ARCHIVES OF RAZI INSTITUTE
Year:2005 | Volume:- | Issue:59|Papers Count:11

To construct of an eukaryotic expression vector encoding herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2), an Iranian isolate of HSV-2 was propagated in HeLa cell line and its DNA was extracted and used as template in polymerase chain reactions (PCR), to amplify gD2 gene. Primers were designed and the restriction enzyme sites for EcoRI and XhoI were considered at their 5′ ends respectively. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector (pBsc) and then sequenced. The fragment encoding gD2 was obtained by digestion using appropriate enzymes and extracted on agarose gel using a commercial kit. The gene of interest was subcloned in the named sites of an eukaryotic expression vector (pcDNA3) to construct pcDNA-gD2. An endotoxin free column was applied to prepare pure pcDNA-gD2, which was transfected into mammalian cells using lipofectamine. Protein expression was confirmed using indirect immunofloerscent test. The results indicated that the construct expresses in mammalian cell lines effectively and it can be used as a DNA vaccine in animal models.  

101 isolates of Brucella spp. including the reference (n=7) and the Iranian field strains belonging to last years 1961-2003 (n=94) were biotyped and classified into 9 biotypes. Random amplified polymorphic DNA (RAPD) analysis using a ten mer-AP4 primer was generated 72 reproduciable DNA band. The typeability was 100%. The thirteen isolates were classified into five groups, each containing 2-4 biotypes. The high DNA polymorphisms among the strains suggested that animals in Iran have been infected with genetically diverse isolates however, in the last forty years B.abortus biotype A3 and B.melitensis biotype M1 have been predominantly isolated from brucellosis cases. The results indicated that the RAPD analysis using AP4 primer can be used to attain reproducibility amplify random fragments of DNA from Brucella genomes in order to differentiate at species and sub-species for epidemiological purpose.

During a period of 7 years (1997-2003) we were received pairs of serum and CSF of 27 suspicious clinically cases from neurology department of several hospitals in Tehran. Among them 7 were serologically positive for progressive rubella panencephalitis (PRPE) and 2 were positive for sub acute sclerosing panencephalitis (SSPE). Rubella virus was isolated from 4 of 7 serologically positive cases of PRPE using three different cell culture systems.    

To evaluate the effect of intrauterine cephapirin on treatment of endometritis 39 Holstein dairy cows that affected with postpartum endometritis were selected and randomly assigned to two experimental designs. In experiment 1, 6 out of 14 cows were 25 to 30 days after parturition were treated by intrauterine oxytetracycline. Another 8 cows were treated by intrauterine cephapirin (Metri-Care). In experiment 2, 25 cows that were 55-60 days after parturition received three types of treatment. 6 cows were received only PGF2α, 9 cows were received PGF2 α and procaine penicillin G, and 10 cows were treated by intrauterine Metri-Care. Cervical mucosa was collected before and after each treatment. There was no significant difference between mean (±SD) of neutrophils before and 14 days after treatment with them. Conception rates of cows received oxytetracycline and Metri-Care were 66.66% (4/6) and 37.5% (3/8), respectively. There was significant difference between mean of neutrophils before and 14 days after treatment at all groups. Conception rate of cows received only PGF2 α was 33.33 % (2/6) and in cows received penicillin and PGF2 α was 55.55% (5/9) but in cows received Metri- Care was 90.0% (9/10). Therefore, Metri- care may be good choice for treatment of postpartum uterine infection in cow.  

To detect the presence of infectious bronchitis virus (IBV) in infected allantoic fluid (AF) of SPF embryonated eggs rapid hemagglutination (HA) activity after treatment with neuraminidase enzyme was used. Twenty IBV suspected materials were inoculated in SPF embryonated eggs via chorioallantoic cavity. Harveste AFs were treated with neuraminidase enzyme and the presence of IBV was detected by inducing hemagglutination of chicken red blood cells. The specificity of rapid HA test was examined with three non-hemagglutinating avian viruses including infectious bursal disease virus, infectious laryngotracheitis virus and fowl pox virus. The sensitivity of the test was compared with reversed transcriptase-polymerase chain reaction. The results showed that this test was specific and had a sensitivity of 100% for IBV detection. The results of this study indicate that HA test using neuraminidase treatment is an accurate, sensitive, specific and inexpensive test for rapid detection of IBV.    

