Toward innovative veterinary nanoparticle vaccines

Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.

Preparation and interaction mechanism analysis of single‑chain fragment variables against phenylethanolamine A

This is the first report on the screening,expression,and recognition mechanism analysis of single-chain fragment variable(scFv)against phenylethanolamine A(PEAA),a newly emergedβ-adrenergic agonist illegally used as a feed additive for growth promotion.The PEAA-specific scFv scFv,called scFv-32,was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E.coli system.The ic-ELISA results revealed an IC_(50)value of 10.34μg/L for scFv-32 and no cross-reactivity with otherβ-adrenergic agonists.Homology modeling and molecular docking revealed the key binding sites VAL178,TYP228,and ASP229.One hydrogen bond,two pisigma bonds,and one pi-pi bond maintain the formation of the antibody‒drug complex.Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity,which confirmed the accuracy of the theoretical analysis.These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.

Antibacterial Effects of Chinese Herbal Medicines against Streptococcus iniae in Oreochromis niloticus

In this study, the antibacterial effects of Chinese herbal medicines against Streptococcus iniae in Oreochromis niloticus were investigated by drug sensitiv- ity test. The results indicated that Streptococcus in/ae was extremely sensitive to Calla Chinensis, Fructus Hippophae and Fructus Murne, highly sensitive to Sappan Lignum, Pericarpium Granati, Folium Eucalypti and Radix Scutellariae, moderately sensitive to Rh/zoma Coptidis, Radix et Rhizoma Rhei, Flos Caryophyllata, Cortex phellodendri and Rabdosia serra （ Maxim. ） Hara, and insensitive to Herba Portulacae , Herba Houttuyniae , Polygonum hydropiper L. , Herba Menthae , Radix Glycyrrhizae , Pericarpium Citri Reticulatae , Herba Andrographitis and Rhizoma Acori Tatarinowii. Chinese medicinal herbs which Streptococcus iniae was extremely sensitive or moderately sensitive to were selected and prepared into three Chinese herbal medicine formulae for medicated bath of Oreochromis niloticus infected arti- ficially with Streptococcus iniae. The results showed that medicated bath elicited therapeutic effects on infected Oreochromis niloticus. Formula II exhibited the best therapeutic effect with an effective percentage of 90%.

Observation of Traditional Chinese Medicine's Anti-bacterial Effect on the Main Pathogenic Bacteria of Dairy Cow Recessive Mastitis

[Objective]This paper aimed to study the anti-bacterial effect of traditional Chinese medicine on cow recessive mastitis＇ s main pathogenic bacteria and provide basis for its clinical application.[Method]Plating and test-tube methods were used to determine the anti-bacterial diameter,minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of 20 kinds of traditional Chinese medicine on dairy cow recessive mastitis＇ s clinically isolated main pathogenic bacteria,Staphylococcus aureus and Streptococcus agalactiae.[Conclusion]The results indicated that 7 kinds of traditional Chinese medicine,Herba Taraxaci,Rhizoma Coptidis,Fructus Forsythiae,Herba Andrographis,Radix Scutellar iae,Flos Carthami,Flos Chrysanthemi Indici had strong anti-bacterial effect.

The Effect of Traditional Chinese Medicine on Serum AMS,LPS and XOD of Piglets with Spleen Deficiency

[Objective] The experiment aimed to determine the effect of Guchangcuzhangsan on serum amylase, lipase and xanthine oxidase of piglets with spleen deficiency. [ Method] The model of piglets with spleen deficiency was produced by the methods of injecting reserpine into piglets, and alternating between no feed and good feed. The determinations of serum amylase, lipase and xanthine oxidase of piglets with spleen deficiency were done before injecting reserpine, on the day when reserpine was injected last time, the next day of finishing giving powder, the eighth day after finishing giving powder. [ Result] After the experiment was finished, all 3 enzymes activities of Guchangcuzhangsan group had no significant differ- ence from that of normal contrast group, enzyme activities of spleen deficiency group and smecta group had significant difference compared with that of normal contrast group. [ Conclusion] Guchangcuzhangsan can promote the recovery of serum amylase, lipase and xanthine oxidase of piglets with spleen deficiency which was caused by reserpine.

