Povezanost između polimorfizma gena CXCR1 i rizika od endometritisa u krava holštajnske pasmine

Endometritis is one of the reproductive diseases that can cause disturbances of postpartum uterine health in cattle. Therefore, identification of resistant genotypes to endometritis is essential. The objective of this study was to evaluate the association between the single nucleotide polymorphism in the interleukin-8 receptor-α (CXCR1) gene and the possibility of endometritis in Holstein dairy cows. For this purpose, blood samples were collected from multiparous dairy cows with a history of clinical endometritis (n = 30), and normal, healthy cows as the control group (n = 10). Deoxyribonucleic acid (DNA) was isolated from a blood sample. To determine genotype, the polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) was used. The results indicated the presence of a different proportion of polymorphisms (G > C) in the CXCR1 gene in cows with clinical endometritis, compared to the control group. Statistical analyses showed that there is a significant correlation between the incidence of the disease and the CXCR1 genotype in nucleotide position 956. The incidence rate of clinical endometritis was associated with the CXCR1.956 genotype; cows with genotype GC had a higher incidence of clinical endometritis compared with cows with the GG genotype. Overall, the results showed that CXCR1 polymorphism could be a useful marker for identification of resistant genotypes to endometritis in Holstein dairy cows.Endometritis je reproduktivna bolest koja može poremetiti poslijeporođajno zdravlje maternice u goveda, stoga je važna identifikacija genotipova otpornih na tu bolest. Cilj ovoga istraživanja bio je procijeniti povezanost između polimorfizma pojedinačnog nukleotida na genu interleukin-8 receptoru-α (CXCR1) i vjerojatnosti pojave endometritisa u holštajnskih mliječnih krava. U tu su svrhu prikupljeni uzorci krvi mliječnih krava multipara s kliničkim endometritisom u anamnezi (n = 30) i zdravih krava kao kontrolne skupine (n = 10). Iz uzoraka krvi izolirana je DNA. Genotip je određivan metodom lančane reakcije polimeraze – polimorfizmom dužine restrikcijskih fragmenata (PCR-RFLP). Rezultati upućuju na prisutnost različitih omjera polimorfizama (G > C) gena CXCR1 u krava s kliničkim endometritisom u usporedbi s kontrolnom skupinom. Statistička analiza pokazala je znakovitu korelaciju između incidencije bolesti i genotipa CXCR1 u poziciji nukleotida 956. Incidencija kliničkog endometritisa bila je povezana s genotipom CXCR1.956; krave s genotipom GC imale su veću incidenciju kliničkog endometritisa u usporedbi s kravama s genotipom GG. Općenito, rezultati pokazuju da bi polimorfizam CXCR1 mogao biti koristan biljeg za identifikaciju genotipova otpornih na endometritis u holštajnskih mliječnih krava

Preparation, characterization, and in ovo vaccination of dextran-spermine nanoparticle DNA vaccine coexpressing the fusion and hemagglutinin genes against Newcastle disease

Plasmid DNA (pDNA)-based vaccines have emerged as effective subunit vaccines against viral and bacterial pathogens. In this study, a DNA vaccine, namely plasmid internal ribosome entry site-HN/F, was applied in ovo against Newcastle disease (ND). Vaccination was carried out using the DNA vaccine alone or as a mixture of the pDNA and dextran-spermine (D-SPM), a nanoparticle used for pDNA delivery. The results showed that in ovo vaccination with 40 µg pDNA/egg alone induced high levels of antibody titer (P0.05). Higher antibody titer was observed in the group immunized with 40 µg pDNA/egg at 4 weeks postvaccination. The findings also showed that vaccination with 40 µg pDNA/egg alone was able to confer protection against Newcastle disease virus strain NDIBS002 in two out of seven SPF chickens. Although the chickens produced antibody titers 3 weeks after in ovo vaccination, it was not sufficient to provide complete protection to the chickens from lethal viral challenge. In addition, vaccination with pDNA/D-SPM complex did not induce high antibody titer when compared with naked pDNA. Therefore, it was concluded that DNA vaccination with plasmid internal ribosome entry site-HN/F can be suitable for in ovo application against ND, whereas D-SPM is not recommended for in ovo gene delivery.Ministry of Science, Technology and Innovations (MOSTI), Malaysia, for the research grant (ERGS/1-2012/5527122), and Institute of Bioscience, Higher Institution Centre of Excellence (IBS HICoE) grant from the Ministry of Higher Education, Government of Malaysi

Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure

Development of DNA vaccine containing HN and F genes of Newcastle disease virus and its efficacy following in vivo and in ovo immunizations

