ABSTRACT
Diagnosis of bovine viral diarrhea (BVD) relies on the detection of antibodies against its viral causing agent, bovine viral diarrhea virus (BVDV). Here, we designed a novel competitive ELISA (cELISA) using the most immunogenic part of BVDV nonstructural protein 3 (NS3), as a single ELISA recombinant antigen, along with a monoclonal antibody to detect antibodies against BVDV in sera of infected animals. Hence, 197 serum samples were tested by this cELISA and the results were compared to the results obtained from virus neutralization test (VNT) as the gold standard method for diagnosis of BVD. McNemar's test indicated that there was no significant difference between the results of this newly designed cELISA and VNT. Meanwhile, kappa coefficients showed that there was a high correlation between these two assays. The relative sensitivity and specificity of cELISA with respect to VNT were 93.90% and 100%, respectively, suggesting that this newly designed cELISA could be a useful diagnostic tool for detection of BVDV infection. Moreover, as NS3 is highly conserved among Pestiviruses and the developed ELISA is a competitive one, it could potentially be applied to detect BVDV infection in other domestic and wildlife species.
ABSTRACT
Side effects of chemical preservatives and drug resistance have raised interest in use of herbal products. This study aimed to examine the chemical composition and antibacterial effects of Cuminum cyminum, Mentha spicata, and Mentha longifolia essential oils individually and in combination with sodium benzoate against methicillin-resistant Staphylococcus aureus and Yersinia enterocolitica. The essential oils were analyzed by gas chromatography-mass spectrometry. Disc diffusion and microdilution assays were used for in vitro antimicrobial screening. The main components were cumin aldehyde, carvone, and pulegone in C. cyminum, M. spicata, and M. longifolia essential oils, respectively. Antibacterial data analysis showed significant differences between different antibacterial effects of essential oils individually and in combination with sodium benzoate. In terms of individual effects, antibacterial effect of M. longifolia and C. cyminum essential oils were the highest against methicillin-resistant S. aureus and Y. enterocolitica, respectively. The antibacterial effects of sodium benzoate combined with essential oils showed significant differences with the individual effect of sodium benzoate in most cases. The results indicated that the combined effect of these essential oils with sodium benzoate could reduce the use of sodium benzoate as an antimicrobial agent, which could decrease its possible side effects. Thus, for more significant effects, these essential oils could be combined with other agents for the preservation of drug and food products.
ABSTRACT
This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcus species were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.
ABSTRACT
BACKGROUND: Honey has previously been shown to have wound healing and antimicrobial properties, but this is dependent on the type of honey, geographical location, and flower from which the final product is derived. We tested the antimicrobial activity of a natural honey originating from the Ziziphus spina-christi tree, against selected strains of bacteria. Ziziphus honey among more than a 100 verities of honey is known to have the greatest value of energy and minerals in it. MATERIALS AND METHODS: The aim of this study was to determine the antibacterial activity of Ziziphus honey in 10%, 20%, 30%, and 40% dilutions (v/v) against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus. Viable count enumeration of the sample was investigated after 0, 24, 72, and 120 h postinoculation with any of the bacteria using pour-plate method. RESULTS: The findings indicate that Ziziphus honey was effective against these pathogenic bacteria. In a comparative trial, antibacterial activity of Ziziphus honey was higher after 120 h incubation for each four bacteria in most dilutions. The microbial count showed 3-7.5 log reduction comparing with control after 120 h. CONCLUSIONS: Therefore, it is recommended using Ziziphus honey as a natural preservative and antibacterial agent. Also, it could potentially be used as therapeutic agents against bacterial infection particularly to the tested microorganisms.
ABSTRACT
BACKGROUND: Bovine viral diarrhea (BVD) is an economically important cattle disease with a worldwide distribution. Detection and elimination of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) is essential for the control of BVD and eradication of BVDV. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or virus-induced antigens or antibodies. OBJECTIVES: Erns as an immunogenic protein of BVDV, is genetically and antigenically conserved among different isolates and therefore, is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological studies or identification of PI animals. The aim of this study was to produce a monoclonal antibody (MAb) against recombinant Erns. MATERIALS AND METHODS: For this purpose, recombinant maltose-binding protein (MBP)-Erns protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen in MAb production. Spleen cells of the immunized mice with the recombinant antigen were fused with SP2/0 myeloma cells. Next, culture supernatants of primary clones of fused cells were screened by indirect ELISA. After three rounds of cloning, the reactivity of the MAbs with recombinant and natural antigen was established by Western blotting. RESULTS: Based on our results, MAb against recombinant Erns was produced and reacted successfully with recombinant and natural antigens. CONCLUSIONS: With regards to the role of Erns in the identification of PI animals, it appears that Erns recombinant antigen and the specific monoclonal antibodies produced against it may be suitable for developing BVDV laboratory diagnostic assays.
ABSTRACT
Six consecutive overlapped coding regions (F1-F6) of whole NS3 molecule of bovine viral diarrhea virus (BVDV) were cloned into pMAL-c2X plasmid vector and expressed in Escherichia coli cells (BL21 strain). The recombinant proteins were then purified by amylose resin to determine the most immunogenic domain(s) of the NS3 molecule. Evaluation of the recombinant proteins was carried out by indirect ELISAs using several bovine sera (previously characterized by virus neutralization test, a commercial ELISA kit, and a newly developed NS3-ELISA) and 6 monoclonal antibodies. The experiments showed that the most immunogenic domain of the NS3 protein was the fourth designed fragment (F4), a 122 amino-acid (AA) region of about 13.5 kDa (nucleotide 1003-1368; residue 335-456). Purified recombinant F4 was also evaluated as single ELISA antigen (F4-ELISA) for the detection of anti-BVDV antibodies in sera of infected cattle. Although this small recombinant fragment of NS3 protein was almost completely soluble and expressed more efficient respect to whole NS3 molecule, it did not show enough sensitivity and specificity to be a proper substitute for NS3 as ELISA antigen to detect specific antibodies against BVDV. However, statistical analyses showed a medium correlation between the results of the developed F4-ELISA and virus neutralization test (kappa coefficient=0.63, P<0.001), with the relative sensitivity and specificity of 78.05% and 84.91%, respectively, suggesting the potential use of this fragment as an ELISA antigen along with other antigens or monoclonal antibody(s) in a competitive ELISA.