Effects of Chinese Medicine on the Number of Intraepithelial Lymphocytes and Goblet Cells of Intestinal Villus of Heat Stress Layers

[ Objective] To study the effects of Chinese herbal additives on the number of intraepithelial lymphocyte and goblet cells of intestinal villus of heat stress layers. [Method] 180 healthy 88-day-old ISA brown egg roosters were selected and randomly divided into nine treatment groups, nor- mal temperature control group, high temperature control group, VC group, prescription one high-dose group, prescription one middle-dose group, prescription one low-dose group, prescription two high-dose group, prescription two middle-dose group, prescription two low-dose group, respec- tively. Prescription one and two groups were respectively fed with low, medium and high concentrations of the three doses of Chinese herbal ex- tracts, and VC group was fed with the VC in aqueous solution. Histological sections conventional technology and HE staining method were used to observe the number of intraepithelial lymphocytes and goblet cells in each sections of small intestine of chicken. [ Result] The number of chicken in- testinal epithelial lymphocytes and goblet cells showed a gradually decreasing trend in a high-temperature state. [ Conclusion] Prescription one and two groups could promote the cytopoiesis of goblet cells and lymphocytes, and the effect of prescription two was the best. Moreover, Adding the Chinese herbs had good effects on relieving the heat stress of layers.

Screening of Chinese Herbal Medicines against Avian Infectious Laryngotracheitis Virus and Escherichia coli

[ Objective] This study aimed to screen Chinese herbal medicines against avian infectious laryngotracheitis virus and Escherichia coli. [ Method ] Conventional Chinese medicine plate dilution method, plate punching method and test tube method were applied to screen Chinese herbal medicines against avian infectious laryngotracheitis virus based on chicken embryo inoculation experiment. [ Result ] Forsythia suspensa, Radix lsatidis, Isatis iadigotica, Lonicera japonica, Codonopsis pilosula, Astragalus membranaceus, Rhizoma Atractylodis Macrocephalae, C, tyeyrrhiza uralensis and Pericarpium granati had relatively strong anti-ILTV effect; among the Chinese herbal medicines against avian E. coli, Sanguisorba offwinalis, Fructus Mume, Rheum palmatum, Anemarrhena asphodeloides, Radix Scutellariae and Fagopyrum cymosum had relatively strong effect against avian E. coli Os, while other Chinese herbal medicines had relatively weak or no inhibitory effect on avian E. coli 0s and 05. [ Conclusion] This study laid the foundation for further development of Chinese herbal medicine compound preparations to treat avian infectious laryngotracheitis, avian colibacillosis and other viral diseases and bacterial diseases.

Allelic functional variation of FimH among Salmonella enterica subspecies

Salmonella enterica has a wide diversity,with numerous serovars belonging to six different subspecies with dynamic animal-host tropism.The FimH protein is the adhesin mediating binding to various cells,and slight amino acid discrepancy significantly affects the adherence capacities.To date,the general function of FimH variability across dif-ferent subspecies of Salmonella enterica has not been addressed.To investigate the biological functions of FimH among the six Salmonella enterica subspecies,the present study performed several assays to determine biofilm for-mation,Caenorhabditis elegans killing,and intestinal porcine enterocyte cell IPEC-J2 adhesion by using various FimH allele mutants.In general,allelic mutations in both the lectin and pilin domains of FimH could cause changes in bind-ing affnity,such as the N79S mutation.We also observed that the N79S variation in Salmonella Dublin increased the adhesive ability of IPEC-J2 cells.Moreover,a new amino acid substitution,T260M,within the pilin domain in one subspecies llb strain beneficial to binding to cells was highlighted in this study,even though the biofilm-forming and Caenorhabditis elegans-killing abilities exhibited no significant differences in variants.Combined with point muta-tions being a natural tendency due to positive selection in harsh environments,we speculate that allelic variation T26oM probably contributes to pathoadaptive evolution in Salmonella enterica subspecies llb.