Neospora caninum, an apicomplexan protozoan, is regarded as a major cause of abortion and stillbirth in cattle in countries world-wide. The ability to detection N.caninum in tissue samples can be a useful detection diagnostic tool for use in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection. However, molecular biology is one of the most sensitive tools for detecting protozoa in infected tissue samples. Specific semi-nested PCR was designed based on specific ITS1 and 5.8S rRNA genomic DNA for detection of parasite in infected tissues. The designed PCR detected four of six aborted fetal brain samples infected by N.caninum. Our results revealed that PCR with selected primers gave a 357bp product in examined samples and confirmed the presence of N.caninum DNA in infected fetal brains. This is the first report that demonstrated the reliability of PCRbased assay to identify N.caninum infection in Iran.  

Various commercial vaccines for immunization of broiler chickens against infectious bursal disease (IBD) are available, so it would be appropriate to compare the pathogenicity and immune response of chickens to these vaccines. In this study the pathogenicity and serologic response of four IBD vaccines, cloned D78®, Bursine-2®, Bursimune® and Cevac Gambo-L®, were evaluated in specific pathogen free (SPF) chickens. 100 SPF chicken were divided into five equal groups (one control group and four vaccinated groups) kept in isolator units and vaccinated at 16-day-old via the eye-drop route. At 5, 10 and 20 day post vaccination birds from each group were weighted, bled, and then necropsied. Lesions were recorded and the bursa of Fabricius was taken out, weighted and was fixed in 10% neutral buffered formalin and processed for histopathological examination. The pathogenicity and serologic effects of the IBD vaccines were evaluated by the antibody response, the bursa, spleen and thymus/body weight ratios and histopathological lesions of the bursa. No any clinical signs and mortality was observed in all groups. The results of this study indicate that D78 and Gambo-L vaccines showed to be more pathogenic and caused more severe bursal damages and also induced higher ELISA titers in serological evaluation.  

An indirect enzyme–linked immunosorbent assay (ELISA) was developed for screening of antibody to avian infectious bronchitis virus (IBV). Antigen was prepared from whole-purified IBV Massachusetts serotype (BR 801 strain). Optimum dilution with minimum background for antigen concentration, rabbit anti-chicken conjugate and sera in developed ELISA was determined 0.1μg/ml, 1:3000 and 1:100, respectively. The optical densities (OD) were compared with a commercial ELISA kit. Also, the results of ELISA were compared to hemagglutination inhibition (HI) test. The correlation coefficient of the results of both ELISAs and HI was significant (P<0.05). The developed ELISA can detect antibody against IBV and is more sensitive and suitable for screening of samples in diagnostic laboratories.  

To prepare hemagglutination inhibition (HI) antigen the egg drop syndrome virus (EDSV) was propagated in 10-day-old embryonated duck eggs. The virus antigen was inactivated with two methods including heating (65°C) and adding 0.5% formaldehyde by considering low destroying effect on hemagglutination (HA) and HI titers. The EDSV HI titers of 310 sera and 100 yolks obtained from 23 chicken farms and specific pathogen free (SPF) chickens before and after EDS vaccination, in various ages were evaluated. Specificity, HA activity, stability and electron microscopic observations were similar in the prepared and standard antigens. The HI titers showed an equal sensitivity and specificity and, complete similarity (>99%) between the results obtained by prepared and standard antigens. Detection of antibody against EDSV using HI test showed slightly less sensitivity but more specificity compared with ELISA test. Upon the results it is suggested that inactivated EDS antigen prepared by either heat or formalin treatment could successfully be used in avian diagnosis laboratories for routine flocks monitoring, EDS vaccine controlling and also surveillance of EDS vaccination program in poultry industry.    

An inactivated trivalent fowl cholera vaccine consisted of serotypes 1, 3 and 4 Pasteurella multocida strains was prepared. The vaccine provided 70-100% protection against challenge with homologous starins. ELISA assay showed a considerable increase in antibody titer after towice vaccination of 8 weeks chickens. It was found that the trivalent vaccine can induce immunogenic response in vaccinated chickens.    

To funistic survey of the cercariae of Melanoides tuberulata (fresh water snail) 1540 M.tuberculata were collected from various streams, swamps, ditches and canals in the central area of Khouzestan province in the south west of Iran. Infected snails, 46 (2.9%), were isolated and cercariae were obtained by emerging or crushing methods and then measuring and drawing were made from specimens. In some cases experimental infection was established in the animals for further identification. A total of 5 trematode families were identified as follow: Heterophyidae: Haplorchis pumilio, H.taishui, Stellantchasmus falcatus and Centrocestus formosanus; Echinostomatidae: Echinochasmus milvi; Schistosomatidae; Plagiorchiidae and Philophtalmidae. These results have been recorded for the first time and show the potential of M. tuberculata for transmission of zoonotic disease in the region.