فهرست مطالب

Archives of Razi Institute - Volume:73 Issue: 4, 2018
  • Volume:73 Issue: 4, 2018
  • تاریخ انتشار: 1397/09/10
  • تعداد عناوین: 9
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  • R. Soltani, A. Dalimi * Pages 257-263
    Hepatozoonosis is a protozoal disease caused by various species of Hepatozoon. This parasite is transmitted from tick; the main vector of Hepatozoon canis is usually the brown dog tick (Rhipicephalus sanguineus). However, several species of ticks are disposed as the alternative vectors. Dogs are usually infected by eating the tick or a part of the tick organ infected by the mature oocysts containing infectious sporozoite. In the current study, a total of 145 blood samples were collected from the cephalic vein of pet, stray, and shelter dogs in Tehran. To conduct this study, first thin blood smears were prepared from all the samples and stained with the Giemsa method. Then, after extraction of DNA from the blood samples, in order to trace Hepatozoon canis, the 18S rRNA gene segment of the parasite was amplified using polymerase chain reaction (PCR). To confirm the PCR-positive results, five randomly selected PCR-positive samples were sequenced. According to the results, through direct observation of microscopic slides, no infection of H. canis parasite was observed, but according to the PCR results, 32 out of the 145 blood samples were found to be infected by H. canis. In this study, infection to H. canis in older dogs was higher than in young dogs, and more male dogs were found to be infected by the parasite compared to female dogs; but no significant difference was observed in this regard (P > 0.05). Moreover, stray dogs showed a significantly higher rate of infection, compared to the pet and shelter ones (P < 0.05).
    Keywords: Hepatozoon canis, Dog, PCR, Tehran
  • P. Panahi, S. A. Pourbakhsh *, T. Zahraei Salehi, M. Esmaelizad, R. Madani Pages 265-275
    P48 protein of Mycoplasma agalactiae is used to diagnose infection and was identified as potential vaccine candidate. According to the genetic nature of mycoplasma and variable sensitivity in P48-based serological diagnosis tests, intra species variation of P48 nucleotide sequence investigated in 13 field isolates of difference province of Iran along with three vaccine strains. Samples were collected from sheep and goat and were cultured in modified PPLO broth. Two pair of primer employed to confirm genus and species of isolates and a pair of primer has developed to amplify the P48 gene. The sequencing results of PCR products were aligned and analyzed besides published sequences in GenBank. T-Cell and B-Cell epitopes and antigenicity of sequence were computationally predicted. The results have shown P48 nucleotide sequences are 99.9% identical in field isolates and vaccine strain of Iran, but analysis of GenBank published sequences have shown divergence up to 5.3% at the nucleotide level and up to 4.9% divergence in protein level of P48 sequences of Iran isolates and other available sequences in GenBank. Single nucleotide polymorphism exists in 89 positions and variable amino acid was observed at 25 residues. Phylogenetic analyses have shown that Mycoplasma agalactiae isolates fall into three main groups based on P48 nucleotide sequences. Immunoinformatics analysis of all available P48 nucleotide sequences have revealed that gene variation lead to differences in immunological properties, but the gene in Iranian isolates are conservative and stable. The sequence variation in epitopes can be underlying source of antigen heterogeneity as a result, affect serological tests accuracy. Due to the high level of divergence in worldwide isolates and high degree of similarity in P48 protein of Iranian isolates, designing recombinant P48 protein based on local pattern can increase the sensitivity and consistency of serological test.
    Keywords: Mycoplasma, Contagious Agalactiae, P48, Variation, Antigen Heterogeneity
  • Z. Boroomand *, R. A. Jafari, D. Gharibi, K. Kazemi Pages 277-285
    Quail is an alternative source of protein for humans. These birds can be affected by common bacterial infections. Bacterial contamination of egg is the most common cause of mortality in Japanese quail chicks. In order to study the role of some members of Enterobacteriaceae responsible for early mortality in Japanese quail chicks, 100 dead or moribund quail chicks were obtained from 10 different farms in Ahvaz, Iran. Samples were taken from the liver and yolk sac of the birds and bacterial isolation from samples was conducted by streaking them on MacConkey, Brilliant Green, Salmonella-Shigella and Xylose Lysine Deoxycholate agar plates. The plates were incubated at 37 °C for 24-48 hours, and by standard biochemical tests bacterial isolates were identified. Final confirmation of Salmonella serotypes was performed by Razi Institute. All the isolates were examined for susceptibility to 12 different antibiotics (Padtan-Teb Co., Tehran, Iran) by the disk diffusion (Kirby Bauer) method. The results showed that 78% of the quail chicks were infected. The isolated bacteria were Escherichia coli (44%), Klebsiella pneumonia (8%), Salmonella serovar ruzizi (5%), Salmonella serovar typhimurium (3%), Enterobacter cloacae (4%), Enterobacter aerogenes (4%), Proteus vulgaris (5%) and Proteus mirabilis (5%). One hundred percent susceptibility was observed to gentamycin, soltrim, tetracycline, fosfomycin, florfenicol, cephalexin and ceftriaxone. E. coli isolates were susceptible to soltrim and ceftriaxone, Salmonella isolates were susceptible to fosfomycin, Enterobacter isolates were susceptible to ceftriaxone and Proteus and Klebsiella isolates showed susceptibility to ceftriaxone. It is concluded that the members of Enterobacteriaceae family, specifically the genera Escherichia and Salmonella, are the major causes of early mortality in newly-hatched Japanese quail chicks.