Newcastle disease is a fatal viral disease which is highly contagious that affects most species of birds and is a major economic threat in the poultry industry. Both the HN and F glycoproteins of Newcastle disease virus (NDV) are essential for pathogenicity and virus infectivity. Though vaccines are available for the control of the disease but the vaccine with more efficacies is still required. The third generation vaccines, which include DNA vaccine, have the potential to be safer and more efficacious. In this study, DNA vaccines developed successfully by using the HN and F genes from a Malaysian viscerotropic velogenic AF2240 NDV strain into the pIRES bicistronic mammalian expression vector separately and combined.Three recombinant plasmids were constructed with the Kozak translation initiation sequences, namely, pIRES/HN, pIRES/F and pIRES/HN/F. The HN and F genes in the pIRES vector has been expressed and tested successfully using the indirect immunofluore scence (IIF) test and Western blotting in the Vero cell line (in vitro). The results of IIF showed that all the DNA-transfected cells exhibited bright cytoplasmic fluorescence, indicating both the F and HN proteins were successfully expressed in the mammalian cell line. Also, Western blotting results revealed the expected bands of size approximately 74kDa, 55 kDa and 12 kDa for HN, F1 and F2, respectively. In vivo experiment I showed that single vaccination with the plasmid DNA (pDNA) was not sufficient to induce antibody titer in specific pathogen free (SPF) chickens. Whereas, single pDNA vaccination and boosted by killed vaccine, showed a significant difference in ELISA antibody levels (p0.05) in vaccinated chickens with pIRES/HN/F and pIRES/HN+pIRES/F which boosted with killed vaccine at one week post booster compared with vaccinated chicken with killed vaccine alone. Overall, it was concluded that the recombinant pDNA vaccine can be used to increase the efficacy of the killed vaccine immunization procedure. In vivo experiment II showed that twice vaccination with pDNA was able to elicit significant antibody titers (p< 0.05) by either monocistronic or bicistronic plasmid, after one week of second pDNA vaccination (booster). The results proposed that DNA immunization of chickens at second vaccination had enhanced the antibody response successfully. As a result, the findings of the present study well demonstrated that vaccination with the co-expression plasmid pIRES/HN/F can induce a stronger antibody response thanvaccination with pIRES/HN or pIRES/F alone. In ovo vaccination with 10 μg pDNA/egg was not sufficient to induce production of antibody in SPF chickens. However, in ovo vaccination with 40 μg pDNA/egg induced high levels of antibody titer (p<0.05) in SPF chickens at four weeks post vaccination. The findings also showed that vaccination with 40 μg pDNA/egg able to confer protection against ND in two out of seven SPF chickens. Although, the chickens produced antibody titres three weeks post in ovo vaccination, it was not sufficient to protect the chickens from lethal viral challenge. However, vaccination with pDNA/Dextranspermine complex did not induce high antibody titer compared with naked pDNA. In conclusion, DNA vaccination with pIRES/HN/F is suitable for in vivo application but not encourage for in ovo use. More studies are required to investigate how to increase the efficacy of the constructed recombinant pDNA vaccines

Developing a vaccine against velogenic sub-genotype seven of Newcastle disease virus based on virus-like particles

Abstract In the present study, for the first time, we released and assembled the particles of three major structural proteins of velogenic NDV (M, HN, and F glycoproteins) as a NDV-VLPs. The ElISA result of the cytokines of splenocyte suspension cells showed that IL2, IL10, TNF-α, and IFN- ˠ titers were significantly higher (p ≤ 0.05) in mice that were immunized only with NDV-VLPs three times with a 10-day interval, in comparison to those that were immunized with NDV-VLPs twice in a 10-day interval and received a B1 live vaccine boost on the third interval. Flow cytometry results showed that CD8 + titers in the group that only received NDV-VLP was higher than other group. However, serum ELISA results did not show a significantly (p ≥ 0.05) higher NDV antibody titer in NDV-VLPs immunized mice compared to the boosted group. Besides, HI results of SPF chickens vaccinated with NDV-VLPs and boosted with B1 live vaccine were significantly (p ≤ 0.05) higher than those that only received NDV-VLPs. Interestingly, after challenging with NDV sub-genotype VII, all the chickens that were solely vaccinated with NDV-VLPs remained alive (six out of six), whereas two out of six chickens that were vaccinated with NDV-VLPs and also received the B1 live vaccine boost died. In conclusion, our results strongly indicated that the T-cell immune response in an NDV host is more important than the B-cell response. Also, the results of the present study revealed that to completely protect chickens against velogenic NDV strains, a vaccine comprising specific epitopes of velogenic strain is needed