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.

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was de-tected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou,Zhejiang Province,China

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.

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.

Immunogenicity of formaldehyde and binary ethylenimine inactivated infectious bursal disease virus in broiler chicks

Infectious bursal disease virus (IBDV) was inactivated by two different chemicals—formaldehyde and binary ethylenimine (BEI). Formaldehyde was used at 0.1% and 0.2%, while BEI was used at concentrations of 0.001 and 0.002 mol/L. These four vaccines were tested for their efficiency in generating humoral immune response in different groups of broiler chicks. Both BEI-inactivated vaccines gave relatively higher antibody titers and were almost twice as efficient as formaldehyde-inactivated ones.

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.

Inactivation of infectious bursal disease virus by binary ethylenimine and formalin

In this experiment conducted to study the inactivation dynamics of infectious bursal disease virus （IBDV） by binary ethylenimine （BEI） in comparison with formalin, IBDV was isolated from the bursa of infected chickens and its confirmation was done by agar gel precipitation test. Viral suspensions were subjected to inactivation with BEI and formalin for pre-set time in- tervals. BEI was employed at concentrations of 0.001 and 0.002 mol/L while formalin was used at 0.1% and 0.2%. Sampling was done at 6, 12, 24, 36 and 48 h of incubation and samples were tested for their inactivation status in 9-day-old embryonated eggs and 3-week-old broiler chickens. IBDV was completely inactivated by 0.001 and 0.002 mol/L BEI after 36 h of incubation at 37℃, whereas formalin at 0. 1% and 0.2% concentrations inactivated IBDV in 24 h.

Role of duck plague virus glycoprotein C in viral adsorption:Absence of specific interactions with cell surface heparan sulfate

Many mammalian herpes viruses utilize heparan sulfate （HS） moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C （gC） homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus （DPV） remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus （DPV-AgC-EGFP）. Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins （gB-DPV, gC-DPV, and gE-DPV） to HS was assessed using a prokaryotic expression system and HJTrapTM HeparJn HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-△gC-EGFP or wild-type strain Chinese virulent duck plague virus （DPV-CHv）. The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on Viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

Nerve growth factor and inducible nitric oxide synthase expression in the mesencephalon and diencephalon, as well as visual-and auditory-related nervous tissues, in a macaque model of type 2 diabetes

The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a macaque model of type 2 diabetes using immunohistochemistry. Results showed that nerve growth factor expression decreased, but inducible nitric oxide synthase expression increased, in the mesencephalon and diencephalon, as well as visual- and auditory- related nervous tissues. These results suggested that nerve growth factor and inducible nitric oxide synthase play an important role in regulating the development of diabetic visual- and auditory-related diseases.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Intragenic and intergenic sequences regulating the expression of the 5'-to-5' linked adult α-and β-globin genes from large yellow croaker Pseudosciaena crocea

One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.

Identification and characterization of adult alpha-and beta-globin genes and their genomic arrangement in Pseudosciaena crocea

The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted： ct39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

Alphaherpesvirus-vectored vaccines against animal diseases: Current progress

Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.

Can natural preservatives serve as a new line of protective technology against bacterial pathogens in meat and meat products?

Contamination of meats and meat products by pathogenic microorganisms is responsible for a significant percentage of outbreaks of foodborne illness.There are also concerns over the carcinogenic potential of dietary nitrate and nitrite in processed meat products.The past few decades have seen an extensive search for novel technologies alternative to synthetic chemical preservatives to reduce the level of contamination of foods by pathogenic and spoilage microbes.This review provides a general overview of natural preservatives with potential applications in the meat industry,including phages and their endolysins,bacteriocins,microbial lipopeptides,antimicrobial peptides of plant or insect origin,and essential oils or extracts of plant origins.Instead of providing summary data from the published literature,we attempt to elaborate the challenges facing the development of novel natural preservatives as antimicrobial hurdles,taking into consideration the sharp contrast between extensive studies in this particular field and very limited industrial use.More specifically,we emphasize the great importance of having streamlined approaches and methodological guidelines in the research and development of natural preservatives so that the journey to their industrial use for safer meats and meat products could be shortened or made easier.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis

Porcine circovirus type 2（PCV2）has recently been reported to elicit the unfolded protein response（UPR）via activation of the PERK/e IF2α（RNA-activated protein kinase-like endoplasmic reticulum（ER）kinase/eukaryotic initiation factor 2α）pathway.This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein.By transient expression,we found that both replicase（Rep）and capsid（Cap）proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eI F2α-ATF4（activating transcription factor 4）-CHOP（CCAAT/enhancer-binding protein homologous protein）axis.Cap expression,but not Rep,significantly reduced antiapoptotic B-cell lymphoma-2（Bcl-2）and increased caspase-3 cleavage,possibly due to increased expression of CHOP.Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression,caspase-3cleavage,and apoptotic cell death possibly by partially rescuing Bcl-2 expression,we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eI F2α/ATF4/CHOP/Bcl-2 pathway.This study,together with our earlier studies,provides insight into the mechanisms underlying PCV2 pathogenesis.

Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin

Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress

The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones.In this study,a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified.The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids.Three characteristic signature regions of the HSP70 family,two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD),and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence.The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting.Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level,and 49.1% to 99.5% at the amino acid level with the known Hsc70s.Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis).The tHsc70 was expressed in the liver,lung,heart,and skeletal muscle.The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C.Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress.Irrespective of different profiles of expression under heat stress,tHsc70 may play roles in protecting turtles from thermal stress.