Among the 389 SPPs, 22 SPPs  caused CSs in the separate isolation room. A total of 219 SPPs (56.30%) were male. The mean age of SPPs was 4.34 ± 3.92 years old, and the mean CT value was 12.44 ± 5.11. In total, 9952 samples with exposure history were taken, including 204 (2.05%) CSs. Among the CSs, the positive rate of flat surfaces was the highest in public areas (2.52%) and separate isolation rooms (4.75%). Disinfection methods of ultraviolet radiation + chemical irradiation significantly reduced the CSs in both the public area (0% vs. 4.56%) and the separate isolation room (0.76% vs. 2.64%) compared with the chemical method alone (P < 0.05). Compared with ordinary SPPs, CRPs were older (6.04 year vs. 4.23 year), and the male proportion was higher (72.73% vs. 55.31%). In particular, it was found that SPPs contaminated their surroundings and therefore imposed risks on other people. Environmental disinfection with ultraviolet radiation + chemical treatment should be emphasized. The findings may be useful to guide infection control practices for the Omicron BA.2.2 variant of SARS-CoV-2.This study aimed to investigate the effect of different environmental disinfection methods on reducing contaminated surfaces (CSs) by the Omicron BA.2.2 variant of SARS-CoV-2 in the fever clinic between March 20 and May 30, 2022, and to analyze the influences and related factors of CSs. This study includes survey data from 389 positive patients (SPPs) and 204 CSs in the fever clinic, including the CS type, disinfection method, length of time spent in the clinic, cycle threshold (CT) value, name, age, weight, mask type, mask-wearing compliance, hand-mouth touch frequency and sex. Associations between study variables and specified outcomes were explored using univariate regression analyses. Mask-wearing compliance had a significant negative correlation with CSs ( As the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rages in parts of China, an outbreak of this pandemic, which has brought great challenges, occurred in Shanghai, the country’s financial hub and the home of many of its top research institutions, from early March to late May, 2022  and has a human-material-human (H-M-H) communication model is still in dispute  of the fever clinic in Shanghai Children’s Medical Center affiliated with the Medical College of Shanghai Jiao Tong University were reviewed, including the CS type, disinfection method, length of time spent in the clinic, cycle threshold (CT) value, name, age, weight, mask type, mask-wearing compliance, hand-mouth touch frequency and sex Fig. .Fig. 1OvParents and/or legal guardians signed written informed consent. All data collection and analysis were approved by the local research ethics and clinical governance bodies.Disinfection Method (used in the vacant environment after SPPs were transferred).Chemical disinfection : The environment was cleaned first, and then 3000 mg of trichloroisocyanuric acid disinfection tablets (Lionser company) was dissolved in 3 L of deionized water (25 ± 2 °C) for 5 min. After being completely dissolved and evenly mixed, the solution was sprayed to disinfect the surfaces. The solution was allowed to sit on the surfaces for half an hour. Finally, the surfaces of the object were wiped with disinfection wipes. Using this method, the fever clinic was disinfected 2 times a day at 5:00 and 17:00.Ultraviolet radiation + chemical disinfection : The environment was cleaned first and then subjected to ultraviolet irradiation for 1 h (Shanghai Yuejin nonmagnetic ultraviolet disinfection vehicle ZXC-ii). The subsequent process was consistent with the chemical disinfection method described above.2, the entire surface was swabbed; if the surface was > 100 cm2, only an area of 100 cm2 was swabbed. The part of the swab that contacted the hand was cut off, and the cotton swab was placed into a test tube containing 10 ml of eluent for the sterility test. For small objects such as door handles, the cotton swab was directly applied to the surface of the object for sampling.A 5 cm*5 cm sterilization specification plate was placed on the surface of the tested object. A cotton swab soaked with sterile 0.03 mol/L phosphate buffer solution or normal saline sampling solution was applied horizontally and vertically to the specification plate 5 times, and then the cotton swab was rotated to continuously sample the area of the 4 specification plates. If the surface to be sampled is less than 100 cmReal time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) is the main diagnostic method of SARS-CoV-2 in clinical practice. Its basic principle is to monitor the growth of the product quantity in real time through the fluorescent labeled-specific probe, tracking the PCR product labeling in the reaction process, and calculating the initial template quantity according to the amplification curve. The SARS-CoV-2 genome contains the 5 'end replicase coding gene  and structural protein coding gene . Among them, ORF1ab fragment and N gene are genus and type specific genes of SARS-CoV-2. A positive case requires that the same sample be positive for both targets.All the object surfaces were divided into three categories, namely, flat surface , hand high-frequency contact surface  and electronic products (Eps). See Appendix P < 0.05 was considered statistically significant.SPSS 20.0 statistical software was used for analysis. The measurement data are expressed as the mean ± standard deviation; the counting data are expressed as the rate. One-way ANOVA was used for comparison of the means within the group, and the counting data were compared by the chi square test. Single-factor analysis was included to further examine the independent influencing factors of CSs with SPPs. All statistics were tested using a two-sided test, and From March 20 to May 30, 2022, a total of 485 SPPs went to the fever clinic, of which 389 were finally included in the study, and 22 SPPs (5.66%) caused CSs in the separate isolation room. A total of 219 SPPs (56.30%) were male. The mean age of SPPs was 4.34 ± 3.92 years old, and the mean CT value was 12.44 ± 5.11. In total, 9952 samples with exposure history (SPPs in the fever clinic on that day or in the separate isolation room) were taken, including 204 (2.05%) CSs . The CT value  and mask-wearing compliance  were both negatively associated with CSs; however, only mask-wearing compliance was statistically significant   of CSs in the FS, HHF and EP groups was 40 (2.52%), 22 (1.89%) and 11 (1.73%), respectively, in the PA and 87 (4.75%), 65 (3.00%) and 35 (1.82%) in the SIR. Among the three categories, FS had the highest positive rate, and EP had the lowest. However, there was no significant difference among the three groups (P < 0.05) , and the waiting time for the results is approximately 2.5 h. If positive, they will enter a separate isolation room (SIR) with a parent for separate isolation and treatment, waiting for transfer. Before the statistical analysis, negative samples with no exposure history (no SARS-CoV-2-positive children in the fever clinic on that day or in that SIR) were excluded. From March 20 to May 30, 2022, the number (rate) of CSs in the PA and SIR was 73 (2.15%) and 131 (2%), respectively. When using the chemical disinfection method, the number (rate) of CSs in the PA and SIR was 73 (4.56%) and 114 (2.64%), respectively. After using ultraviolet radiation + chemical irradiation, the CSs were 0 (0%) and 17 (0.76%), which both decreased significantly (Patients who caused CSs were defined as having contaminated surface-related SARS-CoV-2 patients (CRPs) and all other patients were defined as ordinary SARS-CoV-2-positive patients (OSPPs). The male proportion, age, weight, height, CT value, N95 mask-wearing rate and time spent in the room were 55.31%, 4.23 ± 3.89 years, 20.08 ± 13.72 kg, 101.85 ± 30.64 cm, 12.44 ± 5.15, 7.63% and 16.46 ± 4.65 h, respectively, in OSPPs and 72.73%, 6.04 ± 4.04 years, 24.38 ± 12.36 kg, 115.14 ± 27.67 cm, 12.39 ± 4.52, 9.09% and 14.52 ± 9.38 h in CRPs. Compared with OSPPs, CRPs were older and the male proportion was higher, but none of the differences were significant . Perrine Marcenac et al. question whether the virus can truly spread first through surfaces , high-frequency hand contact surfaces and electronic products . Among the three categories, flat surfaces had the highest positive rate, and electronic products had the lowest positive rate, meaning that a greater surface area rather than the amount of hand contact may affect the risk of transmission in indoor environments.Due to the design of consulting procedures, the Omicron BA.2.2 variant exposure modes in the PA and the SIR are not exactly the same. The PA is open and well ventilated, and each SPP enters while the SIR is separately closed, and each SIR is only accessed by one SPP. Previous studies on the environment of SARS-CoV-2 mainly focused on the confined space, and the exposure mode was similar to the SIR, while there were few studies on the PA, and there were different opinions on the CSs of the confined space .


