Archives of Razi Institute
Volume:67 Issue: 2, Autumn 2012

Hottentotta is one of the most widely distributed genera of the family Buthidae, with species present throughout Africa, the Arabian Peninsula, and in Asia to Pakistan and India. Recently, Kovarik (2007) revised genus of Hottentotta in the world and reported 29 different species-group name in the genus of Hottentotta. Hottentotta is one of the six medical important scorpions of Iran that distributed in almost all parts of country. So, in this article morphological and morphometrical characters of six species of Hottentotta for better distinguishing have been described.

During 2010-2011, Real-time PCR procedure was used to detecting FMDV RNA on 147 epithelium samples from the field. In this survey, for Real-time PCR from 3D gene segment as conserve region selected for tracking all of seven serotypes FMDV. The assay detected the viral RNA in all serotypes of FMDV. The rRT-PCR specifically detected FMD virus in sample with greater sensitivity than our conventional RT-PCR procedure and our routine diagnostic procedures. Sensitivity of this technique was estimated by 10-log serials dilution Asia virus RNA that defined 101.2 TCID50/ml. Also, to assess the specificity of primer pairs related to 3D segment and specific probe was used Swine vesicular disease virus (SVDV) and bluetongue virus (BTV) isolates RNA. The results showed that rRT-PCR is seen as a powerful and valuable tool concomitant with routine diagnostic methods for FMD virus diagnosis.

Understanding genetic structure and status of genetic variation of Fasciola hepatica isolates from different hosts, has important implications on epidemiology and effective control of fasciolosis. Random amplified polymorphic DNA (RAPD-PCR) was used to study the genetic variation of F. hepatica in sheep and cattle. DNA was extracted from adult helminthes removed from livers of each infected animal in slaughterhouse at East-Azerbaijan province, North-West of Iran. DNA template amplified by the polymerase chain reaction, using three oligonucleotide decamers with arbitrary DNA sequences as primers. RAPD patterns showed the specific but different pattern DNA patterns for each primer. The intraspecific similarity coefficient within two isolates of F. hepatica was ranged between 69 to 100%. Present findings showed that the interspecific genetic distance was higher than intraspecific genetic distances (19-47% compares to 0-19%). Pair wise similarity matrices generated from each isolates-primer combination were totaled and the similarity coefficient between strains were calculated both manually (Nei and Li method) and software analysis (Free-Tree-Freeware program). The inferred phylogenetic tree on the fingerprinting of these isolates clearly demonstrated the existence of population genetic diversity sub structuring within F. hepatica of sheep and cattle of Iran, raising interesting questions on the host specificity, epidemiology (e.g., zoonotic transmission) and ecology of this fluke. RAPD-PCR is useful for both individual identification and epidemiological investigations in endemic regions.

The venoms of Buthidae scorpions are known to contain basic, single-chain protein -toxins consisting of 60-70 amino acid residues that are tightly cross-linked by four disulfide bridges. Total RNA was extracted from the venom glands of scorpion Mesobuthus eupeus collected from the Khuzestan province of Iran and then cDNA was synthesized with the modified oligo (dT) primer and extracted total RNA as template. By applying the Semi-nested RT-PCR using homologous primers, the fragments of 273 bp was amplified and sequenced. The coding region encodes a polypeptide of 85 amino acid residues with a calculated molecular weight of 9. 337 kDa and theoretical isoelectric point of 6. 52. It was designated as MeNaTx-4. This peptide with homology to the «Scorpion long chain toxin-like domain» is belongs to the Toxin-3 superfamily. Multiple alignment of the putative amino acid sequence of MeNaTx-4 exhibited 95% sequence identity with lesser Asian scorpion M. eupeus venom sodium channel -toxin-3 but interestingly displayed only 63% identity with other -toxins from this species. The sequence was also identical to the counterpart sequences from M. martensii (92%) and Buthus occitanus israelis (80%). This high sequence similarity together with completely conserved in the positions of all 8 Cys indicated that MeNaTx-4 is a new member of -toxin from the Iranian M. eupeus which may originate from a common ancestor.

