Objective To investigate the temporal changes in pathological damage and macrophage polarization in liver and spleen tissues of mice infected with Angiostrongylus cantonensis, and to preliminarily unravel the peripheral immune responses during the early stage of A. cantonensis infection. Methods Forty female BALB/c mice at ages of 6 to 8 weeks were randomly divided into four groups, including the control group and 7-, 14-, and 21-day infection groups, with 10 mice in each group. Each mouse in the infection groups was inoculated with 30 third-stage (L3) larvae of A. cantonensis by oral gavage, and five mice were randomly selected from each infection group on days 7, 14, and 21 post-infection, while mice in the control group were given the same volume of physiological saline and five mice were randomly selected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled. The histopathological changes of mouse liver and spleen tissues were observed using hematoxylin and eosin (HE) staining, and the percentage of positive staining area and the co-localization positive rates of the macrophage surface antigens F4/80, CD86, and CD206 were quantified in mouse liver and spleen tissues using immunohistochemical and immunofluorescence staining. In addition, five mice were collected from each infection group on days 7, 14, and 21 post-infection, and five mice were collected from the control group on the day of oral gavage. Mouse liver and spleen tissues were sampled for detection of macrophage markers CD86 and CD206 and macrophage phenotyping using flow cytometry, and the expression of M1 macrophage markers, including inducible nitric oxide synthase (Nos2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and M2 markers, including arginase 1 (Arg1), mannose receptor C-type 1 (Mrc1) and chitinase-like protein 3 (Chil3) was quantified in mouse liver and spleen tissues using real-time quantitative PCR (RT-qPCR) assay. Results Proliferative lesions of the hepatocyte were observed in mouse liver tissues and the follicular structures of the mouse spleen white pulp were disrupted 21 days post-infection with A. cantonensis. Immunohistochemical staining showed that there were significant differences in the percentages of F4/80, CD86 and CD206 positive staining areas in the liver and spleen tissues among the four groups of mice (F = 242.40, 197.14, 183.19, 157.65, 242.35 and 146.24; all P values < 0.001), and the percentages of positive staining in the liver and spleen tissues of mice in the 14-day infection group [(4.45 ± 0.51)%, (3.74 ± 0.67)%, (8.32 ± 0.72)%, (16.56 ± 1.14)%, (11.62 ± 0.52)%, and (8.29 ± 0.72)%, respectively] and the 21-day infection group [(3.70 ± 0.11)%, (3.22 ± 0.43)%, (11.53 ± 1.03)%, (12.59 ± 1.05)%, (9.02 ± 0.83)%, and (11.67 ± 1.10)%, respectively] were higher than in the control group [(0.35 ± 0.16)%, (0.40 ± 0.02)%, (0.93 ± 0.05)%, (2.78 ± 0.26)%, (2.33 ± 0.20)%, and (1.85 ± 0.20)%, respectively] (all P values < 0.05). Immunofluorescence staining showed significant differences in the positive rates of F4/80 co-localization with CD86 and CD206 in mouse liver and spleen tissues among the four groups (F = 24.42, 25.28, 54.51 and 130.55; all P values < 0.001). Flow cytometry detected significant differences in the proportions of CD86⁺ and CD206⁺ macrophages in mouse liver and spleen tissues among the four groups (F = 67.98, 18.41, 29.77, 172.80; all P values < 0.001), and the proportions of CD206⁺ macrophages in the liver and spleen of the 21-day infection group were significantly higher than those in the control group [(9.25 ± 2.55)% vs (3.83 ± 0.72)%, and (4.22 ± 0.56)% vs (0.47 ± 0.18)%, respectively] (both P values < 0.05). In addition, RT-qPCR assay quantified significant differences in the relative mRNA expression of M1 macrophage markers (IL-1β, TNF-α and Nos2) and M2 macrophage markers (Arg1, Chil3 and Mrc1) in mouse liver and spleen tissues among the four groups (F = 41.30, 31.82, 199.33, 19.96, 62.01, 119.76, 23.67, 95.90, 72.27, 82.59, 123.41 and 29.75; all P values < 0.05). Conclusions A. cantonensis infection may cause progressive pathological damage in mouse liver and spleen tissues, accompanied by dynamic temporal changes in macrophage polarization. M1 macrophage polarization predominates at the early stage of A. cantonensis infection and shifts towards M2 polarization at the later stages, suggesting that M2 polarization may participate in immune regulation at late stages of A. cantonensis infection by suppressing excessive inflammatory responses and promoting tissue repair.

