–30% for M. arvalis13 and 8% for M. agrestis53. The prevalence value reported in this study  is in agreement with these studies. Importantly, high prevalence of zoonotic CNM in Microtus spp. may be of greater significance than high prevalence in Apodemus spp. or M. glareolus, because Microtus spp. voles can live in close proximity to humans, inhabiting any kind of open areas  Microtus spp. populations can reach high densities, thus constituting an important wildlife reservoir of infection for ticks and humans. Low genetic diversity of these bacteria3, derived from human cases, ticks and rodents, supports a significant role of rodents as the source of infection for humans. Interestingly, in two previous studies carried out in Poland, similar high prevalences of CNM were detected in M. glareolus (18–30%) in the same region of the country (North-Eastern Poland)57, in two murine species in Warsaw (23% in A. flavicollis and 11% in A. agrarius) and in 24–50% of rodents  from South-Western Poland, near Wrocław57.Bartonella spp.43 and Babesia microti42 among three species of voles. In the present study we have extended this route of transmission to other pathogens, verifying that vertical transmission is also a key feature of infections with CNM and Hepatozoon sp. The DNA of CNM was detected in embryos and pups from infected Microtus females. In total, the DNA of CNM was detected in 21% of pups born to CNM infected dams and in 7.3% of embryos obtained from infected female voles. Thus, we have confirmed the occurrence of vertical transmission in two Microtus spp. Our findings support the previous report of vertical transmission of CNM to embryos and neonates of three rodent species from Germany10 and the discovery of a CNM-positive foetus in a litter of an A. flavicollis female from Slovakia7.The present study was planned to investigate the phenomenon of vertical transmission of vector-borne pathogens, bacteria and protists in naturally-infected rodent populations. In our previous papers we documented the occurrence of vertical transmission for 10: congenitally infected offspring were identified in 60% (9 out of 15 ) of litters, with a CNM prevalence of 34%  in rodent foetuses and neonates from positive dams. Among those 15 litters, congenitally infected offspring were found in: 7/12 litters of M. glareolus, 1/1 litter of A. flavicollis, and 1/2 litters of M. arvalis10.Moreover, the successful detection of vertical transmission of CNM in our study supports results presented by Obiegała et al. (2014)Bartonella in this vole community43. The highest prevalence in the youngest voles is consistent with vertical transmission of CNM in vole populations. Furthermore, as stated earlier, a prevalence of CNM in rodents up to 10 times higher than in tick populations supports the existence of transmission routes other than tick-borne and confirms the key role of rodents as reservoir hosts16.In the present study we observed also a declining prevalence of CNM infection with increasing age in the free-living voles sampled in 2013. A similar pattern has been described for prevalence of Hepatozoon spp. have been reported in at least nine rodent species in Europe63 but by far the majority of studies concern M. glareolus (as the main host of Hepatozoon) and other host species have been studied rarely. Also among the studied species, prevalence of Hepatoozoon sp./H. erhardowae has been reported to be highest in M. glareolus in Europe, ranging 17–88%64. In our long-term study on haemoparasites in Masuria, North-Eastern Poland21, prevalence of H. erhardovae oscillated in the range 40–70% in bank voles during an 11 year period. Interestingly, two main genotypes of H. erhardovae (BV1 and BV2) seem to be highly conserved and distributed across distant regions of Europe- Spain, South Germany, Poland52.Infections of Hepatozoon is generally lower in Apodemus spp., ranging 5–28% for A. flavicollis and 18–30% for A. sylavaticus63. There are few studies of Hepatoozoon in Microtus spp. voles, and prevalence, determined solely based on microscopy, has been reported mostly as zero64 or as a single positive individual40. Prevalence of Hepatoozoon sp./H. lavieri, with identification based on molecular techniques, has ranged 3–9% in M. arvalis61; 2–10% in M. agrestis62 and 7% in M. oeconomus from North-Eastern Poland40. In the current study, we detected Hepatozoon infection only in two species of voles, in M. arvalis (14%) and M. oeconomus (9%), but with slightly higher prevalence than reported previously.Prevalence of Hepatoozoon sequences that we obtained were very similar (95–100% homology), and based on the topology of the phylogenetic tree, they are likely to constitute either a Microtus-adapted variant/genotype of H. erhardovae or, less likely, a different species (H. lavieri?25). However, based on 18S rRNA there is apparently little diversity among Hepatozoon isolates obtained from various rodents, amphibians and reptiles26.In our study all the Hepatozoon in rodents by the detection of DNA in embryos and pups. Success of vertical transmission was high for pups, close to 50% both in six litters of M. arvalis and in one litter of M. oeconomus. However, no Hepatozoon infection was detected in dams. The lack of detection of Hepatozoon in dams may have been due to the low burden of parasites, confirmed also by a failure to detect Hepatozoon gamonts by microscopy in PCR-positive animals. Low burdens of parasites are typical for chronic infections and it may be pertinent that we have recently described successful vertical transmission of B. microti from chronically infected BALB/c mice to their offspring, while no such transmission occurred during the acute phase of infection66.One of the main findings of the present study is confirmation of vertical transmission of Hepatozoon griseisciuri was described in naturally-infected grey squirrels kept until partitution in a laboratory, under ectoparasite-free conditions67. Prevalence of infection was 92% in free-living grey squirrels and different life stages of Hepatozoon were then observed in 19 out of 21 pups (90%) (36 h to 4 weeks in age) but no Hepatozoon stages were detected in histological sections of different organs of a single two-week-old embryo67. Interestingly, similar high success of vertical transmission has been observed for H. canis in Beagle dogs (23/29 [79%] of infected pups in six litters from 3 infected bitches35) and in red foxes (2/3 positive foetuses [67%] from an infected vixen37). Thus, vertical transmission appears to be an established route of transmission for these vector-borne parasites in different host species and our study of Hepatozoon in rodents is consistent with this idea. Vertical transmission is likely to significantly contribute to the maintenance and spread of Hepatozoon spp., even in areas where competent vectors do not occur. Further investigation is needed to examine the viability of the agents found in the offspring, and the exact route of transmission .More than 60 years ago, vertical transmission of Microtus spp. community may be the result of a relatively high rate of vertical transmission of CNM in three species of naturally infected voles. Vertical transmission was demonstrated also for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.The high prevalence of CNM infection in our Supplementary Information.