Enzootic epidemiology of Brucella in livestock in central Gansu Province after the National Brucellosis Prevention and Control Plan

Brucellosis remains one of the most common zoonoses spread worldwide,inducing enormous economic losses to the livestock industry and posing serious health threats to humans.Brucellosis re-emerged in China in the mid-1990s and reached a historically high level in 2015.The National Brucellosis Prevention and Control Plan(NBPCP)was initiated from 2016 to 2020.However,the present epidemiological status in livestock has not been elucidated,and whether Brucella variation occurred remains unclear.This study performed an extensive serological investigation in ruminant livestock from 2019 to 2021 in central Gansu Province,China.In total,11,296 samples from 337 farms were collected to detect the specific antibodies of Brucella.The yearly average serological prevalence of Brucella at the flock level and individual level declined from 11.32%to 8.26%and 1.17%to 0.57%,respectively.The apparent individuallevel seroprevalence of small and large ruminants was 0.89%and 0.52%,respectively.The brucellosis distribution has shifted from pastoral areas to agro-pastoral areas.Flock size and gender may be major risks of Brucella infection.Then,the B.melitensis TZ strain was isolated from female Tibetan sheep blood cell lysates.Phonotypical characterization demonstrated that it belongs to B.melitensis.biovar 3,and multilocus sequencing typing results indicated that it belongs to ST8.The whole genome and subsequent phylogenetic analysis demonstrated that the B.melitensis TZ strain is genetically more closely related to the B.melitensis QH61 strain.The B.melitensis TZ strain has similar growth characteristics to the B.melitensis 16 M strain.Overall,our study suggests that after strengthening control and prevention measures based on the NBPCP,there is a very low prevalence or absence of B.melitensis in the central Gansu Province of China,and the genotype of an epidemic strain of Brucella in Northwest China is relatively stable.

Altered gene expression in human brain microvascular endothelial cells in response to the infection of influenza H1N1 virus

Influenza viruses not only cause respiratory illness,but also have been reported to elicit neurological manifestations following acute viral infection.The central nervous system(CNS)has a specific defense mechanism against pathogens structured by cerebral microvasculature lined with brain endothelial cells to form the blood–brain barrier(BBB).To investigate the response of human brain microvascular endothelial cells(hBMECs)to the Influenza A virus(IAV),we inoculated the cells with the A/WSN/33(H1N1)virus.We then conducted an RNAseq experiment to determine the changes in gene expression levels and the activated disease pathways following infection.The analysis revealed an effective activation of the innate immune defense by inducing the pattern recognition receptors(PRRs).Along with the production of proinflammatory cytokines,we detected an upregulation of interferons and interferon-stimulated genes,such as IFN-β/λ,ISG15,CXCL11,CXCL3 and IL-6,etc.Moreover,infected hBMECs exhibited a disruption in the cytoskeletal structure both on the transcriptomic and cytological levels.The RNAseq analysis showed different pathways and candidate genes associated with the neuroactive ligand-receptor interaction,neuroinflammation,and neurodegenerative diseases,together with a predicted activation of the neuroglia.Likewise,some genes linked with the mitochondrial structure and function displayed a significantly altered expression.En masse,this data supports that hBMECs could be infected by the IAV,which induces the innate and inflammatory immune response.The results suggest that the influenza virus infection could potentially induce a subsequent aggravation of neurological disorders.

Protection of Carassius auratus Gibelio against infection by Aeromonas hydrophila using specific immunoglobulins from hen egg yolk

Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.

White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus

Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5-80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer （ITS） of its ribosomal DNA （rDNA）. Detailed morphological examinations were also conducted to confirm the white-spot disease.

