Archives of Razi Institute
Volume:74 Issue: 3, Summer 2019

There are many challenges in the field of public health sciences. Rational decisions are required in order to treat different diseases, gain knowledge and wealth regarding research, and produce biological or synthetic products. Various advances in the basic laboratory science, computer science, and the engineering of biological production processes can help solve the occurring problems. Bioinformatics is defined as a field of science combined of biology, mathematics, physics, chemistry, and computer sciences. Recently, bioinformatics has been extensively used in the designing of the epitope, vaccines, antibodies, adjuvants, diagnostic kits, and therapeutic purposes (e.g., proteins, peptides, or small molecules). Moreover, bioinformatics includes chemoinformatics that has been employed to produce various biological or chemical products to target and combat pathogens. Bioinformatics is involved in other areas of data analysis and prediction, such as structural biology, system biology, phylogeny, population genetics, and next-generation data sequencing. To the best of our knowledge, no published study coherently described the benefits of bioinformatics fields applied for medication development or diagnostic aims in bio-productive and pharmaceutical/vaccine companies. Therefore, in the current review, we attempted to present the available bioinformatics resources, practical experiences, and other findings in the mentioned field along with providing a harmonized and applied model(s). The key points presented in the current review may help to elevate production and reduce the costs for the development of novel vaccines, medicines, and antibodies. In addition, these methods can facilitate the identification of organisms and may guarantee the quality of biological products.

Brucellosis is primarily a worldwide zoonotic disease caused by Brucella species. The genus Brucella contains highly infectious species that are classified as biological threat agents. In this regard, the identification of Brucella can be a time-consuming and labor-intensive process posing a real risk of laboratory-acquired infection to the laboratory staff. This study aimed to present a novel conventional and real-time polymerase chain reaction (PCR) assay for the identification of Brucella abortus strains. Regarding this, two primers (bru ab2) were designed based on the unique loci encoding autotransporter-associated beta strand repeat-containing protein (ID:YP00113760). A total of 56 Brucella strains (e.g., reference, vaccinal, and field isolates) and Yersinia enterocolitica, as a non-Brucella isolate, were evaluated in conventional and real-time PCR systems. The results of the study indicated that 0.4 ng and 400 FG of genomic DNA of B. abortus strains can be detected by conventional and real-time PCR, respectively. The primers, bru ab2, were suitable for both PCR methods. Both methods were specific for the detection of all strains of the bacterium; however, real-time PCR assay was 1000-fold more sensitive than the conventional PCR method. Therefore, this new detection system could be a suitable selective modified method for the accurate identification of all B. abortus strains.

Avian Influenza (AI) H9N2 is endemic in Iran; therefore, it is necessary to estimate the disease prevalence among birds in live bird markets (LBMs) and assess the risk spread across the country. Accordingly, this study aimed to estimate the prevalence of AI subtypes in LBMs, bird gardens, and zoos during October and November 2015 in Iran and investigate the associated risk factors. Data related to independent variables for birds and premises were collected using a prepared questionnaire which included items about previously known potential risk factors associated with avian influenza infection. Serological testing was carried out to detect the antibodies against H5, H7, and H9. Regarding H5 and H7, the antigens H5N2 and H7N1 were used in this study. Positive samples on the first test were examined with the second antigens, namely H5N1 and H7N7. Moreover, sera with titers ≥4 (i.e. log2) were considered positive and premises with at least one positive bird were considered as positive units. In total, 87 premises were included in this cross-sectional study. Serum samples were examined utilizing hemagglutination inhibition, and RT-PCR was conducted on swab samples. Regarding the molecular test, the RNA was extracted using the High Pure Viral RNA Kit (Roche, Germany). In addition, real-time RT-PCR was conducted based on the described method. The seroprevalence rates of H9N2 were 83.9% and 31.8% at the premises and bird levels, respectively. Totally, 9.2% of pooled swab samples were positive for H9N2. However, all sera and swab samples were negative for H5 and H7. Hot and humid weather (OR=0.13, 95% CI 0.02 – 0.78) as well as bird-keeping condition (i.e., enclosed area) (OR=0.11, 95% CI 0.012 – 1.02) were protective factors for H9N2. High seroprevalence rate of H9 indicates that the disease is endemic in Iranian LBMs. Active surveillance must be carried out in LBMs, especially in the northern provinces of Iran. In addition, cleanliness and improved hygiene would be useful to prevent the spread of disease in LBMs.