To evaluate microcirculation and endothelial glycocalyx (eGC) variables using sidestream darkfield (SDF) videomicroscopy in canine cardiopulmonary bypass (CPB).Dogs undergoing CPB for surgical correction of naturally-occurring cardiac disease were prospectively included. Variables collected included patient demographics, underlying cardiac disease, red blood cell flow (Flow), 4-25 μm vessel density (Density), absolute capillary blood volume (CBVabs), relative capillary blood volume (CBVrel) and eGC width assessed by perfused boundary region (PBR). Anesthetized healthy dogs were used as control. Microcirculation and eGC variables were compared at baseline under anesthesia (T0), on CPB prior to cross clamping (T1), after cross clamp removal following surgical correction (T2) and at surgical closure (T3).p = 1.00). Median Density was 173.3, 118.4, 121.0 and 155.4 mm/mm2 at T0, T1, T2, and T3, respectively, (p = 1.00). Median CBVabs decreased over time: 7.4, 6.6, 4.8 and 4.7 103μm3 at T0, T1, T2, and T3, respectively, (p < 0.01). Median CBVrel increased over time: 1.1, 1.5,1.1, and 1.3 103μm3 at T0, T1, T2, and T3, respectively, (p < 0.001). Median PBR increased over time: 1.8, 2.1, 2.4, 2.1 μm at T0, T1, T2, and T3, respectively, (p < 0.001). Compared to control dogs (n = 8), CPB dogs had lower CBVabs at T0.Twelve dogs were enrolled, including 10 with a complete dataset. Median Flow was 233.9, 79.9, 164.3, and 136.1 μm/s at T0, T1, T2, and T3, respectively, (Alterations in eGC thickness and microvascular occur in dogs undergoing CPB for naturally-occurring cardiac disease. The microcirculation is defined as the terminal vascular network of the systemic circulation consisting of micro vessels with diameters <20 μm . The micChanges in microcirculation parameters and eGC shedding have been demonstrated during cardiopulmonary bypass (CPB), a procedure commonly performed in people, but seldomly in clinical veterinary medicine . Using SThe goal of our study was to investigate changes in microcirculatory parameters using SDF microscopy and assess PBR in client-owned dogs undergoing CPB for correction of a naturally occurring cardiac defect. We hypothesize that dogs undergoing CPB will have changes in microcirculatory parameters and an increased PBR.Dogs that presented to the James L. Voss Veterinary Teaching Hospital at Colorado State University from November 2020 to June 2022 were prospectively enrolled in the study if their naturally occurring cardiac disease required surgical correction under CPB. The study had prior ethical approval by IACUC and the Clinical Review Board (KP IACUC 1342). Informed written consent was obtained from the owner or legal custodian of all dogs described in this study for all procedure(s) performed.At the discretion of the board-certified anesthesiologist, dogs were premedicated with a pure μ opioid . Some dogs also received atropine (0.02 mg/kg subcutaneous or intramuscular) or glycopyrrolate (0.01 mg/kg intramuscular) as clinically indicated and at the anesthesiologist’s preference to maintain heart rate above 60 beats per minute. Anesthesia was induced with fentanyl , midazolam (0.2–0.3 mg/kg/h IV CRI), and lidocaine (30–120 μg/kg/min IV CRI). Blood pressure support was provided with dopamine (0.5–7 μg/kg/min IV CRI), dobutamine (1–10 μg/kg/min IV CRI) and/or phenylephrine (0.5–1 μg/kg/min IV CRI) as clinically indicated to maintain a mean arterial pressure above 60 mmHg prior to going onto CPB. Cefazolin (22 mg/kg IV) was administered every 90 min during surgery. Positive pressure ventilation was maintained throughout the procedure except during full CPB. During this period, isoflurane was administered via vaporizer placed in the gas line supplying the membrane oxygenator .2, pulse oximetry and esophageal temperature were monitored continuously during anesthesia. Arterial blood gasses, acid–base status and electrolytes, activated clotting time,A 7-Fr triple lumen catheterDogs were positioned in lateral recumbency for CPB and surgery. Prior to cannulation for CPB, heparin sulfate  was administered to achieve complete anticoagulation, defined as an activated clotting time over 480 s . AntifibSurgery was performed through a right  or left  fifth intercostal thoracotomy and pericardiotomy. For surgical repairs requiring a right atriotomy, tapes with Rommel tourniquets were placed around the cranial vena cava, caudal vena cava and azygous vein. Bicaval venous cannulation of the cranial and caudal vena cava was accomplished with 20 Fr and 22 Fr malleable single-stage venous cannulae,2/min measured by a flow meter placed on the arterial line. Timepoint T1 was defined as dogs under CPB.The primary CPB circuit consisted of a centrifugal pump, reservoir/membrane oxygenator, heat exchanger, and circulating heater/cooler water bath . The CPBWith the CPB pump on, the periaortic fat was dissected from the ascending aorta and a tape was passed around the ascending aorta. Two sutures were placed in the ascending aorta and passed through Rommel tourniquets. A 5 Fr cardioplegia cannulaThe ascending aorta was cross-clamped distal to the cardioplegia cannula. Cold  sanguineous cardioplegia solution was administered and repeated as appropriate during aortic cross-clamp, approximatively every 45 min .Metabolic acidosis during CPB was corrected by administration of sodium bicarbonate (0.5 to 1 mEq/kg) into the CPB circuit as needed to maintain a pH between 7.35 and 7.45 .After the specific surgical procedure was complete and cardiotomy closed, the aortic cross-clamp was removed, and coronary circulation was reestablished. The heart was electrically defibrillated, if necessary, with direct current (20 to 50 J), using internal paddles. Timepoint T2 was defined as the heart began beating on its own. Dogs were warmed to 38°C [100.4°F] and gradually weaned from CPB. During this period, calcium gluconate  was administered to keep the pH-corrected ionized calcium above 1.2 mmol/L. Dobutamine  and phenylephrine (0.5–3 μg/kg/min IV CRI) were administered as necessary to maintain a mean arterial pressure above 60 mmHg.During weaning from CPB, the left heart was vented by suction via the cardioplegia cannula. After weaning from CPB, at the discretion of the primary surgeon (CO), arterial–venous modified ultrafiltration was performed for 15–20 min with an ultrafiltration deviceOnce the dog had been weaned from CPB and was hemodynamically stable, cannulae were removed in the reverse order that they were introduced, and protamine sulfate  was administered slowly (over 30 min) until the activated clotting time returned below 120 s. Thoracotomy was closed in routine fashion, and defined T3.Surgical procedures included tricuspid valve repair , artificDemographic variables recorded included sex, breed, age, body weight and underlying cardiac disease. Macrocirculation variables reported were heart rate (HR), invasive mean blood pressure (MBP) and serum Lactate. Microcirculation variables were assessed using a SDF video microscope with proprietary software to calculate PBR as a surrogate for eGC thickness. All variables were recorded at the following timepoints: T0 (baseline), defined as the patient stable for at least 5 min under general anesthesia; T1 (on pump), defined as a patient on CPB prior to the aortic cross clamp being applied; T2 (after clamp), defined as after the aortic cross clamp was removed after the surgical procedure was performed, while still on CPB; and T3 (closure), during surgical closure when the patient was weaned off CPB.At the timepoints defined previously, a SDF video microscope was placed on the sublingual mucosa (see text footnote 1). The video microscope uses a 540 nm green light emitting stroboscopic diode to detect hemoglobin of passing red blood cells. Using a 5x objective with 0.2 numerical aperture, images were captured, providing a 325-fold magnification in 720 × 576 pixels.The software records movies of 1 s that consist of 23 frames. Recording is initiated automatically when the software deems the images of sufficient quality, meaning that the intensity and focus are sufficient for calculations and that the camera is held sufficiently still. Vessels are automatically detected, and measurement points are defined at 10 μm intervals. The software limits its calculations to vessels with a width between 5 and 25 μm. A measurement is complete when 3,000 measurement points have been acquired.Two researchers (DD and JG), both experienced in performing sublingual measurements with this device, performed one video acquisition for each dog at each timepoint. Care was taken to limit pressure artifacts by ensuring erythrocytes could be seen traveling through the blood vessels during the measurements. Images were recorded only in the absence of air bubbles, excessive amounts of saliva, excessive loops of the vessels and excessive amounts of large venules. All measurements in each dog at each timepoint were performed within 5 min.The analysis of all images was performed using a proprietary software.The PBR was calculated by the software as the dynamic lateral movement of red blood cells into the permeable part of the eGC. The inner layer of the glycocalyx is penetrable to red blood cells and, hence, also called PBR . An intaAs no data has been published using this specific SDF video microscope, microcirculatory variables and PBR measurements were performed for normal dogs under general anesthesia. (KP IACUC 2237). In brief, 8 healthy purpose-bred and ethically sourced Beagles were sedated with hydromorphone (0.1 mg/kg IM), induced with propofol (5–10 mg/kg IV to effect), and intubated. General anesthesia was maintained with isoflurane. Once stabilized under general anesthesia with an invasive mean arterial pressure of 70–80 mmHg for 10 min, the SDF video microscope was placed on the sublingual mucosa. Images were captured and the analysis of all images was carried out with a proprietary software to acquire the aforementioned variables.p ≤ 0.05. All statistics were performed using commercially available software.Descriptive statistics were used to describe patient demographics, underlying cardiac disease and surgical procedure performed. Because of the small sample size, data were reported as median (range) and non-parametric tests were used. Impact of the CPB and surgical procedures on HR, MBP, Lactate, Flow, Density, CBVabs, CBVrel and PBR between each time point was tested with Friedman test for repeated measures. Differences between specific timepoints in the CPB group were tested with a Wilcoxon test for paired samples. At T0, each measured variables  was compared to the control group using an unpaired Mann–Whitney test. Significance was set at A total of 13 dogs were prospectively enrolled. A device malfunction did not allow appropriate recording for all time points in one dog. Data were not recorded on T1 for 1 dog due to device malfunction, and on T4 for one dog due to cardiopulmonary arrest with unsuccessful cardiopulmonary resuscitation. Therefore, 12 dogs were included in the study, and 10 dogs had complete data sets available to be analyzed using Friedman test for repeated measures.n = 12), dogs had a median age of 1.8 years (0.7–6.9) and a