Microarray-Based Detection and Differentiation of Virulent and Attenuated Pseudorabies Virus

DNA microchip used in this study was formed from miniature arrays of pseudorabies virus （PrV） gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip （microarrays） was used for differentiation between virulent and attenuated PrV. The presence of four gene segments （gB, gD, gE~, and gE ） encoding conservative glycoprotein B （gB）, D （gD）, and E （gE） of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE~ gene fragments was shown in virulent （gE~ genotype） and attenuated PrV （gE genotype）, whereas gE- gene （deleted domain in gE gene） was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus （PRRSV）, porcine parvovirus （PPV）, Japanese encephalitis virus （JEV）, and porcine circovirus type 2 （PCV-2）. The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.

The Influence of Chinese Medicinal Herb on Chicken Immune Organs and Small Intestinal Mucosa Immune Organization

[Objective] The aim was to explore the mechanism of Chinese medicinal herb to enhance the body＇s immune. [Method] The quantitative distribution of immunocytes in chicken small intestinal mucosa lymphoid tissue-secretory type immune globulin cell A were dynamic observed to research chicken immune organ growth with histology conventional slice technology and immunohistochemistry dye. 1 day age healthy roosters were divided into 3 groups： the group 3 was control group. 1% and 0.5% concentration of Chinese herbal medicine immunopotentiator drinking water were added in the group 1 and 2 in continuous 60 d. The immune organ index was determined every 12 d and the histotomy of chicken small intes- tine in group control and 1% were taken for histological observation on day 24, 36 and 48. [ Result] Treatment group immune organ index was significantly higher than that of the control group and 1% group of small intestinal villus inherent intraformational immune cells number significantly increased （P〈0.01） compared with controls. Day 36 age group and day 48 group immune cells were higher than day 24 group of cell number （P〈 0.01 ）. [ Conclusionl Chinese medicinal herb had obvious role in promoting chicken immune organ growth and obvious influence on the quantity change of the intestinal mucosal immune cells.

Detection of K88 Fimbriae Gene of Escherichia coli from Mink

[Objective] This paper aimed to study the mechanism of diarrhea of mink caused by Escherichia coil [Method] Through the detection of K88 fimbriae gene of E. coli, cloning of gene fragments and identification, then PCR amplification was used to detect adhesion factor K88 gene, which was connected to T-vector and transformed into competent cells, and positive clones were selected. [ Results] E. coli 078, 029 and 038 were isolated from organs and feces of mink died of diarrhea in 3 mink farms, respectively, the 3 serotypes of E. coliwere detected in carrying K88 fimbriae gene and 3 positive clones were screened, respectively. [ Conclusion] The E. coli causing mink diarrhea carry K88 fimbriae gene.

Effects of fishing handling stress on juvenile red crucian carp(Carassius auratus red variety)

The essay studies the influence of the different intensities of fishing stress on body weight （7.30 ± 1.48） g of red crucian carp juvenile body length, weight, fatness, feeding rate, specific growth rate, food conversion rate, intestinal protease, amylase and immunoglobulin. The result showed that body length, weight, fatness, feeding rate, specific growth rate of red crucian carp juvenile were significantly inhibited from fishing stress compared with the control group, and the magnitude of the inhibitory effect increased with the time going and increased. Fishing stress could increase the food conversion rate of red crucian carp juvenile, and the different intensity stress had no significant different influence. The intestinal protease activity decreased in the beginning, then increased, and was consistent with the level of the control group ultimately. High-stress group accelerated the decline of intestinal amylase activity of the juvenile. The stress increased the number of immunoglobulin of juvenile red crucian carp significantly, but the increase disappeard with the stress time extended.