In the present study, the potency and immunogenicity of the inactivated bivalent vaccine of Newcastle disease (ND) and avian influenza (AI) produced by Razi institute in Iran were compared with a similar imported vaccine administered by standard methods to specific-pathogen-free (SPF) chicken. A total of 150 twenty-one-day-old SPF chickens were used for evaluating Razi and imported inactivated H9N2/ND vaccines. The chickens were divided into eight groups. The subjects in groups 1 and 3 were vaccinated with 0.3 ml/bird by subcutaneous route in the back of the neck with Razi and imported vaccines, respectively. Chickens in group 2 received Razi vaccine based on the recommended dose of the manufacturer (0.2 ml/bird) by the same route. The birds in groups 4 and 5 received 0.01 ml/bird of Razi and imported vaccine, respectively. Groups 7 and 8 were considered for studying the safety of the two vaccines and received a double amount of the full dose of vaccines. Moreover, group6 was regarded as the negative control. Sera were collected weekly from each chicken for antibody analysis against Newcastle disease virus (NDV) and AI virus (AIV) and the study continued for 15 weeks after vaccination. An immunological evaluation was carried out using haemagglutination inhibition (HI) antibody test against NDV and AIV. The results showed that up to 15 weeks after vaccination, the Razi vaccine induced a higher level of protective antibody against AI and ND in comparison with the imported ones at the dose of 0.3 ml/bird. The mean HI titer was significantly different between Razi vaccine and imported vaccine at the dose of 0.3 ml/bird. There was no statically significant difference between Razi vaccine (0.2 ml/bird) and the imported vaccine (0.3 ml/bird) against NDV and AIV. According to the findings, 15 weeks after vaccination, HI titers were still detectable at a high level. The mean HI titer was found as 5.2 log2 against NDV and 5 log2 against H9N2 with Razi vaccine (0.3 ml/bird). In addition, the mean HI titer with Razi vaccine (0.2 ml/bird) was 4.1 log2 and 4.7 log2 against ND and AI, respectively. In summary, our results indicated that Razi inactivated vaccine (0.3 ml/bird) induced a strong and rapid antibody response in vaccinated chickens and is more effective in chicken against AIVs and NDVs, in comparison with the imported vaccine.

Salmonellais a foodborne zoonotic enteric bacterium able to infect both humans and animals. This study aimed to identify the antimicrobial resistance of Salmonella serovars isolated from human, cattle, and poultry. Moreover, we investigated the probable transmission trends of antibiotic-resistant Salmonella isolates from food animals to human. A total of 242 Salmonella isolates collected from various human and animal sources were serotyped. The polymerase chain reaction was performed to detect the invA virulence gene. The isolates were subsequently tested against 14 antimicrobials and the resistance rates among the isolates from three sample sources were statistically analyzed by the Chi-Square test. Serotyping revealed the isolates belonged to various serovars with the dominance of Enteritidis (37%), Typhimurium (35.3%), and Infantis (21.1%). A high frequency of resistance to streptomycin was observed followed by tetracycline, trimethoprim, sulfonamides, spectinomycin, chloramphenicol, florfenicol, ampicillin, kanamycin, ceftazidime, and cefepime. In addition, multidrug resistance was observed in more than 40% of the isolates. The results of the statistical analysis showed a significant relationship (P 0.001) between the rate of antibiotic resistance among the three sources of Salmonellaisolates. Furthermore, the antibiotic resistance had a statistical relationship between the different serotypes isolated from different sources. These findings demonstrate the possible transmission of resistance to human from animal sources. The prevalence of the Typhimurium, Enteritidis, and Infantis serovars in both human and animals suggested that Salmonella contamination in chicken and cattle may be the major source of salmonellosis in human. The high incidence of antibiotic resistance in Salmonellaisolates along with the close relationship between the antimicrobial resistance of animal and human isolates indicate the role of food animal products as an important source of resistance.

In order to evaluate the healing effect of eugenol and other nanofibers, 100 male Wistar rats (200±10 g) were used with 14-15 weeks of age in this study. All of the male rats were transferred in the standard cages under controlled exposure conditions in a 12:12 h light/dark cycle with a constant temperature about 22±2 oC. In addition, the male rats were fed with pellets. Firstly, anesthesia process was performed by 2% xylazine hydrochloride (10mg/Kg/IP) and 10% ketamine hydrochloride (100mg/Kg/IP), and then the rats were placed on the operating table. Then the dorsal surfaces of the rats’ skin to ileum were scrubbed and prepared as the next step. A circular wound (with a 7 mm diameter) was created by a 7 mm sterile biopsy punch. All 100 rats were divided into four groups (n=25) randomly named as control, nano zinc oxide (ZnO), eugenol nanofibers, and polycaprolactone groups. After that, they were divided into five groups regarding the wound closure rate in days 3, 5, 7, 14, and 21. Then, the wound dressings were placed on the wounds and renewed every 24 h. At the end of days 3, 5, 7, 14, and 21, the relevant tests, such as histopathology, were conducted by removing the tissue volume using a biopsy punch, and then decapitation process was performed on the rats. It was obvious that eugenol nanofiber showed the best granulation tissue by the production of collagen. Further studies are being performed on wound healing by eugenol nanofiber.