median body weight of 29.6 kg (12.4–54.0). There were 7 females (4 spayed) and 5 males (1 neutered) included. Mixed breed dogs were the most frequently represented dog breed (n = 3). Other breeds included: Labrador Retrievers (n = 2), Australian Shepherd (n = 2), German Shepherd (n = 2), and one of each of the following breeds: Doberman, Rottweiler, and Great Dane.In the CPB group (n = 5), degenerative mitral valve disease (n = 3), Tetralogy of Fallot (n = 1), peri membranous ventricular septal defect (n = 1), mitral valve dysplasia (n = 1), and partial atrioventricular septal defect (n = 1). Surgeries performed under cardiopulmonary bypass included: tricuspid valve repair (n = 5), Alferri Stich of the mitral valve and annuloplasty (n = 2), ventriculotomy with patch graft (n = 1), ventricular septal defect repair with eight coronary cusps repair (n = 1), mitral valve repair with aortic root tear repair (n = 1), mitral valve repair and annuloplasty (n = 1) and partial atrioventricular septal defect patch (n = 1). The macrocirculation variables showed significant changes over time for HR and Lactate, but not for MBP , dogs had a median age of 5 years (n = 8). They were all female spayed Beagles.In the control group . Median time between the start of the CPB and T1 was 11.1 min (5.3–42.5). Median time between T0 and T2 was 206 min (158–263). Median time between T0 and T3 was 254 min (184–333).n = 10). Specifically, median Flow values were 233.9 μm/s at baseline (T0), and then decreased by 65.8, 29.8 and 41.8% at T1, T2, and T3, respectively, . Median Density values were 173.3 mm/mm2 at baseline (T0) and then decreased by 31.7, 30.2 and 10.3% at T1, T2 and T3, respectively, , and then decreased by 10.8, 35.1, and 36.5% at T1, T2, and T3, respectively, , increased by 36.4% at T1, returned to baseline at T2 (0% change), and increased by 18.2% at T3 , and increased by 16.7, 33.3, and 16.7% at T1, T2, T3, respectively, (n = 8) and CPB cases (n = 12) except for CBVabs. Specifically, the median Flow values were 298.7 μm/s (89.7–2313.9) and 278.7 μm/s (103.3–419.1) for the CPB and control group, respectively (p = 0.68). The median Density values were 173.3 mm/mm2 (70.4–582.7) and 237.1 mm/mm2 (103.5–334.3) for the CPB and control group, respectively (p = 0.68). The mean CBVabs were 18.3 103μm3 (4.4–21.9) for the control group compared to 7.4 103μm3 (1.5–18.3) for the CPB cases (p = 0.02). The median CBVrel were 1.08 103μm3 (1.22–3.44) and 1.26 103μm3 (0.94–1.38) for the CPB and control group, respectively (p = 0.39). The median PBR were 1.80 μm (1.22–3.44) and 2.37 μm (1.77–2.59) for the CPB and control group, respectively (p = 0.13).At T0, the microcirculation variables and PBR did not differ between the control population  down to a median of 79.9 μm/s at the start of the CPB. This can be explained by the fact that the extracorporeal blood flow of the CPB is significantly lower compared to anesthetized dogs. A normal anesthetized dog generates a cardiac output of 4.3 L/m machine , 27. How machine . It shou machine .In our study we were unable to detect a statistically significant decrease in vessel density in dogs undergoing CPB, although the lack of significance may be attributed to our small sample size, and contrasts with available literature in people which documents a progressive decrease in vessel density during CPB and a return to baseline. Specifically, in a study on 30 adults undergoing CPB and elective cardiac valve surgery evaluated using the same SDF device showed a significant decrease in vessel density from baseline to splitting of the sternum. The same study found this decrease intensified during CPB but returned to baseline in 24 h . The decThe capillary blood volume variables are new features available after a software update in the Fall of 2020. This represents the physical space available for blood flow (CBVabs) as well as that space relative to the actual size of the red blood cells (CBVrel) . Our stuOur study found changes in macrocirculatory parameters HR and Lactate over time. The MBP was unchanged over time, probably related to the efforts from the anesthesiologist team to maintain a normal blood pressure. Changes in HR, lactate and Flow between 2 specific time points are consistent with a hemorrhagic shock model using a different SDF video microscope . The dis3μm3 was fairly similar to values in healthy human adults, with a median of 16.5 103μm3  compared to control dogs. In our study, the median CBVabs in healthy control dogs, measured at 18.3 105 103μm3 . Therefo4 103μm3 . Given nThe variable CBVrel was relatively unchanged between CPB dogs and control dogs at T0. As the difference between CBVabs and CBVrel is that CBVabs investigates specifically capillary blood volume whereas CBVrel uses a comparison between capillary blood volume and larger blood vessels, those two parameters are not necessarily related. It is possible that the blood volume in capillary and larger vessels changes in unison, therefore CBVrel stays relatively unchanged. This is the first time this variable has been reported in published literature.Although our study adds to the growing literature of eGC and microcirculatory variables in veterinary medicine and is the first to report full microcirculatory variables and eGC thickness surrogate in dogs, it is not without limitations. Most importantly, our results should be considered pilot due to our small sample size, and our study likely suffers from a type II error . AlthougCPB induced changes in eGC thickness and microvascular changes in dogs suffering from naturally-occurring cardiac disease. The correlation between eGC and microvascular changes with clinical outcome is unknown in CPB, although there is evidence of a correlation between eGC degradation and disease severity and mortality in sepsis .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by IACUC committee, Colorado State University Clinical Board Review Committee, Colorado State University. Written informed consent was obtained from the owners for the participation of their animals in this study.DD: study design, data collection, manuscript draft writing, and final version editing. EO and MR: study design and final version edition. KZ: study design, data collection, and final version editing JG: study design, data collection, statistical analysis, and manuscript draft writing and final version editing. All authors contributed to the article and approved the submitted version.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.


Clostridium kluyveri. Recently, other chain elongators have been isolated that deserve further research, such as Megasphaera hexanoica.Chain elongation technology, which involves fermentation with anaerobic bacteria, has gained attention for converting short and medium chain substrates into valuable and longer-chain products like medium chain fatty acids (MCFAs). In the recent past, the focus of studies with pure chain elongating cultures was on species of other genera, mainly M. hexanoica: (a) a medium rich in different sources of nitrogen and (b) a medium whose only source of nitrogen is yeast extract. Also, batch bioreactor studies at pH values of 5.8, 6.5 and 7.2 were set up to study the fermentation of lactate  and acetate  by M. hexanoica.In this study, batch studies were performed in bottles with two different media to establish the optimal conditions for growth of n-caproate rapidly up to 2.62 ± 0.24 g/L. Subsequent bioreactor experiments at pH 5.8, 6.5, and 7.2 confirmed consistent production profiles, yielding C4-C8 fatty acids. A fourth bioreactor experiment at pH 6.5 and doubling both lactate and acetate concentrations enhanced MCFA production, resulting in 3.7 g/L n-caproate and 1.5 g/L n-caprylate. H2 and CO2 production was observed in all fermentations, being especially high under the increased substrate conditions. Overall, this study provides insights into M. hexanoica’s behavior in lactate-based chain elongation and highlights optimization potential for improved productivity.Batch bottle studies revealed the yeast extract (YE) containing medium as the most promising in terms of production/cost ratio, producing  Likewise, different combinations of carboxylates as electron acceptors were studied using fructose as electron donor , acetate (6 g/L) and propionate (7.4 g/L), whereas 0.3 g/L n-valerate, 9.7 g/L n-caproate and 0.6 g/L n-caprylate were detected from fructose (20 g/L), acetate (6 g/L) and n-butyrate (11.6 g/L). Finally, this strain was also investigated in a co-culture with n-butyrate-producing Clostridium strains, which would be elongated into n-caproate by M. hexanoica  and a n-caproate yield of 0.55 g/g sugar.In recent developments, pure strains belonging to the  process . On the on cycle . For inscontents  and its  sucrose . The goot decade . Moreove5 to 7.5 . Its maiptanoate . In a meon donor . 5.7 g/Lexanoica . For thin-butyrate and longer chain acids . Thus, this work aimed to examine the growth and production of MCFAs from a pure culture of M. hexanoica using only lactate and acetate as electron donor and acceptor and thereby contributing to the state of the art. For this purpose, the behavior of the strain was assessed in a bottle study using two media with different degrees of enrichment and afterwards, the growth at pH values of 5.8, 6.5 and 7.2 in bioreactors was evaluated. Likewise, after verifying the effect of pH, an attempt was made to optimize n-caproate production by changing the fermentation strategy.However, since only one study is available to date that analyzes chain elongation with this microorganism from lactate , it is ain acids . For thiluconate . Convers2.2.1.M. hexanoica DSM 106893 was acquired as a freeze-dried pellet from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH . Initially, the strain was activated in an enriched medium containing fructose and later, after successive inoculations, it was adapted to a medium with lactate and acetate, and the composition described below. All incubations were done at 37°C and under constant shaking conditions at 100 rpm.M. hexanoica contained (per liter distilled water): yeast extract, 10 g; tryptone, 5 g; meat peptone, 5 g; meat extract, 5 g; K2HPO4, 2 g; cysteine-HCl, 0.5 g; saline solution, 40 mL; Na-Acetate·3H2O, 5.04 g; L-(+)-lactic acid 90%, 9.25 mL.The enriched medium used to grow In the only YE-medium, tryptone, meat peptone and meat extract were omitted. In the medium with no enrichment, yeast extract was also omitted along with the other components just mentioned before.2·2H2O, 0.25 g; MgSO4·7H2O, 0.5 g; K2HPO4, 1 g; KH2PO4, 1 g; NaHCO3, 10 g; NaCl, 2 g.The composition of the saline solution (per liter distilled water) was as follows: CaClThe pH of the medium was always adjusted to 6.5, unless otherwise indicated.2.2.M. hexanoica and the effect of the addition of certain nutrients, two media with different degrees of enrichment were tested. To do so, after dissolving all the components in 40 mL of distilled water, the serum bottles were purged with pure nitrogen both in the liquid and in the headspace for at least 6 min. Simultaneously, the pH of the solution was adjusted to 6.5, using either 2 M HCl or 2 M NaOH. Then, the bottles