Cloning of catalase gene and antioxidant genes in Scophthalmus maximus response to metalloprotease of Vibrio anguillarum stress

Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic eff ects in turbot(Scophthalmus maximus).The toxicological eff ect partly results from oxidative damage due to the production of excessive reactive oxygen species(ROS).Catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx)are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells.To evaluate the eff ects of metalloprotease-induced ROS on the antioxidation defense mechanism of S.maximus head kidney cells,the cDNA of CAT gene(designated as SmCAT)was cloned and characterized.SmCAT comprises a 1584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A)tail.Bioinformatics analysis revealed an active site signature sequence,a heme-ligand signature sequence,and three catalytic amino acid residues.The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%-92.4%with those of other species.Phylogenetic analysis revealed that SmCAT is classifi ed with CAT of other fi shes.Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues,especially in blood.The expression of SmCAT,SmMnSOD,and SmGPx were inhibited signifi cantly in head kidney cells treated with metalloprotease from 12 to 24 h.In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group,it was found that the production of ROS was markedly increased,and the mitochondrial membrane potential was decreased considerably.Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0μg/mL metalloprotease for 12 and 24 h.Hence,the toxic eff ects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels,which trigger the activation of apoptosis in the head kidney cells of turbot.Our fi ndings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fi sh.

Studies on acute toxicity of three heavy metal ions to Artemia

Studies on acute toxicity of Cu2+,Mn2+and Zn2^to Artemianauplii(36h)and adults were carried out at15±3°C in the natural seawater where salinity is30using single-factor acute toxicity;on this basis,equi-toxicity test was used to evaluate the effect of joint toxicity of Cu2+,Mn2+and Zn2+to Artemia nauplii(36h)in the same conditions.Results show that Artemia has different tolerances for the three heavy metal ions when it is in different growth period,and the interaction of heavy metal ions varied with growth period,adults are more sensitive of the three heavy metals;The48h LC50of these three heavy metal to the Artemia nauplii is4.0,27.19and115.95mg/L,and to the adults is1.31,6.86and34.23mg/L;the order of toxicity of cuprum,manganese and zinc for Artemia nauplii and adults was Cu2^>Mn2+>Zn2+;the safe concentration of the three heavy metal ions(Cu2+,Mn2+and Zn2+)to Artemia nauplii(36h)and adults were0.4,2.72,11.6,0.13,0.69and3.42mg/L.The results of combined toxicity test showed that Cu-Mn,Cu-Zn and Mn-Zn were all antagonistic to the Artemia naupli.

Constructing and Analyzing Fusion Promoter of Partial Sericin 1 and Bombyx A3 Cytoplasmic Actin

Previous report showed that the 209 bp DNA sequence upstream of the sericin 1 transcriptional start site （-586 to -378 bp） is involved in promoting transcription and responsible for the tissue specificity of sericin 1 promoter in silkworm Bombyx mori. In the present study, this 209 bp sequence exhibited enhancive effect by assembling in two different locations of ubiquitous Bombyx A3 cytoplasmic actin promoter. Sf-9 cells were transfected with recombinant plasmids using Cellfectin reagent. Firefly luciferase gene located downstream of fusion promoter was considered as a reporter, whereas the activity of the co-transfected Renilla luciferase gene （pGL2-SV40） provides an internal control. This 209 bp region up-regulates the strength of A3 promoter significantly （P〈0.01） when it enters into A3 promoter with respect to the position in sericin 1 gene promoter. This 209-bp fragment was almost functionless when being located upstream of A3 promoter.

The Detection of Pathogenic Porcine E.coli Virulence Gene (astA,stb)

[ Objective] This paper aimed to find out the relationship between pathogenic procine E. coli virulence gene and pathogenicity, and ex- plore the pathogenic mechanism of E. coil [ Methed] The detection of two E. coil virulence genes was performed. PCR method was taken to test the virulence genes, astA and stb, from 39 strains of typical serotype O porcine E. coli which had been separated and identified. [ Result] It＇s found that of the 39 isolates of porcine E. coli, 22 carried virulence gene astA which represented 56.41%, and 27 carried virulence gene stb which repre- sented 69.23%. [ Conclusion] This study has provided scientific data for future E. coil pathogenicity researches.