Poliomyelitis, an acute viral infectious disease caused by poliovirus, still remains a public health problem in developing countries. Despite the global effort to eradicate polio, continuing the polio immunization with a potent and safe vaccine is essential. For accurate vaccine evaluation, three types of cell lines including Hela, Hep2C and Vero were evaluated and compared using two methods of polio vaccine potency tests (micro & macro). For cells comparison, five different batches from polio vaccines were tested and to develop the test, five variables including viruses, cells, serum, media and Co2 were studied. For validation, the titer of which has been well established as a working reference preparation (WRP) was applied to control the accuracy and reproducibility of the testing system. Multiple comparisons were performed by analysis of variance (ANOVA) followed by Tokey HDS and LSD. No significant differences were found between the potency of vaccine batches and between macro and micro methods. Reduction in cells sensitivity and potency of vaccines was found with increasing passage number. Significant differences were found between the sensitivity of the cell lines. The highest potency of polio vaccines was obtained using Hela cells (GMT in macro and micro test = 10 6.35); Hep2C cells were afterwards (GMT in macro= 10 6.01 and in micro test= 10 5.94); Vero cells were lowest (GMT in macro= 10 5.78 and in micro test= 10 5.72). So, the sensitivity and accuracy of the potency test for evaluation of the polio vaccine in immunization program in Iran will be assured using the Hela cell line with low passage number in macro and micro methods.

Clostridium perfringens is an important cause of enteric diseases in both human and animals. The bacteria produce several toxins which play key roles in the pathogenesis of diseases and are classified into five toxin types, on the basis of the differential production of Alpha, Beta, Epsilon and Iota toxins. In this study a single PCR assay was developed and used for detection of cpb2 gene to identify the Beta2 harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. It was found that cpb2 presents among C. perfringens isolates types A, B, C and D with 54.5% (6/11), 62% (13/21), 42.8% (6/14), 69.25% (9/13), respectively. Totally 34 of 59 (56.7%) isolates screened by PCR were cpb2-positive. This is the first report of cpb2 positive isolates of C. perfringens causing enteric diseases of animals in Iran. Further studies to demonstrate the exact role of Beta2 toxin in pathogenesis of the bacterium is suggested.

Tetanus is an important disease which created by tetanospasmin toxin of Clostridium tetani. In this study we surveyed six important factors including LF, KF, MLD, pH, OD and total protein assay of Harvard 52 (H52), G5 (HG5) and 49205 (H49205) strains of the bacterium to determine which of them were more suitable for use in vaccine production. The mentioned strains were seperatedly reactivated in thioglycolate medium, 2ml of this suspension was used to inoculate the Muller-Miller medium of each of sixteen 500 ml Erlenmeyer flasks (7,6 and 3 flasks for H52, HG5 and H49205 respectively), where the fermentation runs were performed. Over a period of seven days of experiment, several tests for evaluation of the six mentioned factors on samples of medium cultures were carried out. Results revealed that H52 strain had significantly lower values in LF and OD compared to strains HG5 & H49205 (P<0.001 and P<0.01 respectively) while its MLD and pH were better than other strains (P< 0.05 and P< 0.02 respectively). In conclusion, it seems that HG5 & H49205 strains have been greater toxin producer than H52 strain and as a result, we hope that with some complementary works, these two strains such as H52 strain, be used for routine tetanus vaccine production.

