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: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.

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.

The characteristics of immune factors in somatic cells from lactating dairy cows and their association with commensal bacteria in normal milk have not been clarified. This study investigated the relationship between the pathogenic bacteria in milk and somatic cell immune factors in healthy lactating cows. In total 44 healthy Holstein cows were studied on one farm. Milk samples were collected aseptically using a cannula and these samples were cultured for detection of bacteria and analysis of mRNA of immune factors expressed by somatic cells. Cows were divided into two groups based on the microbial status of their milk samples: 12 cows showed bacteria in cultures (positive group), and the other 32 cows did not (negative group). The mRNA levels of IL-6, lactotransferrin, and cathelicidin expressed by somatic cells after milking decreased significantly compared to those before milking in both groups (P < 0.05). There were significantly lower mRNA levels of IL-6 and cathelicidin in the positive group compared to those in the negative group before milking. These results suggest that mRNA levels of IL-6 and cathelicidin expressed by the somatic cells may be affected by the presence of bacteria in healthy lactating dairy cows.

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.

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.

Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis. Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR. The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity. OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.

The purpose of this study was to investigate the protective effect of hydrogen-rich saline (HRS) against liver ischaemia-reperfusion combined resection injury. Eighteen miniature pigs were randomly divided into three groups: a sham operated group (sham group, laparoscopic liver ischaemia-reperfusion combined resection injury group (IRI group), and a hydrogen-rich saline intervention group (IRI + HRS group). Samples of hepatic tissue and serum were collected at the time of reperfusion and then 3 h, 1 d, and 3 d post reperfusion. Liver function, oxidative stress, autophagy-related mRNA genes, and protein expression were evaluated. Changes in cell and tissue ultrastructure were examined by transmission electron microscopy. Compared with the sham group, the level of autophagy of hepatocytes increased in the IRI and IRI + HRS groups, corresponding to high oxidative stress and severe liver function injury. Liver function, antioxidant content, autophagy levels, and liver injury were improved after intervention with HRS in the IRI + HRS group compared with the IRI group. Intervention with hydrogen-rich saline could exert a protective effect against liver ischaemia-reperfusion combined resection injury through the reduction of oxidative stress and hepatocyte autophagy.

Introduction: Bovine postpartum metritis causes great losses. Mast cell (MC)-released mediators participate in uterine inflammation and immune response, but their role in postpartum metritis in cows has not been reported. This study investigated the effect of endometrial MC on the disorder.Material and Methods: Ten dairy cows, at 6 to 10 days postpartum and with acute purulent metritis made up the experimental group, and 10 comparable healthy cows the control group. Endometrial histamine and IgE levels were determined by ELISA, and the MC particle state and expression of histamine H₁ (H₁R) and H₂ (H₂R) mRNA receptors were examined by transmission electron microscope and real-time quantitative PCR, respectively.Results: Endometrial histamine and IgE levels were significantly higher in the experimental group. In the control group, homogenously distributed size-varied granules were seen in MC cytoplasm of endometrium of lamina propria. In the experimental group however, these showed degranulation with features of reduction. The level of H₁R mRNA was lower in the experimental group, but that of H₂R mRNA was higher.Conclusion: The results suggest MC type I hypersensitivity characteristics during metritis, and histamine provocation of local inflammation. High expression of H₂R and low expression of H₁R inhibited the inflammatory response and prevented excessive uterine tissue damage.