    Keywords: Japanese quail, Mortality, Enterobacteriaceae, Antibiotic Susceptibility
  • M. H. Motedayen, G. Nikbakht Brujeni, M. J. Rasaee, A. Zare Mirakabadi *, A. Khorasani, H. Eizadi, M. M. Ranjbar, S.M. Azimi , M. Esmaelizad Pages 287-294
    Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.
    Keywords: Echis carinatus, Fab fragment, Gene library, Antivenom, Polyclonal
  • M. Ebrahimi, H. Hamidinejat, M. R. Tabandeh *, M.H. Razi Jalali, A. Rasouli Pages 295-303
    Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in Iran. The immunogenic region of EMA-1 gene was amplified using the blood of infected horses. EMA-1 gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent E. coli BL21 (DE3) cells. Cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme assays, and DNA sequence analysis. The recombinant protein was expressed using isopropyl β-D-1-thiogalactopyranoside as an inducer, purified using nickle-nitrilotriacetic acid column, and then confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot analysis utilizing Anti-His Tag antibody. Furthermore, the immunoreactivity of recombinant protein against the serum of the infected horses was evaluated using dot blot analysis. The PCR product analysis showed a 750-bp band belonging to immunogenic regions of EMA-1 gene. Sequence analysis revealed that cloned EMA-1 and protein had 94% and 97% homology to EMA-1 sequences submitted to GenBank from different countries, respectively. Restriction enzyme and sequence analyses confirmed the subcloning and correction of the orientation of inserted gene. The SDS-PAGE analysis confirmed the expression of EMA-1 protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against T. equi could react with the purified recombinant protein. Purified EMA-1 protein can be used as a reliable tool for the future development of diagnostic tests or vaccines.
    Keywords: Theileria equi, EMA-1, Cloning
  • J. Gharekhani email *, M. Yakhchali, B. Esmaeilnejad, K. Mardani, G. Majidi, A. Sohrabei, R. Berahmat, M. Alaei Pages 305-310
    Toxoplasma gondii and Neospora caninum are Apicomplexan intracellular protozoa with global distribution. Small ruminants play an important role as intermediate hosts for N. caninum and T. gondii, parasites of great public health concern. The main goal of the current survey was to evaluate N. caninum and T. gondii infection rate in sheep and goats of Khuzestan Province, southwest of Iran, using enzyme-linked immunosorbent assay (ELISA). In this cross-sectional study during February-April 2016, whole blood samples were taken randomly from 735 animals from 37 herds. The animals were reared under the traditional husbandry system in different parts of the province. Among 550 sheep and 185 goats, 37 (6.8%) sheep and 20 (10.8%) goats were seropositive for N. caninum and 59 (10.8%) sheep and 37 (20%) goats were seropositive for T. gondii. The incidence rates of mixed infection with N. caninum and T. gondii were 3.2% and 5.4% in sheep and goats, respectively. Seroprevalence rate of N. caninum was significantly higher in goats at <1 years of age. There was a significant association between the number of sheep infected with T. gondii and abortion (18.2%). Also, a significant correlation was detected between seroprevalence of N. caninum and T. gondii and mixed infection in goats with a history of abortion. This is the first report of IgG antibody production against N. caninumand T. gondii co-infection in small ruminants in Iran. Our findings indicated that neosporosis and toxoplasmosis may be responsible for abortion in small ruminants in this region. Therefore, further investigations are needed to improve sanitary strategies in animals’ husbandry and launching control programs.