were sealed with butyl rubber septa and aluminum crimps and autoclaved for 20 min at 121°C. This trial was carried out in triplicate.To establish the optimum growth medium for M. hexanoica in the early exponential growth phase as follows: from a seed inoculum containing the enriched medium, two consecutive transfers were made to a bottle with YE-only medium. Once the bacteria entered the early exponential growth phase, a third inoculation was made to each of the bottles under study. This way, the additional carbon and nitrogen sources in the enriched medium were diluted 1,000-fold and the same seed inoculum was also used for all bottles.Each bottle was inoculated with 10% (v/v) seed culture of 600 nm determination and HPLC analysis and 2 mL of gaseous sample (headspace) for H2 and CO2 analysis by gas chromatography.All bottles were sampled daily, at least once. Thus, 1 mL of the liquid phase was withdrawn for pH measurement, OD600nm data points of the exponential growth phase were taken and plotted on an exponential scale versus time. The growth rate (μ) was estimated in h−1 as shown in Eq. 1.For the calculation of the growth rate, at least three OD2.3.M. hexanoica (5.5–7.5), it was decided to study the behavior of the strain under conditions of pH 5.8, 6.5, and 7.2  to determine whether M. hexanoica maintains a constant activity over a wide pH range. The pH adjustment was done by adding HCl 1 M or NaOH 1 M via peristaltic pumps. The control unit used was programmed so that the pH setpoint would only admit an error of 0.02 units, so that when this limit was exceeded both above and below, the peristaltic pumps were activated, and pH was corrected. A seed inoculum of M. hexanoica, in early exponential growth phase , was introduced into the reactor. The pH was continuously monitored and adjusted throughout the experiment. To prevent overpressure in the system, a gas outlet port was used, from which gaseous samples were also taken. Whenever excessive foaming occurred during the purging process, a few drops of antifoam were dispensed into the synthetic medium, which made the foam disappear in a matter of seconds. In addition, a MilliGascounter (Ritter) was placed at the outlet of each reactor to measure the amount of H2 and CO2 flow at the outlet.The experiments were conducted using 2 L Eppendorf BIOFLO 120 bioreactors, with a working volume of 1.2 L, and at a constant temperature of 37°C . For all experiments, the previously described medium containing only yeast extract was introduced into the bioreactor, excluding D-(+)-lactic acid, and then autoclaved at 121°C for 20 min. After cooling down to room temperature (20°C), the reactor medium was subjected to 1.5-h flushing with pure nitrogen using a sparger, while stirring at 100 rpm, to create anaerobic conditions. Subsequently, D-(+)-lactic acid was added aseptically and anaerobically to the reactor through a 0.22 μm PTFE sterile filter. Based on the theoretical pH range of growth for 600nm) and for analysis of metabolites. Also, gas samples of 1 mL were taken from the outlet of the bioreactors, in duplicate, to determine the levels of H2 and CO2.One or two liquid samples were aseptically collected from the bioreactors on a daily basis. These samples were then used for optical density measurements at a wavelength of 600 nm  as follows (Eq. 2).The 2.4.600nm) of the non-filtered samples with a cuvette (pathlength of 10 mm) using a UV–visible spectrophotometer . These measurements, obtained in absorbance units, serve as indicators of the bacteria’s growth phase, with higher values corresponding to increased bacterial activity (and consequently higher MCFAs productivity).Bacterial growth was estimated by measuring the optical density at a wavelength of 600 nm (OD2-C6) were determined using a High Performance Liquid Chromatography (HPLC) with an HP1100 system . The HPLC system was equipped with an Agilent Hi-Plex Column (300 × 7.7 mm) and both a diode array detector (DAD) and a refractive index detector (RID) maintained at 50°C. A 0.005 M H2SO4 solution was utilized as mobile phase, with a flow rate of 0.80 mL/min and a column temperature of 45°C. prior to analysis, all liquid samples were filtered through 0.22 μm PTFE syringe filters.The concentrations of carboxylates (C7) and caprylate (C8) concentrations were detected on a gas chromatograph equipped with a flame ionization detector (GC-FID), with the internal standard method, using 4-methylpentanoic acid as the internal standard. Prior to analysis, the aqueous samples from the reactor were subjected to an extraction process with dichloromethane in 10 mL tubes, which was the solvent used for gas chromatography in this case. For this, 2 mL sample, 2 mL dichloromethane, 0.4 g NaCl, 0.4 mL of the internal standard solution, and 0.5 mL H2SO4 50% were introduced in the tube. They were then shaken at 2000 rpm for 10 min and centrifuged at 3000 rpm for 3 min. Finally, the organic phase was withdrawn and placed in the corresponding vial. The equipment used was a Thermo Scientific TRACE 1300 GC equipped with a DB-FATWAX Ultra Inert column with a length of 30 m, diameter 0.250 mm and film thickness 0.25 μm was used. The temperature of both the detector and the injector was maintained constant at 270°C and 280°C, respectively, whereas the program used in the oven was as follows: first, the temperature was held at 120°C for 1 min; then it was increased, with a ramp of 5°C/min, up to 140°C and held for 3 min; then, the temperature was increased again, with a ramp of 15°C/min, up to 230°C and held for 1 min. Air, makeup gas (nitrogen) and hydrogen flow rates were, respectively, 350 mL/min, 40 mL/min and 35 mL/min. The carrier gas was helium at a flow rate of 62 mL/min with a split ratio of 50.Heptanoate . The GC used for H2 measurements was composed of a thermal conductivity detector (TCD) and a 15-m HP-PLOT Molecular Sieve 5 A column . The equipment used for CO2 determinations was a HP 5890 gas chromatograph , containing a Porapak Q 80/100 (inox) column (2 m × 1/8′) connected to a thermal conductivity detector (TCD). The injection, oven and detection temperatures were kept at 90, 25 and 100°C, respectively. In both cases, helium was used as carrier gas.H3.3.1.M. hexanoica. Therefore, the strain was inoculated in two media with different substrates as carbon source: (a) enriched medium: with yeast extract, tryptone, meat peptone, meat extract, lactate, and acetate; and (b) only YE-medium: with yeast extract, lactate, and acetate.This study aimed at evaluating the effect of the presence or absence of certain nutrients in the culture medium on the growth and production of MCFAs by M. hexanoica to ferment lactate and acetate to produce mainly n-caproate , 11 g/L n-butyrate (0.12 mol) and 6 g/L acetate (0.10 mol), which corresponds to an electron donor:acceptor ratio of approximately 0.5. On the other hand, other authors have reported that an excess of lactate over acetate is necessary to efficiently accomplish the chain elongation process towards the production of MCFAs with other pure cultures, such as Caproicibacterium lactatifermentans  occurred at a lactate:acetate molar ratio of 3:1 and that ratios below this led to an accumulation of shorter chain carboxylates . This phenomenon was also previously described in ethanol-based chain elongation with C. kluyveri  due to economic considerations, as stated before; (b) stemming from the desire to yield the most elongated products feasible from the shortest carbon source ; and (c) given that acetate can be produced from acetogenic bacteria utilizing C1 gases, which offers the potential for a synergistic coupling of syngas fermentation and chain elongation with M. hexanoica. Conversely, the production of n-butyrate is confined to a very limited number of acetogenic bacteria, such as C. carboxidivorans , which is then further oxidized to acetyl-CoA (2 carbons) by pyruvate decarboxylase, with the consequent generation of CO2 , 10.5 ± 0.94% and 18.4 ± 1.7% .After inoculation of h. 600nm  and the n of CO2 . In thesa of 3.86, compared to the pKa value of the MCFAs produced, 4.76 , while the final pH remained around 7.3. This pattern of gradual increase in pH is normal during lactate fermentation, given the lower lactic acid pKed, 4.76 . This is2, CO2) compounds, and in view of the fact that the addition of meat extract, meat peptone and tryptone do not improve any of these aspects compared to a medium with only YE as nitrogen source, it was decided to choose the YE-only medium to grow M. hexanoica in the subsequent bioreactor experiments. It is advisable to use a medium with the lowest possible degree of enrichment, in order to reduce costs in the event of scaling up the process  and gaseous . From this point on, n-butyrate production reached a plateau, and its concentration started to decrease, a sign that part of it was being used for n-caproate accumulation. This is a typical behavior in chain elongation in which there is an excess of electron donor over acceptor  and n-valerate (0.12 g/L). Trace concentrations of iso-caproate were also found (data not shown). On the other hand, there is an acrylate pathway that directly competes with the chain elongation that steers carbon flow from lactate to propionate and thus decreases the selectivity of MCFAs , the initial concentrations of lactate and acetate were adjusted again, adding them under sterile and anaerobic conditions, so as to avoid decay of bacterial activity and to further stimulate the production of MCFAs. Nonetheless, after doing this the ODkluyveri  and may 3.2.2.M. hexanoica hardly showed any lag phase, gradually increasing the OD600nm up to a maximum of 2.0, after 35 h . In this case, the highest concentrations of n-butyrate, iso-valerate, n-valerate, n-caproate, n-heptanoate, and n-caprylate were, respectively, 0.66 g/L, 0.26 g/L, 0.10 g/L, 2.1 g/L, 0.13 g/L, and 0.62 g/L. Likewise, trace concentrations of iso-caproate were measured in the fermentation broth.In case of the reactor working at the most alkaline pH of those tested, = 61.5 h . LactateM. hexanoica maintains a high level of activity in a pH range of at least 1.4 units. This offers great versatility and opens up a wide range of possibilities for co-culturing M. hexanoica in a microbiome, performing chain elongation in mildly acidic conditions (pH ~ 5.5) in order to inhibit acetoclastic methanogens .If the results obtained at three different pH values are evaluated jointly and compared, it can be stated that hanogens . In contaround 5 , and itsaround 5 . So far,n-caproate, are quite similar at the studied pHs of 5.8, 6.5 and 7.2. Also, the strategy of reestablishing substrate concentrations just before depletion was not satisfactory in any of the three reactors as indicated above. The main dissimilarities are due to slight kinetic differences and small fluctuations of OD600nm, the maximum OD600nm value being nevertheless of the same order in all cases. Besides, in the three pH conditions studied, the production of odd-chain MCFAs from lactate and acetate, such as n-valerate, iso-valerate (C5), and n-heptanoate (C7), could be observed. In other investigations with the same strain, high concentrations of n-valerate (9.48 g/L) and n-heptanoate (2.48 g/L) have also been detected, whereas the substrates used were fructose, acetate and propionate  are high enough to exert an inhibitory effect on the microorganism. Therefore, keeping the same lactate:acetate molar ratio of 3:1 as in previous experiments, the initial substrate concentrations were doubled, thus starting from 17.2 g/L and 4.2 g/L of lactate and acetate, respectively . Rapidly studies , and then-caproate achieved from lactate and acetate by M. hexanoica are not as high as those achieved by C. kluyveri from ethanol and acetate, reaching 11.8 g/L, with the latter , using a large number of substrates  . Therefore, the exploration of production from shorter-chain substrates  should remain under investigation, as demonstrated here, where its utilization has proven effective in achieving relatively high concentrations of n-caprylate. On the other hand, up to 1.2 g/L of n-caprylate was detected with this strain but using fructose as electron donor and a combination of acetate and n-caproate as electron acceptors (n-caprylate in the fermentation broth when using fructose and acetate and/or n-butyrate. In a study carried out by n-caprylate concentrations of 3.77 ± 0.61 mM (0.544 ± 0.088 g/L) were found, which are comparable to those obtained in this study. Besides, in a continuous bioreactor study using waste activated sludge, with a sludge retention time of 15 days, 1.48 g COD/L (0.606 g/L) of n-caprylate was detected (It is noteworthy that the concentrations of e latter . Howeveraproate) . This evcceptors . Kang etdetected .iso-valerate and n-heptanoate) have been found in the four conditions explored in bioreactor. These concentrations may be the result of amino acid metabolism due to the use of such high concentrations of yeast extract (10 g/L), since M. hexanoica cannot use the acrylate pathway with the consequent formation of odd-chains.On the other hand, relatively low but considerable concentrations of odd chains . Also, the CO2 and H2 produced by the strain can be used for other bioprocesses, such as fermentation by acetogenic bacteria  to form acetate and then be reduced with H2 to yield ethanol, which could be used, respectively, as electron acceptors and electron donors for the formation of n-caproate and n-caprylate. Interestingly, the authors who isolated M. elsdenii found that the use of an electron donor slightly more oxidized than lactate, such as pyruvate, changes the metabolism of the strain from H2-producing to H2-consuming  and show serious difficulties to grow if deviating much from this value. For example, this strain was shown to be able to produce n-butyrate and n-caproate from ethanol and acetate in co-culture with C. aceticum at pH 7.5, although it failed to maintain its activity in a prolonged manner, whereby OD600 nm decreased abruptly and not even a pH reset to 6.8 was able to recover the activity of C. kluyveri  in the pH range of 5.0 to 6.6, whereas at a lower pH of 4.65 growth decreased substantially (μ = 0.17 h−1)  . Therefoacceptor .2 as a co-electron donor is another intriguing avenue for research. Still, it is worthy to note how M. hexanoica produces odd-chains even in small concentrations, from only lactate and acetate. Considering that the YE concentration used is quite high , the assumption that the odd-chain fatty acids obtained after fermentation may come from amino acid metabolism can be made. In this context, it is conceivable that reducing the YE concentration could enhance selectivity for even-chain fatty acids by minimizing the presence of amino acids with odd-carbon chains. This reduction would potentially decrease the release of odd metabolic intermediates that favor the production of compounds like n-valerate and n-heptanoate.As future research with this topic, it is proposed to try to improve the selectivity of the process towards a specific product. For example, it has been proven that higher electron donor:electron acceptor ratios favor the appearance of longer chains . It is t4.M. hexanoica as a chain elongator, using lactate and acetate, to produce MCFAs. The most cost-effective medium included YE, lactate and acetate as carbon sources. M. hexanoica performed well over a wide pH range  with high conversion efficiencies, making it versatile for co-culture. This distinguishes M. hexanoica from other chain elongators, e.g., C. kluyveri, which is very susceptible to pH changes. From 17.2 g/L lactate and 4.2 g/L acetate, the major compound obtained was n-caproate (3.7 g/L), followed by n-caprylate (1.5 g/L), and n-butyrate (0.5 g/L), along with minor amounts of iso-valerate, n-valerate, iso-caproate, n-caproate and n-heptanoate. This is the first time that concentrations of n-caprylate and iso-caproate have been quantified from lactate and acetate with this strain. Thus, it is a microorganism that deserves further research and that allows upgrading lactate, which can be a by-product of different food and beverage industries, as well as cosmetics, towards MCFAs, such as n-caproate and n-caprylate, which are of great interest in the food, chemical, pharmaceutical and biofuel production industries. Also, the H2 and CO2 produced by M. hexanoica during these fermentations could be utilized in the formation of short-chain fatty acids and bioalcohols by other acetogenic strains. In this way, a better use of available resources could be achieved and waste or by-products from other industries could be converted into high value-added products.This study highlights the potential of M. hexanoica, thus contributing to the state of the art.In summary, this work aims to better understand the behavior of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CF-B: Data curation, Investigation, Writing – original draft, Writing – review & editing. MV: Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing. CK: Data curation, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing.


Pseudokabatana alburnus is a xenoma-forming fish microsporidium, firstly described from the liver of the Culter alburnus from Poyang Lake in China. In the present study, P. alburnus was firstly reported from the ovary of 6 other East Asian minnows, including Squaliobarbus curriculus, Hemiculter leucisculus, Cultrichthys erythropterus, Pseudolaubuca engraulis, Toxabramis swinhonis, and Elopichthys bambusa. Genetic analysis revealed high sequence diversity in the ribosomal internal transcribed spacer region (ITS) and the largest subunit of RNA polymerase II (Rpb1) loci of P. alburnus isolated from different hosts and locations. The variation of Rpb1 mainly occurred in the 1,477–1737 bp regions. The presence of a wide variety of Rpb1 haplotypes within a single fish host, together with evidence of genetic recombination suggested that P. alburnus may have the intergenomic variation and sexual reproduction might be present in other hosts (possibly freshwater shrimp). Phylogenetic analysis and population genetic analysis showed that there was no geographical population divergence for P. alburnus. Homogeneity and high variability of ITS sequences indicates that ITS may be a suitable molecular marker to distinguish different P. alburnus isolates. Our data confirm the broad geographical distribution and host range of P. alburnus in the middle and lower reaches of the Yangtze River. Additionally, we emendated the genus Pseudokabatana to exclude the infection site, liver as one of the taxonomic criteria, and proposed that fish ovary was be the general infection site of P. alburnus. Therefore, a comprehensive taxonomic approach of integrating morphological characters, ultrastructural data, host, and molecular phylogeny of based on SSU rDNA sequence data has been reached a consensus for the classification of the microsporidia . General species . If thisbryconis . The swibryconis . Therefobryconis . So, somPseudokabatana to discriminate the teleost skeletal muscle-infecting genus Kabatana, with P. alburnus as the type species which infects the liver of an East Asian minnow Culter alburnus  collected from Lake Poyang, the biggest freshwater lake in China  of P. alburnus were investigated based on the sequence comparisons of ITS and Rpb1 which were widely employed molecular markers for population genetics of microsporidia  in July 2018. Six specimens of Hemiculter leucisculus and 9 specimens of Cultrichthys erythropterus were captured by gill nets from Lake Weishan located in the Southern Shandong province, China  in October 2020. One specimen of Pseudolaubuca engraulis, 5 specimens of Hemiculter leucisculus, 15 specimens of Cultrichthys erythropterus and 97 specimens of Toxabramis swinhonis were captured by gill nets from Lake Luoma located in Northern Jiangsu province, East China  in October 2020. Two specimens of Elopichthys bambusa was collected from Lake Gehu located in Southern Jiangsu province, East China  in May 2022. All fish specimens were immediately transported to the local laboratory in cooled plastic bags for the preliminary parasitological examination. Gross observations were conducted on scales, skin, and visceral organs, including gill, liver, heart, spleen, kidney, intestine, ovary, gallbladder, urinary bladder, and mesentery to find the suspected microsporidian infection for the presence of the whitish cysts which were subjected for the further wet mount preparation. A single cyst isolated from the infected tissues was ruptured with a fine needle on a slide for morphological observation. Spore images were captured using an Olympus BX 53 microscope equipped with an Olympus DP72 digital camera . The cyst-containing tissue samples were preserved in 95% ethanol for further molecular characterization, and in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for electron microscopic observation, as well as in 10% neutral buffered formalin for histological examination, respectively. In addition, the previously collected isolate from Culter alburnus from Lake Poyang was also used for molecular analyses  and examined under Olympus BX 53 microscope .2.3.Glutaraldehyde-fixed cysts were washed three times for 10 min in sodium cacodylate buffer and then fixed with 1% osmium tetroxide (OsO4) in the same buffer for 1 h. After dehydration through a gradually ascending series of ethanol and propylene oxide, samples were embedded in Spur resin . Ultrath2.4.2, 1.25 units Taq polymerase, 20 pmol each primer, and 2 μl DNA template. The partial SSU rDNA was amplified using the primer pair V1f/1492r, and the PCR reaction conditions consisted of an initial denaturation step at 95°C for 4 min, followed by 35 cycles at 95°C for 1 min, 50°C for 30s, 72°C for 2 min, and a final extension at 72°C for 10 min. The 3′ terminal partial SSU rDNA, complete ITS, and the partial LSU rDNA sequence was amplified using the primer pair HG4F/ILSUR, and the PCR cycle comprised of an initial denaturation step at 95°C for 4 min, followed by 35 cycles at 95°C for 30s, 53°C for 30s, 72°C for 2 min, and a final extension at 72°C for 10 min. For some taxa, to which the primer pair HG4F/ILSUR did not work, the primer pair HG4F/HG4R was applied, with thermocycler parameters as follows: an initial denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 54°C for 