BACKGROUND: Ticks are one of the most important ectoparasites in animals that cause economic losses in livestock industry. So, removal or reduction of ticks on animals is necessary. Cysteine proteases are among the compounds that play an important role in the physiological action of ticks and are a good candidate for the anti-tick vaccine. Cathepsins is one of the most important cysteine proteases. OBJECTIVES: The aim of this study was cloning and expression of recombinant cathepsin L gene of Rhipicephalus annulatus in order to evaluate its immunogenicity. METHODS: After collection the ticks were cultivated. Then RNA was extracted from ticks, cDNA was synthesized by using specific primer of cathepsin and amplification by RT-PCR. The desired genes were cloned into expressional pQE30 plasmid. Further, a shorter sequence of the cathepsin gene (654 bp) was prepared as a synthetic plasmid. The expression of the protein produced by both recombinant plasmids in the E.coliBL21 prokaryotic expression system is carried out and the immunity of the recombinant proteins was evaluated by Dot Blot and Western Blot using serum of challenged rabbits with recombinant protein and calves infected with ticks were examined and compared. RESULTS: The results of this study indicate that the protein derived from recombinant plasmid No. 2 had higher expression and purity due to its solubility. Also, the challenge of rabbit serum with these proteins was able to identify both recombinant proteins. But the serum of challenged calves with ticks did not show a satisfactory response with recombinant proteins. CONCLUSIONS: Although the sera reaction of calves infested with ticks was lower than the challenged rabbits sera with cathepsin L, this result was expected, because L cathepsin protein is considered as a concealed antigen.  Overall, the recombinant cathepsin L could be an appropriate candidate for immunizing calves against Rhipicephalus annulatus, although it seems further investigations are necessary.

BACKGROUND: Leucine is one of the subgroups of amino acids, which play an important role in the anabolism of muscles, adipose tissue, and the liver by stimulating insulin secretion.OBJECTIVES: Effects of different levels of leucine were investigated on carcass yield, characteristics, and quality, and expression of insulin-like growth factor (IGF-1) and insulin genes in male broilers.METHODS: Five levels of L-leucine (100, 110, 120, 130, and 140 % of Ross strain requirements) were tested with 250 male one-day-old chicks in a completely randomized design with five treatments and five replicates (containing 10 chicks each). On day 42 of their age, the blood samples of two birds from each replicate (10 birds per treatment) were taken to determine serum IGF-1 gene expression. Subsequently, these birds were slaughtered for analysis of carcass characteristics and quality, and collecting the samples of liver and breast for expression of IGF-1 and insulin genes.RESULTS: Body weight increased by consumption of 140 % of leucine as compared to 100 %. Reduction in feed conversion ratio was observed by feeding 140 % of leucine level. The IGF-1 gene expression of breast and liver increased by 110 % of leucine level. Moreover, feeding 110 % of leucine level caused a higher expression of insulin gene in breast and liver. Consumption of 130 % of leucine improved the meat protein, fat, and ash contents. Furthermore, consumption of 110 % of leucine increased the serum IGF-1 concentration.CONCLUSIONS: Consumption of leucine in broiler diets was found to increase the expression of IGF-1 and insulin genes and consequently, improve the performance and carcass quality. It also decreased the abdominal fat in broiler chickens.

BACKGROUND: High grain diets in ruminants increases the risk of digestives disorders such as acidosis which may lead to high economic loss. OBJECTIVES: This experiment was conducted to determine the effects of an unsaturated and polyunsaturated fatty acid and monensin on gene expression of enzymes involved metabolic pathway of cell proliferation and rumen epithelial intracellular pH regulation. METHODS: Twenty two male Afshari lambs with live body weight of 45 ± 8 kg and six month age were used in a completely randomized design with 3 treatments replicates for 77days including 21 days adaptation period. Experimental diets were consisted of a basal high concentrate diet (16% CP and 2.75 Mcal/kg ME) and 1) no additive (control, C= 8 lambs), 2) 30 mg monensin/day/head during the whole experimental period (T1= 8 lambs), and 3) (polyunsaturated fatty acidduring the whole experimental period (T2 = 6 lambs). Lambs were killed after 77 days on the treatment diets. RESULTS: Compared with the C treatment, relative abundance of mRNA of monocarboxylate transporter isoforms MCT1, MCT4 and the ketogenic enzyme 3-hydroxy-3 methyl-glutaryl CoA-synthase (HMGCS2) were higher for the T1 treatment. The expression of cholesterolgenic enzyme HMGCS1 was down-regulated for the T1 treatment and that of HMGCS1 was up- regulated for the T2 treatment. The expression of MCT1 and MCT4 were down-regulated for the T2 treatment. Monensin had an additional impact on the mRNA abundance of epithelial SCFA- and acid-base transporters with concurrent changes in rumen epithelial thickness. CONCLUSIONS: The results suggest that adding monensin and oil as nutritional means to reduce acidosis cause changes in mRNA expression of VFA transferring proteins and limiting enzyme in the synthesis of cholesterol and Ketone bodies in the rumen epithelium.

BACKGROUND: Betaine is a derivative of three methylates of glycine amino acids, found in the body of many animals. Objectives: The study was conducted to investigate the effects of betaine supplementation in the diet on gene expression and activities of lipogenic enzymes, and lipid levels of blood and liver in broilers. Methods: A total of 320 broilers were evaluated (Ross 308) in a completely randomized design with 4 treatments and 4 replicates, with the diets consisted of the control treatment which did not use the betaine supplementation. The second, third and fourth treatments contained 0.05, 0.08 and 0.11% betaine hydrochloride 98%, respectively. Results: Betaine supplementation to the diet has no significant effect on lipoprotein lipase gene expression, and the activities of enzyme Acetyl-CoA carboxylase and fatty acid synthase (p> 0.05). Betaine supplementation caused a significant decrease in liver cholesterol and triglyceride chicks fourth treatment (containing betaine hydrochloride 0.11 percent) compared to the control group (p<0.05). Conclusions: In general, the results show that in broilers lipoprotein lipase gene expression and the activities of enzyme Acetyl-CoA carboxylase and fatty acid synthase are less influenced by betaine.

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.

Bovine immunodeficiency virus (BIV) is found worldwide in cattle under natural conditions. However, the effect of BIV infection on immune functions has not been fully characterised.

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.