ARCHIVES OF RAZI INSTITUTE
Year:2000 | Volume:- | Issue:51|Papers Count:16

Double stranded cDNA coding for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type O1/Iran from the virion RNA was prepared .Two primer pairs for VP1 gene of virus were designed by comparison of published sequences and statistically analyzed using oligo and DNasis software. A PCR program was developed and optimized to amplify a segment of about 645 bp. Noninfected cells were used as negative control. The synthesized cDNA was then cloned into the NdeI and EcoR1 restriction enzymes site of pEt-23a+ plasmid using already attach linkers. The plasmid has been successfully propagated in and isolated from Echerishia coli bacterial culture and cloning of VP1 gene was confirmed by Dot Blot hybridization.

The immunologic efficacy of inactivated oil-emulsion vaccines against Newcastle disease (ND) and avian influenza (AI) by use of embryo vaccination technology was evaluated. The vaccine antigens were prepared from LaSota strain of ND and H9N2 strain of AI viruses. These antigens were emulsified with an oil adjuvant. 18-day-old embryonated eggs from Aryan broiler breeder were divided into three groups and were delivered inactivated oil-ND and -AI. Group 3 was considered as unvaccinated control. After incubation period, no significant difference (P<0.05) was observed in hatchability between two treatment and control groups. At intervals of experimental procedure up to 6 weeks the haemgglutination-inhibition antibody titer of groups 1 and 2 were higher than control group. The results of our study indicate that vaccination of chicks against ND and AI vaccines by in ovo technique, were successful under laboratory conditions.

In August 1998, the avian influenza (AI) outbreak appeared in one of the layer farms in Tehran province. Birds on the affected farm exhibited respiratory infection and reduction of egg production with very low mortality. An agar gel precipitation (AGP) test using AN antiserum revealed the presence of the virus in the samples from the affected birds. The sera collected from the surviving birds of the affected flocks also showed the presence of antibodies against AIV by AGP test. An avian influenza virus H9N2 subtype was isolated in embryonated eggs from the tracheal and clocal swabs as well as pooled visceral organs of the affected birds and coded Alchicken/Iran/259/1998/H9N2.Virulence testing of the low passage virus was undertaken at the time of the outbreak in specific pathogen free (SPF) chickens. The experimentally infected chickens showed significant clinical signs and recovered gradually without any death. The isolated virus was characterized as nHPAI on the basis of pathotyping studies in SPF chickens. It is the first report on isolation of AIV from the chicken flocks in Iran.

The immunogenicity of mass campaign doses of oral poliovirus vaccine (OPY) was evaluated by measuring serum neutralizing antibodies (NAs) in two groups of children. The first group included 215 children before and after two mass vaccination campaing conducted in 1994"s national immunization days (NIDs) and the second, included 420 children after receiving two supplemental doses of OPY in 1996.In order to measure the NAs against poliovirus types, 1,2, and 3 transferred to the neonates 412 sera collected from umbilical cord blood and tested. Precampaign seroprevalences to the virus types were compared with those of the post campaign in the first group. The titers of NAs against all three types of the virus had raised significantly in postcampaign serum samples. The NA prevalence against poliovirus types was also high in the serum samples collected from the second group. Four weeks after the second supplementary dose of OPY, 96%, 94% and 91% of these children had immune titers of NAs against poliovirus types 1,2 and 3 respectively. Of 412 umbilical cord blood samples, 82.3%, 80.8% and 68.9% showed NAs at dilution of>1:8 against poliovirus types 1,2,and 3 respectively. These findings suggest that NIDs should be continued to maintain the level of herd immunity at highest standard level, among susceptible and at risk hosts, against all three types of the virus.

Because of immunotherapeutic effects of Mycobacterium vaccae (M. vaccae) by inducing Th1 responses, we tried a heat-killed suspension of M. vaccae in mice models (C57BL/6J), which had already been challenged by tumor cell lines B16-F10. The Immunization consisted of subcutaneous injection of irradiated B16-F10 cells, as an antigen, plus M. vaccae into the tail base at two inguinal sites of mice, which were injected by live wild type B16-F10 cells. Survival and tumor growth was recorded every three days for a period of three months. Increase in survival was observed whilst decrease in tumor growth was recorded. The result indicates that the heat-killed suspension of the bacterium is a powerful immunological adjuvant.

