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Archives of Razi Institute - Volume:74 Issue: 1, Winter 2019

Archives of Razi Institute
Volume:74 Issue: 1, Winter 2019

  • تاریخ انتشار: 1397/12/10
  • تعداد عناوین: 10
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  • R. Akbari, M. H. Sekhavati *, A. Bahrami, R. Majidzadeh Heravi, S. Yousefi Pages 1-6
    Brucella bacterium causes Brucellosis, an infectious disease spreading from animals to human. Brucella lumazine synthase (BLS) is a highly immunogenic protein with adjuvant properties, which has been introduced as an effective protein carrier for vaccine development. This protein also plays a significant role in inducing immune system. This study aimed to clone, express, and purify the BLS gene from Brucella melitensis Rev1. The BLS gene was amplified by particular primers with the restriction enzyme sites as a linker and it was inserted into pTZ57R/T vector. Subsequently, it was ligated into pET32(a)+ expression vector. Recombinant expression vector containing coding sequence of BLS was transformed into E. coli BL21 (DE3) host gene expression and stimulated by 0.1mM IPTG. The results of sequencing showed that there were not any mutations in BLS encoding sequence. The expression results were set by sequencing and endorsed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and western blotting that showed 35 kDa protein band appropriately.
    Keywords: Brucella melitensis Rev1, BLS, Gene Expression, Recombinant Protein
  • S. Ataei *, M. M. Ranjbar, N. Motamed, S. Ataei Kachooei, F. Amini Njafi Pages 7-20
    The haemolytic biovar of Gallibacterium anatis (G. anatis) is responsible for urogenital, gastrointestinal, and respiratory diseases in chickens. There are numerous reports on the resistance of G. anatis to antibiotics and recurrence of the disease, which raise concerns about antimicrobial treatment efficiency. Vaccination has been considered as the most feasible procedure of prevention in high risk farms. Subunit vaccines containing immunogenic components can have practical protective value in preventive measures regarding the infection. The present study aimed to introduce a polytopic vaccine candidate based on epitope detection. All registered sequences of four immunogenic proteins, includig Flfa, GTxA, Gab_1309, and Gab_2348 were retrieved and directed for variational analysis. A vaccine isolate was selected for each protein and tested for B-cell epitope mapping using different tools. Furthermore, consensus selected immunogenic regions with special patterns fused together by flexible linkers were integrated into two constructs and checked for the best status of proteasomal cleavage sites, as well as hydropathy plot. Moreover, back translations, along with codon optimization were performed, and then some tags were added to the constructs. The selected consensus B-cell immunogenic epitopes were for 12656: AA114-181, 7990: AA114-181, Avicor: AA42-77, 134-197, and IPDH: 61-155 for Flfa protein, AA185-235, AA372-457, and AA807-941 for GtxA-N, AA260-305, AA340-400, and AA110-146 for Gab-1309, and AA125-AA175 for Gab-2348. Two suitable patterns of attachment were selected from the different fusion patterns of epitopes in B-cell polytopic vaccinal constructs. Finally, the examination of these constructs showed their effect and efficacy for immune system stimulation. Based on bioinformatics results, these immunogens could be utilized as potential candidates to develop polytopic protective vaccines and design diagnostic kits.