30s, 72°C for 1 min, and a final extension at 72°C for 10 min. For amplification of the largest subunit of RNA Polymerase II (Rpb1), the general microsporidian primer pair RPB1-F1/RPB1-R1 was used to obtain preliminary sequence. The preliminary sequence was then used as the template to design specific primers using Primer Premier 5.0. Then, the desired Rpb1 fragement was assembled with three overlapped amplified fragments using the primer pairs AF3/PR1, PF1/RPB1-R1, and PF2/GR1. The PCR amplification was performed under the following conditions: an initial denaturation for 4 min at 94°C, 35 cycles of 30s at 94°C, 30s at 46°C, 1 min at 72°C, and a terminal extension of 10 min at 72°C. All desired PCR products were excised from an agarose gel, purified using a PCR purification kit , and cloned into the PMD-18 T vector system . For each gene and isolate, five positive clones were randomly selected for sequencing in both directions with the ABI BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI 3100 Genetic Analyzer.Ethanol-fixed cysts were washed with distilled water two times to remove ethanol remnants. Multiple cysts from a single host were used to extract the genomic DNA. The genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit  with the assistance of a Fast Prep cell disruptor  for 2 min at 8 m/s following the manufacturer’s instructions. The primer sets used for rDNA and Rpb1 gene amplification were shown in 2.5.Gurleya daphniae (AF439320) was used as an outgroup. Pairwise genetic distances/similarities of these species based on SSU, ITS, and LSU were calculated using the Kimura-2 parameter model distance matrix for transitions and transversions. Phylogenetic analyses were conducted using the maximum likelihood (ML) method in PhyML 3.0 and Bayesian inference (BI) in MrBayes 3.2.4, respectively. The optimal evolutionary model was determined to be GTR + I + G by ModelTest 3.7 using the Akaike information criteria. Two independent runs were conducted with four chains for one million generations for BI. Phylogenetic trees were sampled every 100 generations. The first 25% of the samples were discarded from the cold chain (burninfrac = 0.25). Bootstrap confidence values were calculated with 100 repetitions for ML. The tree was initially examined in Figtree v1.4.4The all obtained sequences fragments were assembled by BioEdit  to produ2.6.w) by using DnaSP v5.10 (D (Fs (Fst) were calculated to evaluate population genetic differentiation using Arlequin 3.5. Molecular variance analysis (AMOVA)  and the average number of nucleotide differences per site   was condThe genetic recombination was analyzed with RDP4  and SimP3.3.1.Squaliobarbus curriculus  mm long, and 1.11 (0.71–1.41) mm wide (N = 10). The smallest cysts from S. curriculus collected from Lake Poyang were spherical to oval, measuring 0.45 (0.34–0.60) mm long and 0.40 (0.30–0.56) mm wide (N = 10) (T. swinhonis collected from Lake Luoma measured 3.54 ± 0.17 (3.20–3.85) μm in length and 1.90 ± 0.11 (1.60–2.05) μm in width (N = 40). The smallest spores isolated from H. leucisculus collected from Lake Weishan were pyriform, and measured 2.96 ± 0.21 (2.65–3.27) μm in length and 1.66 ± 0.09 (1.46–1.82) μm in width (N = 40)  . Spores (N = 10) –N. The l = 10 . S(N = 40) . Histolo(N = 40) . The wal3.2.T. swinhonis from Lake Luoma, H. leucisculus from Lake Weishan, C. erythropterus from Lake Luoma, P. engraulis from Lake Luoma, and E. bambusa from Lake Gehu were subjected to the TEM investigation. For being almost the most advanced development stage, ultrastructural characteristics of mature spores from the above 5 specimens were solely presented here which are highly similar, but also of slight difference in spore size  recently identified from the liver of Culter alburnus in Lake Poyang, followed by that of Kabatana rondoni , Inodosporus octosporus , and K. takedai . Therefore, based on the morphological and high SSU rDNA sequences similarity, the microsporidian from the ovary of all sampled 6 East Asian minnows along the middle and lower reaches of the Yangtze River are conspecific to P. alburnus. The pairwise distances/sequence similarities between the obtained SSU rDNA sequences of all P. alburnus isolates and 3 highly genetically related Kabatana species ranged from 0.0008/99.92%  to 0.1340/86.60% (between K. rondoni FJ843105 and K. takedai AF356222)  . Given lmilarity , howevermilarity . Geneticectively . SlidingNA locus . HaplotyNA locus . Bayesiac signal .P. alburnus isolates were successfully amplified to further explore their possible genetic variation which were deposited in GenBank under accession numbers ON049422–ON049429, ON087855–ON087869, and OP150918, but failed for other isolates. In detail, five Rpb1 sequences were obtained from H. leucisculus individual from Lake Luoma. Four Rpb1 sequences were obtained from S. curriculus individual from Lake Poyang and of T. swinhonis individual from Lake Luoma, respectively. Three Rpb1 sequences were obtained from C. erythropterus individual from Lake Weishan. Two Rpb1 sequences were obtained from P. engraulis individual from Lake Luoma, of C. erythropterus individual from Lake Luoma and of C. alburnus individual from Lake Poyang, respectively. Totally, 24 P. alburnus haplotypes referring from Rpb1 sequence variation were detected  among some fish-infecting microsporidian species, such as Spraguea spp.  might involve the meiosis and sexual reproduction. Inodosporus, the closest branch relative of the genus Pseudokabatana, have been suggested to have a multi-host life cycle between fish and crustacean hosts where different developmental patterns occurred  can be found at: The animal study was reviewed and approved by The Animal Research Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.MW collected the samples, performed the parasitological examination, data analysis, and helped write the manuscript. XZ, ZX, SX, QZ, and AL performed the morphological comparisons and helped write the manuscript. JZ designed this study and drafted the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (32173019), Young experts of Taishan Scholars in Shandong Province (tsqn201909133), Initiative grant for high-level personnel recruitment in Qingdao Agricultural University, JSPS Bridge Fellowship (BR221304) awarded to JZ and the “First Class Fishery Discipline” programme in Shandong Province, China; the Talent plan “One Thing One Decision (Yishi Yiyi)” in Shandong Province, China.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.


Vibrio splendidus is an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to the Apostichopus japonicus breeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of the V. splendidus fur (Vsfur) gene in the pathogenesis of V. splendidus remains unclear. Hence, we constructed a Vsfur knock-down mutant of the V. splendidus strain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence on A. japonicus. The result showed that the growth curves of the wild-type V. splendidus strain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related gene Vshppd mRNA were 3.54- and 7.33-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly gene Vsflic was downregulated 0.56-fold in MTVs at the OD600 of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reduced A. japonicus mortality. The median lethal doses of WTVs and MTVs were 9.116 × 106 and 1.658 × 1011 CFU·ml−1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid of A. japonicus were significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis of V. splendidus by regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities. Vibrio splendidus can cause severe vibriosis in numerous aquatic animals   , 4. Sea uroidea) . In receuroidea) , 7, compuroidea) . Unfortuprotease , 10, lacfur gene also plays a critical role in the expression of virulence-related genes, such as oxidative stress response, quorum sensing, swarming motility, and biofilm formation in numerous pathogens during host invasion  showed high similarities to that of the Furs from other Vibrio sp. (Vsfur gene was knocked down by antisense RNA interference. The Vsfur knock-down mutant of V. splendidus (MTVs) was assessed for growth curve and expression of virulence-related genes compared with wild-type V. splendidus (WTVs). The WTVs and MTVs cells were collected to analyze swarming motility and biofilm formation in normal, iron-replete, and iron-starved conditions. Furthermore, the colonization ability and pathogenicity of WTVs and MTVs in A. japonicus were analyzed and compared.Our previous study confirmed that the iron uptake pathway of ore-IutA . Alignmebrio sp. . HoweverV. splendidus strain was dissociated from the focus of sea cucumbers with SUS and preserved in our laboratory. Zhang et al. tested the strain’s pathogenicity in sea cucumbers by means of a reinfection experiment . Escherichia coli S17λpir and DH5α  were grown in Luria–Bertani (LB) broth or agar at 37°C. The antimicrobial agent was purchased from a commercial source Sangon . Antibiotics were added to the medium at the following concentrations: kanamycin (Kn) 50 μg·ml−1, gentamicin (Gm) 100 μg·ml−1, and ampicillin (Ap) 100 μg·ml−1. The vectors used in the experiment were pMD19-T, which was purchased from Takara ; vector pBBR1MCS-5, which was purchased from Fenghui Biotechnology ; and vectors pET28a and pBT3, which were kept in our laboratory. Restriction endonucleases were purchased from New England Biolabs and used according to the manufacturer’s instructions.The wild-type periment . The bacV. splendidus strain LGP32 with accession number FM954973.2 and listed in Trc was generated to construct the MTVs with the reverse Vsfur sequence. The antisense RNA fragment of Vsfur was amplified with the forward primer VsfurF (Xho I) and reverse primer VsfurR (BamH I) and ligated into pMD19-T for subsequent restriction digestion. Plasmid pBT3Vsfur was constructed by ligating Vsfur into pBT3, between the BamH I/Xho I sites. The reverse Vsfur fragment fusing Trc promoter was obtained after pBT3Vsfur was digested with Swa I. The PTrc-Vsfur fragment was inserted into the blunt end site EcoR V of pET28a and then transformed into S17λπ to construct S17λπ/pET28aVsfur. To transform the recombinant plasmid pET28aVsfur into wild-type V. splendidus, bacterial conjugation was conducted as described previously  and used for further research.To complement the Vibrio splendidus strains, including WTVs, MTVs and Cfur were coated to 2216E agar at 28°C for 24 h. Single colonies were transferred into 10 ml of fresh 2216E broth and allowed to reach the optical density at 600 nm (OD600) of 1.0. The culture was diluted proportionally to the same concentration. One hundred microlitre aliquots of WTVs, MTVs, and Cfur were reinoculated into flasks with 100 ml of fresh 2216E broth, 2216E broth supplemented with 100 μM iron chelator 2,2′-dipyridyl (DP), and 2216E broth supplemented with 50 μM FeCl3 and grown at 28°C with agitation at 180 rpm. The OD600 was recorded at different time points with an ultraviolet spectrophotometer . The experiment was repeated three times for each sample.600 values. Data were standardized with the endogenous reference gene 16S rDNA (933F and 16SRTR1). The PrimeScript RT