Objectives: . This study aimed to investigate the regulatory functions of gamma-aminobutyric acid (GABA)ergic neural circuits from the inferior colliculus (IC) to the medial geniculate body (MGB) in salicylate-induced tinnitus. Methods: . Mice were treated with salicylate to induce tinnitus, and tinnitus-like behaviors were evaluated via gap prepulse inhibition of acoustic startle. Using combined viral tracing methodologies, we identified and mapped the pathways and connections from the IC to the MGB. Furthermore, we employed Gq-coupled human M3 designer receptors exclusively activated by designer drugs (DREADDs) and Gi-coupled human M4 DREADDs to achieve targeted excitation or suppression of GABAergic neurons in the IC and MGB. Following the administration of clozapine N-oxide, which binds to these receptors, we modulated these neural circuits to assess their impact on tinnitus severity in a mouse model. Results: . Our findings demonstrated that mice exposed to salicylate exhibited tinnitus-like behaviors. GABAergic neurons projecting retrogradely from the MGB to the IC were primarily concentrated in the external nucleus of the IC. After clozapine N-oxide administration, chemogenetic activation of IC-MGB GABAergic neurons aggravated salicylate-induced tinnitus. Additionally, activation of GABAergic neurons between the IC and MGB induced the perception of tinnitus even without salicylate. However, chemogenetic inhibition of the IC-MGB GABAergic circuit did not reverse salicylate-induced tinnitus. Conclusion . These findings suggest that activation of the IC-MGB GABAergic neural circuit may contribute to tinnitus generation through a mechanism distinct from that of salicylate-induced tinnitus. This study provides novel insights into the mechanisms underlying tinnitus.

The aim of this paper was to investigate the serotypes,virulence factors and drug resist-ance of clinical isolates of Pasteurella rnultocida of porcine origin in recent years.Morphological screening and polymerase chain reaction(PCR)were used to isolate and identify 119 isolates from nasal swabs and lung tissue samples sent from swine farms in Zhejiang Province from 2010 to 2024.The isolates of Pasteurella multocida were subjected to capsular polysaccharide serotyping,lipopolysaccharide serotyping,virulence factor detection and drug resistance analysis by PCR and Kirby-Bauer disc agar diffusion method(K-B).The results showed that there were 64 strains(53.7％)of A-type,54 strains(45.3％)of D-type and 1 strain(0.9％)of F-type among the capsu-lar polysaccharide serotypes,and 10 strains(8.4％)of L1-type,20 strains(16.8％)of L3-type,86 strains(72.2％)of L6-type,and 3 strains(2.6％)of undetermined type among the lipopolysaccha-ride serotypes.The amplification results of 10 virulence genes showed that the detection rate of virulence genes hgbA,higbB and fimA was over 86.0％,the detection rate of toxA was 8.4％,while the virulence gene tbpA was not detected.There were also differences in the distribution vir-ulence genes in different capsular polysaccharide serotypes.Virulence factor pfhA was detected in type A and F but not in type D.The detection rate of adhesin gene tadD in serotype A(92.2％)was significantly higher than that of type D(9.3％),and,on the contrary,the detection rate of ad-hesin gene hsf-l in serotype D(90.7％)was significantly higher than that of type A(20.3％).Drug resistance analysis revealed that Pasteurella multocida showed high susceptibility to antimi-crobial drugs such as amoxicillin,ampicillin,cephalosporins,doxycycline,fosfenicol and ciprofloxa-cin,and showed strong resistance to antimicrobial drugs such as lincomycin,cotrimoxazole,genta-micin and amikacin,and there were 54 multi-drug resistant strains(78.3％).In summary,capsular polysaccharide serotypes were dominated by type A and D,lipopolysaccharide serotypes were dom-inated by L6,the distribution of some virulence genes varied greatly among different serotypes,and the proportion of multi-resistant strains was high,which provide reference for the prevention and control of this disease.

This study was aimed at preliminarily investigating the genetic and antimicrobial resistance characteristics of Salmonella Alachua isolates through whole-genome analyses.Five Salmonella Alachua isolates from various sources(both hu-man and non-human)were collected and identified.Phenotype and serotype verification,antimicrobial susceptibility testing,and whole-genome sequencing were performed.Virulence genes,antimicrobial resistance genes,and plasmid replicons were predicted according to globally available Salmonella Alachua genomic data.A phylogenetic tree was constructed to explore the genetic background.The first report of Salmonella Alachua in China emerged in Shanghai in 2015,and patients presented pri-marily with diarrhea.The isolates have been found predominantly in the eastern and southern coastal regions.Among the five i-solates analyzed,four belonged to sequence type(ST)2061,and one belonged to ST1298.All isolates were susceptible to most commonly used clinical antibiotics.Whole-genome analyses revealed that two ST2061 strains carried the blaKPC-2 gene,and one ST1298 strain carried the fosA7 gene.Phylogenetic analysis of global Salmonella Alachua populations indicated that the ST2061 clone belonged to the C1 clade,which was closely related to strains from the UK,whereas the ST1298 clone was found in the C4 clade,a globally disseminated fosA 7-positive lineage.This study provides initial insights into the genetic and antimi-crobial resistance characteristics of Salmonella Alachua in China,highlighting the presence of strains carrying blaKPC-2 and fo-sA7 genes.These findings may provide a reference for future large-scale molecular epidemiological surveillance and source-trac-ing efforts,and they underscore the importance of enhanced resistance monitoring for Salmonella Alachua.