    Keywords: Gallibacterium anatis, Vaccine, Polytopic, In-silico
  • A. Salarpour, R. Toroghi *, R. Momayez Pages 21-31
    Infectious bronchitis (IB) virus genome codes for four structural proteins, among which the S1 subunit of spike glycoprotein comprises the major epitopes to induce neutralizing antibodies. This study involved the comparison of the full S1 sequences of five IB viruses, namely two Massachusetts and three 793/B serotypes, isolated from IB outbreaks during 2001-2002, with all other Iranian and foreign 793/B isolates and 10 known serotypes. Analysis of S1 subunit showed three unique amino acid changes at positions 349 (V to L), 392 (T to N), and 393 (Q or R to T or K or S) for the Iranian 793/B isolates, compared to those of the foreign 793/B isolates reported before 2006 (onset of vaccination with 793/B vaccine in Iran). They were used as amino acid markers for the differentiation of Iranian 793/B isolates for years. Sequence alignment of the Iranian isolates with those of the foreign ones reported after 2006 demonstrated that amino acids 392 and 393 were no longer considered as amino acid markers, and only the change in amino acid 349 still remained specific to the Iranian 793/B isolates. Phylogenetic tree sequence analysis revealed that the Iranian 793/B isolates were closely related indicating that they came from a single source, more probably from France. There was a very close correlation between the first detection of 793/B serotype and the time of French chicken meat importation. Moreover, it was shown that one of the Massachusetts isolates was completely identical with the H120 vaccine strain. Furthermore, the other Massachusetts isolate with two amino acid changes at positions 64 (G to E) and 95 (S to R) was very similar to this vaccine strain. It seems that the latter isolate is a passaged chicken H120 vaccine strain.
    Keywords: Infectious bronchitis virus, 793, B serotype, S1 gene, Amino acid marker, Phylogenetic analysis
  • A. Yousefi *, M. R. Chaechi Nosrati, A. Golmohammadi, S. Azami Pages 33-38
    The genus Anaplasma is an obligated intracellular Rickettsia and among its species, Anaplasma phagocytophilum (A. phagocytophilum) is a zoonotic agent that infects host neutrophils. The aim of this study was molecular detection of A. phagocytophilum infection based on MSP4 gene in owned and stray dogs in Tehran, capital of Iran. One hundred and fifty blood samples were collected from dogs in Tehran and suburbs of Tehran, Iran. Firstly, the thin blood smears were prepared and Giemsa staining method was conducted. Then, the samples were examined under oil immersion objective and 0.67% of them were observed infected with A. phagocytophilum. The DNA was extracted from blood samples using a DNA isolation kit (MBST, Iran), and MSP4 gene extraction was performed by Polymerase chain reaction (PCR) and nested-PCR. Finally, 2% of the samples were positive for A. phagocytophilum. The data were analyzed using SPSS software (version 19.0) and Chi-square test was performed. There was no significant relation between infection and age, as well as sex and ectoparasitic infestation (P>0.05). This article was a report of A. phagocytophilum infection in dogs and their potentials as host carriers of this important microorganism in Tehran, Iran.
    Keywords: Anaplasma phagocytophilum, Tick-borne, Dog, Tehran, Iran
  • H. Morovati, S. J. Seyyed Tabaei *, M. Gholamzad, K. Omidfar, A. Ahmadi, Z. Arab Mazar, A. Eshaghi, F. Sheikhsofla Pages 39-49
    Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis and is of medical importance in pregnant women and immunosuppressed patients. In recent years, many methods have been developed for the detection of infection caused by this parasite; however, most of the developed methods are not adequately sensitive. The dense granular antigen (GRA) 7 is a highly immunogenic protein that is used as a specific antigen for the diagnosis of toxoplasmosis. This study was designed to produce recombinant GRA7 (rGRA7) antigen in bacterial system in order to be applied as an antigen for developing a simple and rapid lateral flow immunoassay test strip using a gold nanoparticle-pAb conjugate probe to detect Toxoplasma IgG-specific antibodies in human sera. After the extraction of genomic DNA from RH strain tachyzoites, polymerase chain reaction (PCR) was performed using specific primers considering restriction sites and BglII and XhoI enzymes. Subsequently, the GRA7 gene was cloned in pET-32a (+) expression vector, and then pET-32a(+)-GRA7 was transformed into E. coli Rosetta (DE3). The induction of protein production was accomplished by IPTG, and the product was finally purified by Ni-NTA affinity chromatography. In order to make the strip test, the anti-human gold nanoparticle conjugate was applied on conjugate pad, rGRA7 antigen was immobilized to a nitrocellulose membrane as the capture agent, and sample and absorbance pads were assembled on a backing card. For the analysis of the sensitivity and specificity of the assay, the selected patients’ sera samples were tested by standard chemiluminescence immunoassay (CLIA) method, and then compared with TOXO-IgG strip results. The findings showed that the use of rGRA7 is an accurate, sensitive, and inexpensive technique for the rapid detection of anti-Toxo plasma IgG in human sera. Therefore, rGRA7 can be applied as a diagnostic agent in laboratories.