To Comparison between the local tuberculin produced by Razi institute and RT23, 86 people were participated. They were confirmed tuberculosis patients and apparent healthy students that randomly divided in two groups. For the first group the local tuberculin and RT23 respectively injected to right and left hand simultaneously and for the second group the reverse was done. To compare the agreement of two PPD in different tuberculosis prevalence, which has practical importance, results simulated in populations with different tuberculosis infection rates. The agreement between tuberculins was assessed by weighted kappa coefficient. The weighted kappa coefficient, with 37% tuberculosis prevalence, was 63% (95% CI: 55-71). However, in two simulations in suspected cases of tuberculosis in a referral clinic and apparent healthy people were 48% and 43%, respectively. Based on estimated kappa coefficients the agreement between local PPD and RT23 for clinical and epidemiological proposes was moderate. In addition, with the reduction of the tuberculosis infection in these groups, the agreement between two tuberculin would decrease. The main recommendation is to allocate a tuberculin product in Iran for human usage.

A mannose-resistant hemagglutinating (MRHA) P or P-related fimbriae was purified from three E.coli isolates from chickens with colisepticemia. The polyclonal antiserum against the fimbriae was prepared in rabbits. Examination of the purified fimbriae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated a major fimbriae subunit of approximately 18 kDa. This band was also reacted with anti-F11 serum on Western blotting. The antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae and with F11 fimbriae on Western blotting. The first 21 N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11 fimbriae. The adhesive properties of the avian P fimbriae were similar to that of F11 fimbriae with regard to both MRHA of human erythrocytes and binding to the Gal-Gal class II receptor. These results indicate that these fimbriae on avian E.coli isolates are closely related to F11 fimbriae which are associated with E.coli isolated from human urinary tract infection.

Four antigens consisting of heat killed cells, formalin killed cells, lipopolysaccharides and extracellular products were prepared from Aeromonas hydrophila. The immune response of about 3-year-old Persian sturgeon (Acipenser persicus) that immunized by intraperitoneally injection of the antigens with or without Freund's adjuvant was evaluated. The immune response to the extracellular products was significantly (P<0.05) less than that other antigens. The results indicate that whole cell antigens of A.hydrophila represent protective antigens against motile aeromonad septicemia.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA), indirect immunofluorescent antibody (IFA) and indirect haemagglutination(IHA) techniques were used. For detecting anti-echinococcal antibodies caused by Echinococcus granulosus. 246 sera samples including 32 from surgically proved hydatid cyst patients, 104 of patients suffering from a disease other than hydatidosis and 110 from healthy individuals were tested. The results indicate, the Dot-ELISA with 100% sensitivity and 99.5% specificity was more sensitive and specific than other tests. IFA test with 90.6% sensitivity and 96.3% specificity as well as the IHA test with 78.1% sensitivity and 93.9% specificity was found inferior to Dot-ELISA assay, respectively.

An antigen from the isolated propagated, and purified oocysts of Cryptosporidium bailey was prepared. To evaluate the serologic prevalence of Cryptosporidium in broiler flocks in Shiraz area a Cryptosporidium-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed by using the antigen. Both the sensitivity and specify of the ELISA was compared with an indirect immunofluorescent (IIF) test using the purified oocysts of the isolated C.baileyi as antigen. 125 out of1522 (8.2%) serum samples from 26 broiler flocks were positive against Crytosporidium. Upon the result, previous reports on faecal shedding of Cryptosporidium in poultry farms were confirmed and C.baileyican is added to the list of respiratory pathogens in Iran poultry industry.