Objective To explore the value of amide proton transfer(APT)imaging combined with diffusion weighted imaging(DWI)in the assessment of neurovascular invasion(NVI)of stage T3/T4 rectal cancer.Methods The clinical and MR imaging data of 46 patients with rectal cancer were analyzed retrospectively and divided into NVI positive group and NVI negative group according to the pathological results of NVI.The differences of APT values[maximum APT(APTmax)value,minimum APT(APTmin)value,mean APT(APTmean)value]and apparent diffusion coefficient(ADC)value between the two groups were compared,and the diagnostic efficiency was analyzed by receiver operating characteristic(ROC)curve.Results The APTmax and APTmean values in the NVI positive group were significantly higher than those in the NVI negative group,while the ADC value in the NVI positive group was significantly lower than that in the NVI negative group(P＜0.05).The area under the curve(AUC)of APTmax,APTmean and ADC values for NVI identification were 0.717,0.722 and 0.751,respectively.The AUC of the three indexes combined to identify NVI was 0.858.The combined AUC of the three indexes was larger than that of APTmax,APTmean and ADC alone(P＜0.05).Conclusion APT imaging combined with DWI has a high value in the evaluation of NVI of stage T3/T4 rectal cancer,which can provide guidance for clinical treatment.

Objective To explore the value of amide proton transfer(APT)imaging combined with diffusion weighted imaging(DWI)in the assessment of neurovascular invasion(NVI)of stage T3/T4 rectal cancer.Methods The clinical and MR imaging data of 46 patients with rectal cancer were analyzed retrospectively and divided into NVI positive group and NVI negative group according to the pathological results of NVI.The differences of APT values[maximum APT(APTmax)value,minimum APT(APTmin)value,mean APT(APTmean)value]and apparent diffusion coefficient(ADC)value between the two groups were compared,and the diagnostic efficiency was analyzed by receiver operating characteristic(ROC)curve.Results The APTmax and APTmean values in the NVI positive group were significantly higher than those in the NVI negative group,while the ADC value in the NVI positive group was significantly lower than that in the NVI negative group(P＜0.05).The area under the curve(AUC)of APTmax,APTmean and ADC values for NVI identification were 0.717,0.722 and 0.751,respectively.The AUC of the three indexes combined to identify NVI was 0.858.The combined AUC of the three indexes was larger than that of APTmax,APTmean and ADC alone(P＜0.05).Conclusion APT imaging combined with DWI has a high value in the evaluation of NVI of stage T3/T4 rectal cancer,which can provide guidance for clinical treatment.

Glaesserella parasuis is the causative agent of Gl?sser's disease in pigs.However,the pathogenic mechanisms underlying its virulence is not yet fully understood.The RuvB protein,a member of the AAA+superfamily,is implicated in various cellular processes,yet its specific role in the virulence of Glaesserella parasuis has not been fully characterized.In this study,we con-structed a RuvB gene deletion mutant,designated ΔRuvB,using the serotype 13 Glaesserella pa-rasuis strain ZJ1208 and a suicide plasmid-mediated natural transformation approach.To elucidate the functional role of the RuvB gene,we comprehensively evaluated the biological characteristics of the ΔRuvB strain through a series of assays,including growth kinetics,colony morphology,bac-terial staining,transmission electron microscopy(TEM),osmotic stress tolerance,high-tempera-ture tolerance,heat shock resistance,UV resistance,capsular polysaccharide quantification,serum bactericidal assays,and murine virulence experiments.Our findings revealed that the growth rate of ΔRuvB showed no significant difference compared to the parental strain.TEM revealed a notable increase in bacterial cell length;however,the number of outer membrane vesicles(OMVs)on the surface of ΔRuvB did not significantly increase.Notably,the ΔRuvB strain displayed a significant reduction in capsular polysaccharide production and serum resistance,as well as diminished toler-ance to UV radiation and high temperatures.Significant alterations were observed in its resistance to osmotic stress or oxidative stress.In the mouse toxicity challenge experiment,in com-parison with the parental strain ZJ1208,the mortality rate dropped by 20 percentage points,suggesting that the virulence of ΔRuvB has been weakened to some extent.Collectively,these results underscore the critical role of the RuvB gene in enhancing the environmental adaptability of Glaesserella parasuis.