    Keywords: Toxoplasma gondii, rGRA7, Rosetta (DE3), pET-32a(+), lateral flow immunoassay (LFA)
  • R. A. Jafari *, Z. Boroomand, Annahita Rezaie, M. Mayahi, A. Nejati Saravi Pages 51-57
    Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. The ND accounts for heavy losses in Iranian poultry flocks. There are some reports regarding the epidemiology of this infection in Iran. This study was performed to investigate the infection of turkeys with a Newcastle disease virus (NDV) isolated from a broiler chicken flock in southwestern Iran during 2013. For the purpose of the study, 70 day-old Wishard bronze poults were allocated into two groups of control (n=25) and infected (n=45). At 32 days of age, each bird in the infected group was inoculated with 0.1 mL (50 μL per eye) of NDV-infected allantoic fluid through an ocular route and received 105 EID50 of viral inoculum. On the other hand, the birds in the control group were inoculated with phosphate buffered saline by the same route. Swab samples were taken from both groups at different time points, namely from 1 to 21 days postinoculation, and verified for NDV infection by using reverse transcription-polymerase chain reaction (RT-PCR). Both groups were also examined serologically by haemagglutination inhibition test. Clinically, the infected turkeys exhibited anorexia, severe depression, sitting on the hock joint, white to greenish (sometimes bloody) diarrhea, neurological disorders, and mild respiratory problems. Out of 45 inoculated birds, 9 (20%) cases died. Based on RT-PCR, virus shedding was observed in the challenged birds 3-8 days postinoculation. The NDV was detected more in tracheal swabs (50%) than in cloacal swabs (12.5%). The infected birds showed a high seroconversion. Therefore, the NDV circulating in Iranian chicken flocks has the potential to cause a serious illness in commercial turkeys. The vaccination of turkeys, as well as biosecurity, should be considered carefully to prevent the ND outbreaks in the future.
    Keywords: Newcastle disease, Turkeys, Immune response, pathogenicity, RT-PCR
  • H. Moradtalab, M. Noofeli *, H. Zeighami, F. Haghi Pages 59-67
    Whole-cell pertussis vaccine (wP) has been imperative and highly effective in preventing childhood deaths due to pertussis. Pertussis toxin is one of the virulence factors of Bordetella pertussis in all available pertussis vaccines. wP production in Razi Vaccine and Serum Research Institute is according to bioreactor culture of B. pertussis strains in B2 medium. The aim of this study was to evaluate B. pertussis strain 509 PT production in B2 and Thalen-IJssel (THIJS) media by Chinese Hamster Ovary (CHO) cell and enzyme-linked immunosorbent assay methods (ELISA). In the current study, B. pertussis strain 509 was cultured in B2 and THIJS media. Six samples were taken during the log growth phase within 2-3 h intervals (triplicate). The growth rate was calculated using opacity and the quantification of cell-associated and released PT measured by ELISA and CHO cell assays. THIJS medium was significantly showed an increase in the bacterial growth rate. During the first 29 h, bacterial concentrations in B2 and THIJS culture medium were 19 and 29 IOU, respectively. In THIJS medium, greater amount of pertussistoxin production was cell-associated. In B2 medium, maximum cell-associated toxin by ELISA and CHO cell assays were in the ODs of 1.1 and 0.9 and for THIJS medium in the ODs of 1.6 and 1.1, respectively. B. pertussis strain 509 in THIJS medium produced higher cell mass and cell-associated pertussis toxin than that of B2. It can be used for the production of whole-cell vaccine with higher pertussis toxin and accordingly using lower biomass per dose leading to the reduction of vaccine toxicity.