In recent years, chitosan nanoparticles have been studied widely for protein delivery. In this study, Hemiscorpius lepturus (HL) venom was encapsulated in chitosan nanoparticles. The aim of the present work was to carry out a systematic study for preparing biocompatible and biodegradable nanoparticles for loading HL scorpion venom and to evaluate their potential as an antigen delivery system. In this study, HL venom loaded chitosan nanoparticles fabricated by ionic gelation of chitosan and tripolyphosphate and the factors which may be influenced in the preparation of nanoparticles were analyzed. Also, their physicochemical properties and in vitro release behavior were studied. The optimum encapsulation efficiency and capacity were observed when the chitosan concentration and HL venom were 2mg/ml and 500µg/ml, respectively. The HL venom loaded nanoparticles were in the size range of 130-160nm (polydispersity index values of 0.423) and exhibited the positive zeta potential. Transmission electron microscope imaging showed spherical and smooth surface of nanoparticles. The profiles of the release exhibited a burst releases about 50% in the first 4 hr and then slowed down at a constant rate. The obtained results suggested that the chitosan nanoparticles prepared in this work had the potential for antigen delivery.

The aim of this study was production and partial purification of α-amylase enzyme by Bacillus licheniformis. B. Licheniformis was allowed to grow in broth culture for purpose of inducing α-amylase enzyme. Optimal conditions for amylase production by B. Licheniformis are incubation period of 120 h, temperature of 37 °C and pH 7.0. The α-amylase enzyme was purified by ion exchange chromatography on DEAE-sepharose CL-6B and sephadex G-100 gel filtration with a 19.1-fold increase in specific activity as compared to the concentrated supernatant and with a specific activity of 926.47 U/mg. The α-amylase had the highest activity at pH 7.0 and 65 °C. According to the data on native polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was 72 kDa.

Toxoplasma gondii is an apicomplexan parasite that infects human and almost all warm-blooded animals. The life cycle of the parasite includes an asexual reproduction in intermediate hosts (Mammals and birds) and a sexual reproduction in definitive hosts (Felidae). Cats are both the intermediate and the definitive host for T. gondii. The aim of this study was to investigate anti-T. gondii antibodies in cats by using an immunoblot method based on a major surface antigen; SAG1. Six sero-negative kittens were infected intra-peritoneally by Rh strain of T. gondii. Serum samples were collected and evaluated for the presence of IgG antibodies by using two techniques; immunoblotting and indirect fluorescent antibody test (IFAT).SAG1 based immunoblotting was able to detect anti-T. gondii antibodies at least eight days after infection. Comparative evaluation showed that this method is as sensitive as IFAT to diagnose T. gondii infection in cats.

The purpose of this study was to examine the potential application of Sorbitol, Sucrose and Gelatin in combination with glycerol in order to Theileria annulata infected cell line cryo-preservation. The efficiency of the freezing methods was compared by assessing the viability and plating efficiency of cells after resuscitation, dilution and equilibration in cell culture medium. As a result, the conventional cryo-preservative medium containing 5% PEG treated Bovine Serum and 10% glycerol successfully cryo-preserved cell as efficiently as the standard medium containing 80% FBS and 10% glycerol and was superior to all thirteen different experimental cryo-preservative media. A crucial result was obtained among the thirteen different serum-free media which showed that the 0.2M sorbitol+10% glycerol method yielded cells with viability of 54% and good plating efficiency results. It was concluded that the conventional method would be the best and ideal for the cryo-preservation and storage of Theileria annulata-infected cells for vaccine purposes to date.

Epulis is a benign neoplasms of the oral cavity, derived from the periodontal ligament or connective tissue, occur commonly in dogs and rare in cats. However, it is very rare in horse. The tumor can be very large to interfere with normal mastication. Several forms of epulides can histologically be distinguished in dogs. Fibromatous epulis microscopically characterized by a dense well vascularized stroma populated by expansile mass of stellate fibroblasts surrounded by variable amounts of densely packed fibrillar collagen. The present report is related to a sixteen- years-old mixed breed stallion, had been used for several years in production of snake antivenom. The animal showed abnormality in mastication and difficulties in swallowing food. Therefore, clinical examination was performed. Close examination of the oral cavity revealed that there was a large proliferating mass (measured 15 × 12 cm in diameter) on the gingival of the right mandible. There was no good prognosis, therefore, the animal was euthanized and, subsequently, necropsied. Based on location of the lesion as well as gross and microscopic features, the protruded mass was diagnosed and classified as fibromatous epulis.