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: Diarrhea is a common disease in the neonate calf which imposes significant economic burden on cattle industry around the world. During the first week after birth, Enterotoxigenic E.coli (ETEC) strains carrying F5 fimbria are one of the most important pathogens causing calf diarrhea. F5 fimbria is involved in early stage of pathogenesis and is responsible for attachment of bacteria to enterocytes; this attachment is mediated by FanC protein of F5 fimbria. Antibodies directed against F5 fimbriae play a significant role in prevention and control of the disease. Objectives: Evaluation and expression of recombinant expression of F5 Fimbriae of Enterotoxigenic Escherichia Coli associated with calf diarrhea. Methods: In the present study,  the fanC region of F5 fimbria was cloned in a pET28a plasmid. Results: The recombinant construct was confirmed by sequencing and protein production in Escherichia coli BL21 (DE3) was evaluated by western blotting procedure. Conclusions: Based on our findings, the recombinant FanC protein or the BL21 (DE3) strain are suitable candidates to develop an effective vaccine against calf colibacillosis or use in a diagnostic kit for F5+ ETEC.

BACKGROUND: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D (PLD) is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. OBJECTIVES: The aim of this study was the cloning and expression of the PLD and HSP60 genes of C. pseudotuberculosis, used subsequently to evaluate the protectivity of these recombinant proteins for vaccine development against this bacterium. METHODS: PLD and HSP60 genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH5 strain of E. coli. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleotide sequence analysis. RESULTS: The transformed E. coli strain DH5 expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form. CONCLUSIONS: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed.

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.

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.

Objective-To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. Sample Population-4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. Procedure-Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multitargeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. Results-No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cellcycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. Conclusions and Clinical Relevance-These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

This work reports the first molecular analysis study of chicken anaemia virus (CAV) in backyard chickens in Africa using molecular cloning and sequence analysis to characterize CAV strains obtained from commercial chickens and Nigerian backyard chickens. Partial VP1 gene sequences were determined for three CAVs from commercial chickens and for six CAV variants present in samples from a backyard chicken. Multiple alignment analysis revealed that the 6 % and 4 % nucleotide diversity obtained respectively for the commercial and backyard chicken strains translated to only 2 % amino acid diversity for each breed. Overall, the amino acid composition of Nigerian CAVs was found to be highly conserved. Since the partial VP1 gene sequence of two backyard chicken cloned CAV strains (NGR/Cl-8 and NGR/Cl-9) were almost identical and evolutionarily closely related to the commercial chicken strains NGR-1, and NGR-4 and NGR-5, respectively, we concluded that CAV infections had crossed the farm boundary.

Use of a combination of an effective gE gene-deleted pseudorabies virus (PRV) vaccine with a companion diagnostic kit for PRV glycoprotein gE has proven successful in several pseudorabies-eradication programs. To produce a large quantity of functional gE protein for development of a PRV-gE diagnostic kit, an Escherichia coli expression system containing the distal region of the PRV-gE gene of a PRV strain CF was constructed. The expressed protein contained 134 amino acids of gE protein (amino acids 77-210) fused to a 19-amino acids tag containing 6 histidine residues. After induction, a truncated PRV-gE polypeptide of 18-kd was expressed to about 20% of the total E coli proteins. Results of immunoblot analysis indicated that this E coli-produced PRV-gE protein reacted specifically with serum from PRv-hyperimmunized pigs and from field PRv-infected pigs, but not with serum samples from specific-pathogen-free pigs or pigs inoculated with gE-deleted PRV vaccine. These data indicate that, although the recombinant gE protein is produced in E coli, it still retains the antigenicity of the viral gE glycoprotein. Comparison between the recombinant gE protein, using immunoblot analysis with a commercial gE ELISA containing natural PRV-gE protein, revealed comparable test performance. This finding indicated that recombinant gE protein produced by E coli can be used for development of a companion serologic assay for a PRV-gE gene-deleted vaccine.

A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 microgram of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.