    Keywords: Bordetella pertussis, Pertussis Vaccine, ELISA, CHO cells, Pertussis Toxin
  • H. Hejazi *, G. Abedi, A. Jahandide, A. Asghari, S. Hesaraki Pages 69-75
    Anesthesia and analgesia are important in human and veterinary medicine, especially in surgical procedures. Rodents, avians, and exotic species are required to be anesthetized using an appropriate anesthetic regimen. This study aimed to suggest a new anesthetic drug and method in order to facilitate anesthesia as well as analgesia among rabbits, laboratory animals, and humans. Spinal injection of dexamethasone combined with intramuscular ketamine among rabbits can play the role of premedication agents. A total of 24 healthy white adult rabbits from New-Zealand were equally assigned into four groups. Groups 1, 2, 3, and 4 were subjected to spinal xylazine (5mg/kg) with ketamine (35mg/kg,IM), spinal dexamethasone (0.37mg/kg-four times diluted) with ketamine (35mg/kg,IM), dexamethasone (4mg/kg,IM) with ketamine (35mg/kg,IM), and spinal dexamethasone (0.37mg/kg-four times diluted), respectively. The results showed that there was a significant difference in terms of clinical reflexes recorded for group 2, compared to groups 1 and 3. A significant difference was also observed regarding clinical reflexes between group 2 and the other groups. Furthermore, no abnormality was observed in terms of histological sections within groups 2 and 4. Spinal dexamethasone can be used as a premedication combined with ketamine in rabbit anesthesia.
    Keywords: Anesthesia, Dexamethasone, Premedication, Rabbit, Spine
  • J. Tajik, H. Tavakoli *, D. Soltani Pages 77-82
    The aim of the present was to evaluate the prevalence of H9N2-specific antibodies among water buffaloes (Bubalus bubalis). To this end, blood samples were obtained from 80 randomly selected water buffaloes, 40 cases of which were obtained in the winter months, and 40 cases were sampled in the spring months. The presence of H9N2-specific antibody was determined by hemagglutination inhibition method. The antibody was diagnosed in 14 buffaloes (i.e., 10 males and 4 females). There were no significant differences between the two genders and between different age groups in terms of antibody prevalence. The presence of the antibody had a seasonal pattern; in this regard, all positive cases were found in the winter months (P
    Keywords: Serological, Influenza, H9N2, Bubalus bubalis
  • F. Azadbakht *, S. Shirali, M. T. Ronagh, I. Zamani Pages 83-89
    Bacterial diseases in cultured fish are considered the main problem with aquaculture system in Iran. The gills are multifunctional organs responsible for respiration, osmoregulation, nitrogenous waste excretion, and acid-base balance. Moreover, they are very sensitive to water contamination. Aeromonas hydrophila (A. hydrophila) is an opportunist pathogen responsible for a wide range of diseases in different species of fish. The gill histological alterations were used to assess the effects of A. hydrophila exposure on yellowfin sea bream, Acanthopagrus latus (A. latus). In this regard, 90 A. latus were exposed to the concentrations of A. hydrophila (103 and 106 CFU/ml) for three weeks. The most histopathological alterations in the gill of the exposed fish included hypertrophy and hyperplasia of the epithelial cells, lamellar fusion, club shaping of gill lamellae, lifting of the epithelium and edema of lamellae with large sub-epithelial space, blood congestion, and hypertrophy and hyperplasia of the mucosal cells. The histopathological alterations were observed in the gill of fish exposed to higher levels of A. hydrophila (106 CFU/ml) consisted of aneurysm and hemorrhage with blood congestion. According to the obtained results of this study, A. hydrophila could cause severe histopathological changes in the gill of A. latus and decrease gas change capability in yellowfin sea bream. Furthermore, the findings of the present study suggested that histopathological changes of the gill provide helpful information about the environmental conditions and as particular biomarkers may help the evaluation of fish general health.
    Keywords: Histopathological, Gill, Acanthopagrus latus, Aeromonas hydrophila