Glaesserella parasuis is the causative agent of Gl?sser's disease in pigs.However,the pathogenic mechanisms underlying its virulence is not yet fully understood.The RuvB protein,a member of the AAA+superfamily,is implicated in various cellular processes,yet its specific role in the virulence of Glaesserella parasuis has not been fully characterized.In this study,we con-structed a RuvB gene deletion mutant,designated ΔRuvB,using the serotype 13 Glaesserella pa-rasuis strain ZJ1208 and a suicide plasmid-mediated natural transformation approach.To elucidate the functional role of the RuvB gene,we comprehensively evaluated the biological characteristics of the ΔRuvB strain through a series of assays,including growth kinetics,colony morphology,bac-terial staining,transmission electron microscopy(TEM),osmotic stress tolerance,high-tempera-ture tolerance,heat shock resistance,UV resistance,capsular polysaccharide quantification,serum bactericidal assays,and murine virulence experiments.Our findings revealed that the growth rate of ΔRuvB showed no significant difference compared to the parental strain.TEM revealed a notable increase in bacterial cell length;however,the number of outer membrane vesicles(OMVs)on the surface of ΔRuvB did not significantly increase.Notably,the ΔRuvB strain displayed a significant reduction in capsular polysaccharide production and serum resistance,as well as diminished toler-ance to UV radiation and high temperatures.Significant alterations were observed in its resistance to osmotic stress or oxidative stress.In the mouse toxicity challenge experiment,in com-parison with the parental strain ZJ1208,the mortality rate dropped by 20 percentage points,suggesting that the virulence of ΔRuvB has been weakened to some extent.Collectively,these results underscore the critical role of the RuvB gene in enhancing the environmental adaptability of Glaesserella parasuis.

To identify clinical viral diseases characterized by reproductive disorders and abortion,a quadruple qPCR method was established for simultaneous detection of PRV,PPV,PCV2 and AS-FV.Four pairs of specific primers and probes were designed according to the conserved genes of four viruses in the NCBI gene bank.The annealing temperature,primer concentration and probe concentration of the reaction were optimized,and the specificity,sensitivity and repeatability of the method were tested.The results showed that the method could not detect other pathogens except the target ones.The minimum detection limit of PRV,PPV,PCV2 and ASFV was 10 copies.Intra-group and inter-group repeatability tests showed that the coefficient of variation of C,values be-tween different batches was less than 3％,indicating that the method was highly specific,sensitive and stable.Establishment of an efficient and sensitive quadruple qPCR method provides technical reference for the clinical prevention and control of porcine pseudorabies virus disease,porcine circo-virus disease,porcine parvovirus disease and African swine fever.

This study investigates the effects of pseudorabies virus(PRV)infection on the antiviral immune signaling pathways and type Ⅰ interferon factors in mouse trigeminal ganglion(TG)cells.In this experiment,primary TG cells were infected with PRV at a multiplicity of infection(MOI)of 1,while mice were infected via a drop-nose method using 106,29 TCID50 of PRV.Real-time fluorescence quantitative PCR(qPCR),Western blot and ELISA were used to assess gene tran-scription,protein expression,and the secretion of IFN-α and IFN-β.The results indicated that PRV infection of mouse TG primary cells led to alterations in the gene and protein expression of RIG-Ⅰ,MAVS,and IRF3,as well as the phosphorylation of IRF3 and IKBα both in vivo and ex vivo.ELISA results showed that PRV infection could regulate the secretion of IFN-α and IFN-β in mouse primary TG cells and mouse TGs.The results of RIG-Ⅰ signaling pathway-related proteins and the secretion of IFN-a and IFN-β were analyzed using Western blot after using siRNA to interfere with RIG-Ⅰ expression in TG cells.The results showed that siRIG-Ⅰ successfully inter-fered with RIG-Ⅰ protein expression in TG cells and caused changes in the expression of down-stream proteins such as MAVS and IRF3,and also regulated the secretion of IFN-α and IFN-β in TG cells.Furthermore,the results indicated that PRV infection induced the expression of RIG-Ⅰ in mouse TG progenitor cells,regulating the antiviral immune response of type Ⅰ interferon factors in TG cells through the RIG-Ⅰ-MAVS-IRF3 signaling axis.Notably,PRV inhibited the expression of IRF3 in TG cells while significantly upregulating the expression of IFN-β during the later stages of infection,which may be an important factor in the important reason for the rapid mortality ob-served in mice during the late stages of PRV infection.This experiment elucidates part of the anti-viral immune mechanism mediated by the RIG-Ⅰ-MAVS-IRF3 signaling pathway in regulating type Ⅰ interferon factor after PRV infection of mouse TG cells,as well as the discovery of differ-ent trends of IRF3 protein changes in vivo and ex vivo,laying the groundwork for future in-depth studies.