kit  and the reagent for removing genomic DNA were used for reverse transcription. Real-time polymerase chain reaction (RT-PCR) was performed in an ABI 7500 RT-PCR detection system (Applied Biosystems) with a total volume of 20 μl containing 0.4 μl of Dye-II (ROX), 0.8 μl of each of the forward and reverse primers (10 μM), 8 μl of diluted cDNA template, and 10 μl of 2 × SYBR Green Mix. The experimental procedure was set as follows: 95°C for 5 min to activate the polymerase, 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. Fluorescent signals were collected at the extension stage of each cycle. The primers for RT-PCR are listed in −ΔΔCT method . Meanwhile, the coelomic fluids were filtered through a 200-mesh and spread on 2216E agar plates supplemented with Ap. Single colonies from each plate were identified by 16S rDNA sequencing analysis and the number of colonies was counted after incubation at 28°C for 24 h.The distributions of eviously . Sea cucCfur strains were separately grown in fresh 2216E broth at 28°C with agitation at 180 rpm, and OD600 was adjusted to 1.0. Then, 2 μl bacterial cell suspension was dropped on the center of 2216E, 2216E supplemented with 200 μM iron chelator DP, and 2216E supplemented with 100 μM FeCl3 plates containing 0.3% agar. The 2216E agar plates were then incubated at 28°C for 3 days to observe the diameters of the swarming halos. The iron treatment with different concentrations was repeated three times.Swarming motility was assayed as described previously . WTVs, MCfur were conducted as described by Luo et al.  for 30 min and washed with sterile PBS. The culture plate was then placed upside down on the absorbent paper. The adsorbed biofilm was dissolved with 200 μl of ethanol and the OD570 was measured. The iron treatment with different concentrations was conducted in five independent biological replicates.Biofilm formation assays for WTVs, MTVs, and o et al. . Brieflyhttp://www.softberry.com. The 500-bp upstream sequences of virulence-related genes from the initial codon site were used to search the Fur binding box. All data are expressed as the mean ± SD of at least three sets of independent experiments. Statistical significance was determined by one-way Analysis of Variance (ANOVA) with a Dunnett’s test. The significance level was defined as *p < 0.05, **p < 0.01.Promoter prediction analyses were conducted with online prediction tools at Cfur were measured under different iron concentration culture conditions. The OD600 was measured at 3, 6, 9, 12, 21, 24, 45, and 48 h. Although iron chelator DP inhibited the growth of three strains within 24 h, the growth curves of the WTVs, MTVs, and Cfur strains were almost the same in 2216E broth  and had no obvious influence on the growth rate of V. splendidus.The growth curves of WTVs, MTVs, and 6E broth , 2216E bbroth  in MTVs at the OD600 of 1.0 and 1.5 compared with the WTVs (Vshppd mRNA were 3.54- and 7.33-fold (p < 0.01) in MTVs at the OD600 of 1.0 and 1.5  in MTVs at the OD600 of 1.0 and 1.5 (Vsflic was downregulated 0.56-fold (p < 0.05) in MTVs at the OD600 of 1.0 compared with the WTVs  and Vsm  and downregulated genes such as Vsflic (flagella C) related to flagellum biosynthesis. In summary, Vsfur has both positive and negative regulatory functions.The WTVs and MTVs cells were collected at the ODthe WTVs . Compare and 1.5 . Meanwhi and 1.5 . Under tthe WTVs . These rVshppd, Vsm, and Vsflic were performed to predict the putative Fur binding boxes in the V. splendidus strain. The promoter regions before the ATG initial cordon of the three virulence-related genes were searched in the genomic DNA database. BPROMVshppd, Vsm, and Vsflic showed 52.63, 63.16, and 52.63% homology to the consensus Fur binding site sequence (5′-GATAATGATAATGATTATC-3′), respectively  of WTVs and MTVs calculated by SPSS were 9.116 × 106 and 1.658 × 1011 CFU·ml−1, respectively. These results indicate that Vsfur is crucial to the pathogenicity of V. splendidus.In the artificial infection experiment, WTVs showed stronger pathogenicity than MTVs. Compared with the WTVs group, the of death . The firs of SUS . No deatA. japonicus after immersion infection with WTVs and MTVs is shown in 7, 5.51 × 108, 7.77 × 107, 1.24 × 108, and 2.42 × 108 CFU·g−1, respectively. The colonization amount of WTVs to the coelomic fluid was 2.95 × 107 CFU·ml−1. The colonization quantities of MTVs to all tissues except the body wall and respiratory tree decreased. The colonization quantities of MTVs to the body wall, muscle, respiratory tree, intestine, and tentacle tissues were 1.71 × 107, 8.92 × 107, 4.78 × 107, 2.70 × 107, and 6.71 × 107 CFU·g−1, respectively. The colonization amount of MTVs to the coelomic fluid was 1.02 × 107 CFU·ml−1.The quantitative detection of cell colonization in various tissues of Cfur strains was measured in 2216E agar plates, and the results demonstrated an obvious difference in the swarming motility of WTVs and MTVs (p < 0.01) of that of WTVs in normal conditions  and 22% of WTVs and Cfur, respectively (p < 0.05) of WTVs in iron-replete conditions. Under the iron-starved conditions, the biofilm formation of WTVs and MTVs showed no significant difference  staining assay. The biofilms of WTVs, MTVs, and ectively . The biofference . Taken tfur deletion mutants of V. splendidus failed, leading to the assumption that Fur is an essential protein in V. splendidus. While several attempts to obtain Vsfur in-frame deletion mutants by suicide vector failed, we constructed a Vsfur knock-down mutant of V. splendidus through antisense RNA interference. The recombinant plasmid pET28aVsfur containing promoter PTrc could efficiently express the interference sequence in V. splendidus , Zhejiang Provincial Natural Science Foundation (LY23C190003), and the K. C. Wong Magna Fund at Ningbo University.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.


To explore the direction and SPV (slow phase velocity) of the components of spontaneous nystagmus (SN) in patients with vestibular neuritis (VN) and the correlation between SN components and affected semicircular canals (SCCs). Additionally, we aimed to elucidate the role of directional features of peripheral SN in diagnosing acute vestibular syndrome.A retrospective analysis was conducted on 38 patients diagnosed with VN in our hospital between 2022 and 2023. The direction and SPV of SN components recorded with three-dimensional videonystagmography (3D-VNG) and the video head impulse test (vHIT) gain of each SCC were analyzed as observational indicators. We examined the correlation between superior and inferior vestibular nerve damage and the direction and SPV of SN components, and vHIT gain values in VN patients.p < 0.001). The aVOR gains of A-, L-, and P-SCC on the ipsilesional sides in 31 SVN patients were 0.62 ± 0.24, 0.45 ± 0.16, and 0.96 ± 0.10, while the aVOR gains on the opposite side were 0.96 ± 0.13, 0.91 ± 0.06, and 0.98 ± 0.11, respectively. There was a statistically significant difference in the aVOR gain between the A-, L-SCC on the ipsilesional side and the other SCCs (p < 0.001). In 7 TVN patients, the aVOR gains of A-, L-, and P-SCC on the ipsilesional side were 0.50 ± 0.14, 0.38 ± 0.06, and 0.53 ± 0.07, while the aVOR gains on the opposite side were 0.93 ± 0.17, 0.90 ± 0.16, and 0.89 ± 0.09, respectively. There was a statistically significant difference in the aVOR gain between the A-, L-, and P-SCC on the ipsilesional side and the other SCCs (p < 0.001). The aVOR gain asymmetry of L-SCCs in 38 VN was 36.3%. The aVOR gain asymmetry between bilateral A-SCCs and bilateral P-SCCs for VN patients with and without a vertical upward component was 12.8% and 8.3%, which was statistically significant (p < 0.05). For VN patients with and without a torsional component, the aVOR gain asymmetry of bilateral vertical SCCs was 17.0% and 6.6%, which was statistically significant (p < 0.01). Further analysis revealed a significant positive correlation between the aVOR gain asymmetry of L-SCCs and the SPV of the horizontal component of SN in all VN patients , as well as between the asymmetry of bilateral vertical SCCs and the SPV of torsional component in 26 VN patients . However, there was no significant correlation between the aVOR gains asymmetry of bilateral A-SCCs and P-SCCs and the SPV of the vertical component in 25 VN patients.The median illness duration of between symptom onset and moment of testing was 6 days among the 38 VN patients (17 right VN and 21 left VN). In total, 31 patients had superior vestibular neuritis (SVN), and 7 had total vestibular neuritis (TVN). Among the 38 VN patients, all had horizontal component with an SPV of (7.66 ± 5.37) °/s, 25 (65.8%) had vertical upward component with a SPV of (2.64 ± 1.63) °/s, and 26 (68.4%) had torsional component with a SPV of (4.40 ± 3.12) °/s. The vHIT results in the 38 VN patients showed that the angular vestibulo-ocular reflex (aVOR) gain of the anterior (A), lateral (L), and posterior (P) SCCs on the ipsilesional side were 0.60 ± 0.23, 0.44 ± 0.15 and 0.89 ± 0.19, respectively, while the gains on the opposite side were 0.95 ± 0.14, 0.91 ± 0.08, and 0.96 ± 0.11, respectively. There was a statistically significant difference in the aVOR gain between the A-, L-SCC on the ipsilesional side and the other SCCs (There is a correlation between the three-dimensional direction and SPV characteristics of SN and the aVOR gain of vHIT in VN patients. These direction characteristics can help assess different SCCs impairments in patients with unilateral vestibular diseases. SN is typically horizontal or horizontal-torsional, direction-fixed, and enhanced by removing visual fixation, and its SPV follows Alexander’s law . It is a2.2.1.This retrospective study involved the assessment of 38 patients with unilateral VN or acute unilateral vestibulopathy (AUVP) patients, examined at the Ear, Nose, and Throat (ENT) Department of MY Hospital, Tianjin First Central Hospital between 2022 and 2023. Of 38 patients, 31 had superior vestibular neuritis (SVN), and seven had total vestibular neuritis (TVN). All the subjects provided informed consent to be included in the study. The study procedures were approved by the Ethics Committee of Tianjin First Central Hospital.We included patients diagnosed with AUVP or VN according to the multidisciplinary experts’ consensus on vestibular neuritis  and the We exclude patients with unclear diagnoses or diagnostic controversies. Additionally, we excluded patients with coexistence of other diseases, such as BPPV, Meniere disease, sudden deafness with vertigo, Ramsay Hunt syndrome, labyrinthitis, as well as head trauma, vestibular migraine, stroke, and other central vestibular vertigo and balance disorders.2.2.We obtained a detailed medical history of the onset of symptoms and their severity, illness duration, and associated factors. VN and AUVP were diagnosed with the results of SN, vHIT, and caloric test. The lesion side, superior or inferior vestibular nerve damage, and associated SCCs were determined. SN, vHIT, caloric test, and corresponding parameters were observed and recorded using 3D-VNG .The VOR gain of vHIT is the ratio of eye velocity to head velocity, with a normal values range