Glutathione S-transferase (GST) is an important candidate for vaccination against temperate fasciolosis. For assessment of cellular immunity against GST of Fasciola gigantica (FgGST), sheep was immunized by FgGST combined with saponin and AI(OH)3, as adjuvants. Cellular immune response, after three times immunization,was evaluated by flowcytometry of peripheral blood, lymphocyte transformation test (LTT) and delayed hypersesitivity skin test. Flowcytometry of peripheral blood showed no significant differentiation between treatment and control groups in the percent of CD8+ lymphocyte, MHC class I+ and MHC class II+ leukocyte subsets. Proliferation of T cells against FgGST was detected in vaccinated groups; but stimulation index was lower than 2 in these groups.Erythema and induration of skin against FgGST in immunized groups were significantly greater than control. In conclusion FgGST appears to be only weakly antigenic for sheep T cells and it is therefore an unpromising candidate for inducing resistance to F.gigantica.

Neurotoxin of Clostridium botulinum is one of the most potent known toxins in nature, which can cause an often fatal food poisoning. However, recently the use of binding domain of the toxin has also been proposed as a candidate vaccine. In this study coding region for this domain was amplified from genomic DNA of Clostridium botulinum type A using a pair of primers containing BamHI and HindIII. The amplified l.3kb fragment corresponding to the gene of interest was cloned in pUC18 after verification with different restriction enzymes. The construct was then transformed into competent Escherichia coli DH5α.and the recombinant clone was selected on plates containing ampicillin.The plasmid purified from these clones was used for sequencing by dideoxy chain termination method. The sequence thus obtained was verified by comparison with published sequence, which showed the authencity of the PCR product. This fragment was subcloned in an IPTG inducible expression vector pQE30 using E.coli M15 as a host and expression was analyzed by RT-PCR.    

For diagnosis of Herpes simplex virus (HSV) primary infections by use of complement fixation test, the HSV-CF antigen was prepared. Virus was isolated from herpetic lesions on lips and confirmed by electron microscopy and specific antisera. The results confirmed correct HSV isolation. The isolated virus inoculated to susceptible cell line-Vero-with a high infections dose (MOI=0.1) and incubated at 33°C. When complete CPE appeared, supernatant harvested as source of antigen.93 serum samples were tested by complement fixation test (Kolmer method) using both prepared and imported antigens. The specificity and sensitivity of the test using prepared antigen in comparison of imported one were 100% and 84.7%, respectively. Because of we prepare the antigen using only one of two serotypes of the virus, in some cases that antibody directed against other serotype in patient's serum, false-negative results occur. This led to reduction the sensitivity of test by homemade antigen.

450 paired serum samples were taken before and after administration of an inactivated foot - and - mouth disease vaccine, containing A-Mardabad and O1 virus strains analyzed for presence of neutralizing antibodies. Serum neutralization tests were performed in BA cell culture employing equal volume of serial two-fold dilutions of each of the previously inactivated serum against 100 TClD50 of each virus strain. The results showed that 93.99% and 95.54% of tested sera did not have a protective level of neutralizing antibodies (titer of 16 or more) against A-Mardabad and O1 strains, respectively. A good increase in the antibody titers was observed in 85.36% and 90% of the vaccinated, cattle against the field strains of the above mentioned viruses, respectively. This study indicates that the foot-and- mouth disease vaccine, which produced by Razi Vaccine & Serum Institute, is a reliable one to be used for control of the disease in Iran.

The prevalence of male Ixodidae ticks was studied in Boushehr province. Specimens were randomly collected from domestic ruminants in different villages during four seasons from 1997-1998.Ticks species were identified by means of direct examination using morphological features. Totally 1841 specimens were collected. The collection included Hyalomma anatolicum anatolicum Kock, 1844 (45.22%), Rhipiciphalus sanguineus (Latreille, 1806) (38012%), Hyalomma detritum Senevet, 1922 (8.99%) and Hyalomma dromedarii koch, 1844 (5.36%). Moreover, one female Boophilus annalatus Say, 1821 and one male Haemaphysalis sulcata were identified.

The prevalence of ringworm was studied in 400 horses in different areas of Golestan province. Trichophyton equinum var.equinum was isolated from 6 (1.5%) of them. The infection rate was high in truck horse and the incidence increased during the summer. The majority of lesion was located on the neck. No significant difference was observed between age groups.