Introduction: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Introduction: Due to their immunostimulatory properties TLR ligands are used prophylactically to protect against a variety of viral and bacterial pathogens in mammals. Knowledge of the molecular and functional aspects of TLRs is essential for a better understanding of the immune system and resistance to diseases in birds. For that reason, this study attempted to determine the impact of TLR21 stimulation by its synthetic ligand (CpG ODN, class B) on the chicken immune system. Material and Methods: Sixty embryonated chicken eggs were randomly allocated into three groups (control and two experimental groups). On day 18 of embryonic development, chickens in one experimental group were administered in ovo a low dose of CpG ODN and the birds of the second experimental group were given a high dose of the ligand. Spleens were collected at 1, 2, 5, and 10 days post-hatching (dph) for analysis of IFN-α, IFN-β, IFN-γ, IL-6, and IL-10 expression using qRT-PCR. Results: Significant differences were observed in mRNA expression levels of all the measured cytokines associated with the modulation and regulation of the immune response at different time points. Conclusion: The obtained data clearly demonstrate that immune response induction takes place after in ovo administration of class B CpG ODN, and that the ligand has the ability to induce cytokine responses in neonatal chicken spleen.

Introduction: The effects of Jin-Ying-Tang (JYT) on Toll-like Receptor 4 (TLR4) signalling transduction of lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (MECs) in vitro were examined. Material and Methods: The cytotoxicity of JYT (0.06-62.50 mg/mL) on mouse MECs was determined by MTT assay. The MECs were co-cultured with LPS in the presence or absence of JYT (39.10 μg/mL, 391 μg/mL, 3910 μg/mL). The concentrations of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in the culture supernatants were detected by ELISA. The mRNA expression of TLR4 and downstream TLR4 signalling molecules such as myeloid differentiation factor 88 (MyD88), tumour necrosis factor receptor associated factor 6 (TRAF-6), inhibitor κB (IκB), and nuclear factor κB inducing kinase (NIK) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The results showed that the IC₅₀ of JYT on MECs was 12.25 mg/mL and JYT could significantly decrease the concentrations of IL-6 and TNF-α in LPS-stimulated MECs (P < 0.05). The mRNA expression of TLR4, MyD88, TRAF-6, IκB, and NIK was also significantly decreased when the LPS-stimulated MECs were cocultured at appropriate concentrations of JYT (P < 0.05, P < 0.01). Conclusion: These observations indicate a potential mechanism through which JYT attenuates the systemic inflammatory response to LPS-stimulated mouse mammary epithelial cells by inhibiting the activation of TLR4/MyD88/ TRAF-6/NIK pathway at the mRNA level.

Foot-and-mouth disease is a highly infectious viral disease affecting all cloven-footed domestic animals. The three foot-and-mouth disease virus (FMDV) serotypes A, O and SAT2 are at present the greatest threat to susceptible animals in Egypt. The aim of the present study was, for the host factors associated with different FMDV infections in cattle during the acute phase, to compare these factors’ influence on the expression of the IL-10, TLR-2, TNF-α, CXCL10, CD48, NFATC4 and IFNG inflammatory and immune-related genes. Vesicular fluid and epithelium samples were obtained from at least three infected cattle on the same affected farm during three different FMDV outbreaks and were used for serotyping of the virus and for expression analysis of host genes. A two-step RT-PCR was used for diagnosis of the virus with primers specific for each serotype. In quantitative PCR analysis, the expression patterns of TLR-2 and IFNG were prominent, while NFATC4 expression was absent in all FMDV-infected cattle. The highest expression of CD48 was associated with increased expression of other inflammatory and immune-related genes (IL-10, TLR-2, TNF-α and IFNG), which may be an indication of rapid virus clearance. The use of vesicular fluid and epithelium for investigation of viral and immune-related gene expression levels in acute FMDV infection is possible. Host-dependent variation in the expression of the studied genes was observed in different FMDV serotype outbreaks.

Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen–free chickens were immunised and their humoral and cellular immune responses were analysed. The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4⁺/CD8⁺ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4⁺/CD8⁺ ratio and the increase in IL-4 did not promote anti-HA antibodies. Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

Toll-like receptors (TLRs) play an important role in fast activation of the immune response to a variety of pathogens, including parasites. In this study, we focused on TLR2, because this receptor is one of the best known and most frequently analysed members of the TLR family. The aim of this study was to assess the effect of Trichinella spiralis on expression of TLR2 during the intestinal stage of infection. The experimental material consisted of isolates prepared from the intestines (jejunum and colon) of BALB/c mice infected with T. spiralis taken at 4, 8, and 16 days post infection. Our results based on quantitative real-time PCR showed that the mRNA level for TLR2 was statistically significantly higher in the jejuna of mice infected with T. spiralis than in this tissue of uninfected mice. In addition, the presence of TLR2 protein in the intestinal phase of trichinellosis was confirmed by a strong positive immunohistochemical reaction. Our results indicate that infection with T. spiralis changes the expression of TLR2 in the small intestine of the mouse host and suggest a contribution of these receptors to the host defence mechanisms during experimental trichinellosis.

Introduction: Tiletamine-xylazine-tramadol (XFM) has few side effects and can provide good sedation and analgesia. Adenosine 5’-monophosphate-activated protein kinase (AMPK) can attenuate trigeminal neuralgia. The study aimed to investigate the effects of XFM and its specific antagonist on AMPK in different regions of the brain. Material and Methods: A model of XFM in the rat was established. A total of 72 Sprague Dawley (SD) rats were randomly divided into three equally sized groups: XFM anaesthesia (M group), antagonist (W group), and XFM with antagonist interactive groups (MW group). Eighteen SD rats were in the control group and were injected intraperitoneally with saline (C group). The rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus, and brain stem were immediately separated, in order to detect AMPKα mRNA expression by quantitative PCR. Results: XFM was able to increase the mRNA expression of AMPKα1 and AMPKα2 in all brain regions, and the antagonist caused the opposite effect, although the effects of XFM could not be completely reversed in some areas. Conclusion: XFM can influence the expression of AMPK in the central nervous system of the rat, which can provide a reference for the future development of anaesthetics for animals.

Introduction: Toll-like receptors (TLRs) play a key role in the rapid activation of the innate immune response to a variety of pathogens. The aim of this study was to evaluate the effect of Trichinella spiralis infection on the level of expression of the tlr4 gene in mouse intestines during the intestinal phase of experimental trichinellosis. Material and Methods: The experimental material consisted of the small and large intestines of BALB/c mice infected with Trichinella spiralis sampled at 4, 8, and 16 days post infection (dpi). Results: A statistically significant increase was demonstrated in the tlr4 mRNA level isolated from the infected mice jejunum at 4, 8, and 16 dpi over the uninfected control. Moreover, at 4, 8, and 16 dpi in the jejunum of infected mice, a strong positive reaction for the presence of TLR4 protein compared with that of uninfected mice was observed. Conclusion: Infection with T. spiralis changes the expression of the tlr4 gene in the small intestine of the mouse host.

Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

Enolases are enzymes in the glycolytic pathway, which catalyse the reversible conversion of D-2-phosphoglycerate into phosphoenol pyruvate in the second half of the pathway. In this research, the effects of α-enolase (ENO1) on steroid reproductive-related hormone receptor expression and on hormone synthesis of primary granulosa cells from goose F1 follicles were studied. Primary granulosa cells from the F1 follicles of eight healthy 8-month-old Zi geese were separated and cultured. An ENO1 interference expression vector was designed, constructed and transfected into primary cultured granulosa cells. The mRNA expression levels of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR), oestrogen receptor α (ER α), oestrogen receptor β (ER β), growth hormone receptor (GHR) and insulin-like growth factor binding protein-1 (IGFBP-1) in the cells were evaluated as were the secretion levels of oestradiol, activin, progesterone, testosterone, inhibin and follistatin in cell supernatant. α-enolase gene silencing reduced the expression of FSHR, LHR, ERα, ERβ, GHR, and IGFBP-1 mRNA, potentiated the secretion of oestrogen, progesterone, testosterone, and follistatin of granulosa cells, and hampered the production of activin and inhibin. ENO1 can regulate the reactivity of granulosa cells to reproductive hormones and regulate cell growth and development by adjusting their hormone secretion and reproductive hormone receptor expression. The study provided a better understanding of the functional action of ENO1 in the processes of goose ovary development and egg laying.