of 0.8–1.2 for L-SCCs and 0.7–1.2 for vertical SCCs. Unilateral weakness (UW) ≥25% and directional preponderance (DP) ≥30% indicate an abnormal caloric test.2.3.The main measures in vHIT are the aVOR gain, aVOR gain asymmetry, and the presence or absence of compensatory saccades. The aVOR gain assesses the function of the SCCs and their corresponding nerves on both sides. The aVOR gain asymmetry is a good sensitivity and specificity test for the early diagnosis of VN associated with acute vertigo. We analyzed the 3D direction and SPV (slow phase velocity) of SN and the aVOR gain of vHIT in patients with unilateral VN. The horizontal component of SN is caused by the imbalance of aVOR in the bilateral L-SCCs, while the vertical and torsional components are the comprehensive vectors of bilateral A- and P-SCCs effects.The formula for calculating the aVOR gain asymmetry of vHIT is as follows:The aVOR gain asymmetry of L-SCCs:The aVOR gain asymmetry between bilateral A-SCCs and bilateral P-SCCs:The aVOR gains asymmetry of bilateral vertical SCCs:r) was calculated, and a p-value <0.05 was considered statistically significant.IBM SPSS Statistics 22  and JASP 0.16.3  were used for statistical analyses. The quantitative data are presented as mean ± SD values and plotted using GraphPad Prism version 5 . The strength of correlation (3.3.1.p > 0.05). All VN patients had a median illness duration of between symptom onset and moment of testing of 6 days. And the illness duration was negatively correlated with the horizontal and torsional components, respectively, but not with the vertical components  ranged from 18–68 years (mean 42.21). Seventeen of the 38 patients had right-VN  and their ages ranged from 20–62 years (mean 42.76), while 21 patients had left-VN  with age ranges from 18–68 years (mean 41.76). Thirty one patients had superior-VN , and their ages ranged from 18–68 years (mean 40.68); 7 patients had total-VN  with ages ranging from 43–62 years (mean 49.00). Demographic data for VN are shown in mponents –C.3.2.The 3D direction and SPV of SN in the 38 VN patients were recorded and analyzed. The direction of the horizontal component was toward the opposite side, and the SPV was 7.66 ± 5.37°/s. About 65.7% (25/38) of the patients had a vertical upward component with a SPV of 2.64 ± 1.63°/s, and 68.4% (26/38) had a torsional component with a SPV of 4.40 ± 3.12°/s. Among the 17 RVN patients, all cases had a left horizontal component with a SPV of 11.30 ± 5.42°/s, 12 cases had a vertical upward component with a SPV of 3.47 ± 1.78°/s, and 13 cases had a torsional component with the direction of upper pole of the eye beating toward the right ear (from patients’ perspective) with a SPV of 5.46 ± 3.72°/s. Among the 21 LVN cases, 21 had a right horizontal component with a SPV of 4.70 ± 3.05°/s, 13 cases had a vertical upward component with a SPV of 1.87 ± 1.02°/s, and 13 cases had a torsional component with the direction of upper pole of the eye beating toward the left ear (from patients’ perspective) with a SPV of 3.35 ± 2.01°/s. Of the 31 SVN patients, the SPV of the horizontal component was 7.38 ± 5.49°/s, 23 cases had a vertical upward component with a SPV of 2.64 ± 1.67°/s, and 19 cases had a torsional component with a SPV of 4.41 ± 3.19°/s. In the 7 TVN patients, all cases had a horizontal and torsional components with SPV of 8.87 ± 4.98°/s and 5.54 ± 2.61°/s respectively, and 2 case had vertical component with SPV of 2.50 ± 1.41°/s .3.3.p < 0.001). In the 17 RVN patients, the gains of A-, L-, and P-SCCs on the right side were 0.51 ± 0.21, 0.35 ± 0.11, and 0.85 ± 0.21, and those on the left side were 0.99 ± 0.12, 0.94 ± 0.08, and 0.89 ± 0.09, respectively. There were significant differences between the gains of the A- and L-SCCs on the right side and those of the other SCCs (p < 0.001). In the 21 LVN patients, the gains of A-, L-, and P-SCCs on the right side were 0.92 ± 0.14, 0.88 ± 0.07, and 1.02 ± 0.10, and those on the left side were 0.67 ± 0.22, 0.51 ± 0.15, and 0.91 ± 0.17, respectively. There were significant differences between the gains of the A- and L-SCCs on the left side and those of the other SCCs (p < 0.001). In the 31 SVN patients, the gains of A-, L-, and P-SCCs on the ipsilesional side were 0.62 ± 0.24, 0.45 ± 0.16, 0.96 ± 0.10, and those on the opposite were 0.96 ± 0.13, 0.91 ± 0.06, and 0.98 ± 0.11, respectively. There were significant differences between the gains of the A- and L-SCCs on the ipsilesional side and those of the other SCCs (p < 0.001). Among the 7 TVN patients, the gains of A-, L-, and P-SCCs on the ipsilesional side were 0.50 ± 0.14, 0.38 ± 0.06, and 0.53 ± 0.07, and those on the opposite were 0.93 ± 0.17, 0.90 ± 0.16, and 0.89 ± 0.09, respectively. There were significant differences between the gains of the A-, L-, and P-SCCs on the ipsilesional side and those of the other SCCs (p < 0.001)  .The caloric test showed decreased aVOR function of L-SCCs on the ipsilesional side at low frequency (0.003 Hz) in all the VN patients and DP to the opposite side . The UW and DP were (47.60 ± 20.69) and (89.00 ± 27.11) in the 17 RVN patients and were (66.70 ± 22.02) and (79.15 ± 25.04) in the 21 LVN patients, respectively, consistent with previous research results .3.4.p < 0.05). For VN patients with a torsional component , the aVOR gain asymmetry of bilateral vertical SCCs was 17.0%, while the asymmetry for those without a vertical component was 6.6%, and these differences were statistically significant (p < 0.01).All patients with VN had SN directed toward the opposite side . We analr = 0.484, p < 0.01), as well as the asymmetry of bilateral vertical SCCs and the SPV of torsional component in 26 VN patients . However, no significant correlations were found in asymmetry of bilateral A- and P-SCCs and the SPV of vertical component among 25 VN patients.We further analyzed the correlation between the aVOR gain asymmetry of SCCs in vHIT and the SPV of 3D components of SN in VN patients –F. There4.Spontaneous nystagmus (SN)—present in a static state with the head upright and looking straight forward without any inducing condition—is common in acute unilateral vestibular dysfunction, which is spontaneous, unilateral and caused by asymmetric hypo-function of the angular vestibulo-ocular reflex (aVOR) . In the Ewald’s law, derived from animal experiments, reveals the physiological effects of endolymph flowing in a single SCC, including the plane, direction, and SPV of nystagmus. One of its main connotations is that the direction of nystagmus is the same as the plane of the stimulated SCCs. The law helps in understanding the physiological and pathological characteristics of human SCCs. Nystagmus induced by unilateral L-SCC stimulation was mainly the horizontal component accompanied by a weak vertical upward component. By contrast, nystagmus induced by unilateral P-SCC stimulation was chiefly vertical, upward, and torsional, accompanied by a weak horizontal component. Meanwhile, nystagmus induced by unilateral A-SCC stimulation was vertical, downward, and torsional nystagmus . The nysHowever, peripheral vestibular diseases represented by VN in clinical often involve two or more SCCs pathways, resulting in a combined vector feature of multiple SCCs effects in the direction of SN. This interplay makes it challenging for clinicians to observe and understand SN and its intrinsic pathology. 3D-VNG technology can help analyze the combined vector features of SN. According to Ewald’s law, analyzing the characteristics of SN components using 3D-VNG technology and tracing the damage to the affected SCCs pathway is a new perspective for the clinical diagnosis of vestibular disease. Our study focuses on patients with a confirmed diagnosis of VN or AUVP and combines vHIT with 3D-VNG technology to explore the 3D characteristics of nystagmus after different SCCs aVOR injuries.p < 0.05). And for VN patients with a torsional component, the aVOR gains asymmetry of bilateral vertical SCCs was 17.0%, while the asymmetry was 6.6% for those without a vertical component, and these differences were statistically significant (p < 0.01).VN is a unilateral vestibular lesion mainly affecting the superior division of the vestibular nerve —injuriesr = 0.484, p < 0.01), as well as between the asymmetry of bilateral vertical SCCs  and the SPV of torsional component in 26 VN patients , while there was no significant correlations in asymmetry of bilateral A- and P-SCCs and the SPV of vertical component. However, no effective correlation analysis could be conducted due to the small sample size. Additionally, the intensities of SN components induced by the A- and P-SCCs were 30% and 10% of that of the L-SCCs, respectively . In patients with TVN, the vertical components of the A- and P-SCCs cancel or partially cancel each other, while the torsional components are added up with a stronger SPV than that of SVN , so thatAmong the VN patients included in this study, the superior vestibular nerve was affected in 31, while both the superior and inferior vestibular nerves were affected in 7. The number of cases was relatively small, and there was a lack of patients with a dysfunctional inferior vestibular nerve. It is impossible to describe and analyze the SN characteristics of a single P-SCC lesion. Therefore, the SN characteristics of a single P-SCC lesion and its correlation with VHIT gain need further study. In addition, the lack of VEMPs results makes it impossible to evaluate the impact of otolith damage on SN in VN patients. In contrast to vHIT and caloric testing, VEMPs are much less relevant for the diagnosis of VN/AUVP , while iIn conclusion, we analyzed the correlation between the direction and SPV of SN components and vHIT gain. The results showed that the SPV of the SN horizontal components and torsional components in VN patients were positively correlated with the aVOR gain asymmetry of vHIT, and the direction of SN corresponded to the plane of the excitable SCCs. There is a horizontal-vertical upward-torsional nystagmus in SVN, while horizontal nystagmus with a strong torsional component is found with no vertical component in TVN . Combining Ewald’s law and observing the 3D direction of SN in patients with VN/AUVP, it is of great clinical significance to locate the damaged SCCs and trace the target of the lesion. This study provides an objective basis for investigating the relationship between the SN direction and SCC lesions. It also clarifies the significance of the directional characteristics of vestibular peripheral SN components in diagnosing acute vestibular syndrome through medical history and examination.The original contributions presented in the study are included in the article/WW and TC performed the study design. XZ and QD acquired and analyzed the data. XZ and TC drafted the manuscript. YL, SL, QL, CW, and XH revised the manuscript. All authors read and approved the final manuscript.This study was supported by the Tianjin Key Medical Discipline Construction Project (TJYXZDXK-046A), Tianjin Applied Basic Research Multiple Investment Fund Project (21JCQNJC01780 and 21JCQNJC01670), and Tianjin Health Research Project .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.