    Keywords: Neospora caninum, Toxoplasma gondii, Sheep, Goats, Iran
  • M. Moharrami *, H. Modirrousta Pages 311-318
    The identification of honeybee viruses is of serious importance, particularly considering the lack of information on the natural incidence of viral infections in honeybee populations worldwide. Moreover, the global spread of Varroa destructor in honeybee colonies has a significant effect on the viral infection. In the present study, 160 samples of adult bee from apparently healthy colonies but with a background of parasitic diseases, tremor, and paralysis, were collected during 2011-2012. The samples belonged to 23 different provinces of Iran. They were sent to Razi Vaccine and Serum Research Institute, Karaj, Iran, for further analysis, and examined for the presence of viruses using reverse transcription polymerase chain reaction assay. According to the results, out of 160 samples, 9 (5.8 %), 40 (25.6 %), 12 (7.8 %), 34 (21.8 %), 7 (4.5 %), and 29 (18.5%) cases were positive for acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV). The samples collected from 18 provinces (78 %) were positive for at least one virus. Among all samples, 83 (53.2 %) specimens were infected with at least one virus. The highest prevalent virus was BQCV, followed by DWV, SBV, CBPV, ABPV, and KBV, respectively.
    Keywords: Honey bee viruses (ABPV-BQCV-CBPV- DWV- KBV-SBV), RT-PCR, Iran
  • A. Nazari *, I. Khalili, M. Samianifard Pages 319-324
    Avian influenza (AI) H9N2 is a low pathogenic virus subtype belonging to Orthomyxoviridae family. Given the prevalence of this subtype as an infectious agent in poultry industry, special attention has been always directed toward the development of vaccine production against this infection. The vaccine of this infection is produced by killing the virus and using a mixture of inactivated antigen and oil phase. Egg-based viral antigens have high levels of unwanted proteins that may adversely affect the vaccine formulation. In addition, it is required to raise the antigen concentration for the production of combination vaccines, especially in low doses. This underscores the need to the improvement of the downstream purification process and concentration of antigens. The optimization of downstream processing would decrease the cost of vaccine procurement and maintenance. Regarding this, the present study was conducted to evaluate a downstream procedure for the concentration and purification of avian influenza virus (H9N2) and investigate the immunogenicity of the vaccine containing these antigens. To this end, after harvesting and clarifying virus-containing allantoic fluid, it was concentrated and purified using ultrafiltration and chromatography, respectively. The concentrated and purified samples were checked for their ovalbumin level and emulsified with oil adjuvant to access their immunogenicity. The results showed that one dose of both formulated antigens (i.e., concentrated and purified) was effective in raising the immune response in the vaccinated chicks for a long time. The applied formulation had a one-year stability in the refrigerator. Furthermore, the concentrated antigen showed a high hemagglutination activity through a year when storing in the refrigerator. Based on the findings, the optimization of downstream process of influenza (H9N2) vaccine production and use of new technologies could be considered in the large-scale preparation of a sustainable vaccine without any unwanted risk factors.
    Keywords: Avian H9N2 Influenza Virus, Vaccine, Immunogenicity, Ultrafiltration
  • M. H. Fallah Mehrabadi, A. Bahonar* , K. Mirzaei, A. Ghalyanchi Langeroudi, S. A. Ghafouri, F. Tehrani, A. Hashemi Pages 325-330
    The aim of this study was to determine the seroprevalence of avian influenzaH9N2 subtype in the industrial ostrich farms and its geographical distribution. This cross-sectional study was conducted from January to June 2015. A total of 40 farms were selected from different provinces of Iran, from each of which 11 ostriches (n=440) were sampled. The sera samples were examined using 4 hemagglutination units of H9N2 antigens. A frequency distribution was used to describe the responses to the survey questions. The mean titers between provinces were compared using one-way analysis of variance. According to the results, 21 (47.5%) out of 40 farms and 108 (24.5%) out of 440 ostriches tested positive in the HI-H9N2 test. There were statistically significant differences between the mean titers of samples in different provinces (P<0.001). The current study was conducted on unvaccinated ostriches. The results showed that H9N2 had a high seroprevalence at both farm and bird levels. The findings of this study can be for the further investigation of infection in ostrich farms in order to consider this species in the surveillance programs of the Iranian Veterinary Organization. The detection and isolation of viruses and epidemiological investigation are necessary for the persistent use of H9N2 vaccines in some ostrich farms.
    Keywords: Ostrich, Seroprevalence, H9N2, Avian Influenza, Iran