The present study determined the regenerative effect of bone marrow-derived stem cells (BMSCs) on thioacetamide (TA)-induced liver fibrosis in rats. A total of 30 male Wistar rats were randomly divided into sham control and treatment groups. The rats of the sham control group were subdivided into three groups and sampled on the 14th, 18th, and 20th weeks after fibrosis induction. The rats of the treatment group were subdivided into two groups and sampled on the 4th and 6th weeks after BMSCs treatment. Fibrosis was induced by the intraperitoneal administration of 200 mg/kg of TA twice a week for a period of 14 weeks. All the animals underwent liver function tests and histopathologic evaluation 4 and 6 weeks after BMSCs transplantation. The BMSCs were characterized using osteogenic induction and reverse transcription-polymerase chain reaction. The BMSCs were plastic adherent, spindle-shaped, and positive for osteogenic differentiation. They expressed CD73 and were negative for CD45. The infiltration of inflammatory cells and deposition of collagen fibers were noticed after TA administration. A significant decline in inflammatory cells and a healing process were detected 4 weeks after cell transplantation. The amelioration in hepatic tissue was significant 6 weeks after cell therapy. Following the injection of BMSCs, a nonsignificant decrease was visible in aspartate transaminase level; however, this decline was significant for alanine aminotransferase level. The alkaline phosphatase and albumin levels showed an increasing trend after cell administration. The transplantation of BMSCs resulted in a significant regenerative effect after hepatic injuries. Therefore, it was shown that BMSCs transplantation can open a new window and be a therapy of choice in the amelioration of liver fibrosis.

Numerous pharmaceutical agents can induce adverse reactions in the human body, including toxicity to the liver and the inflammation of intestines. Therefore, nowadays one of the most urgent problems in modern medical science is the prevention and restoration of morphological and dysbiosis disorders caused by numerous medications. With this background in mind, we aimed to evaluate the efficacy of phytobacteria on toxic damage to the structure and function of the liver and ileum, as well as the composition of the large intestine microflora in white rats with intestinal dysbacteriosis due to carbon tetrachloride (CCl4) and ampicillin trihydrate. In order to prevent toxic damage to the liver and ileum of experimental animals, a phytobacterial agent was used. This test agent was composed of a mixture of commercial lactobacteria Lactobacillus helveticus with a water-soluble extract of thyme (Thymus Serpyllum L.) on a sterile milk basis. Our results showed that the introduction of phytobacterial agent led to reduced inflammation, accelerated regeneration of the ileum mucous membrane, and a positive effect on the damaged intestine. The phytobacterial agent increased the resistance of the body to potentially pathogenic microorganisms and toxic compounds by restoring the microflora of the large intestine. It was established that the phytobacterial remedy resulted in the normalization of the intestinal microflora of white rats, which had toxic damage to the liver and ileum caused by CCl4 and ampicillin trihydrate administration. Moreover, the usage of phytobacteria was correlated with improvement in the structure and function of the liver and ileum.

Abortion is one of the most important economic issues in sheep flocks. Chlamydophila abortus is an agent of enzootic abortions in sheep. Mycoplasma agalactiae is the main etiological agent of contagious agalactia, which can cause abortion in sheep. The aim of this study was to investigate the prevalence of M. agalactiae and C. abortus among aborted ovine fetuses in Sistan and Baluchestan, Iran. Sheep owners were asked to transfer their aborted fetuses to a nearby veterinary clinic; furthermore, they were taught biosecurity principles. A total of 78 aborted sheep fetuses were collected from all over Sistan region in the autumn of 2015 and winter of 2016. The samples were then transferred in ice to the Anatomy Laboratory of the Veterinary Faculty of Zabol University, Zabol, Iran. The spleen and abomasum contents of the fetuses were sampled under sterile and safe conditions. Polymerase chain reaction was used to detect M. agalactiae and C. abortus. The results showed that 24 (30.8%) cases were infected with M. agalactiae. However, infection with C. abortus was not detected in any fetuses. There was no statistically significant relationship between such independent variables as the location of livestock, history of abortion, fetal gender and age, age and parity of ewe, and fetal infection with M. agalactiae. The high incidence of Mycoplasma contamination in this study may be due to inappropriate biosecurity measures and lack of vaccination against agalactia in sheep herds in Sistan region.

Staphylococcus aureus is a major pathogen in the transmission of diseases from animals to humans and vice-versa.Various infections, such as mastitis in cattle, sheep and goats, as well as gastroenteritis due to food poisoning in humans are the most frequent problems caused by S. aureus. The bacteria also lead to severe economic losses in dairy industry. A major virulence factor for the organism is encoded by the coagulase (coa) gene. This study aimed to assess the polymorphisms of the coa gene in S. aureus strains isolated from bovine mastitis and dairy product samples in Ahvaz, Iran. The results showed that out of 91 S. aureus, 80 (87.91%) isolates were positive for coa gene(s). In total, nine different polymerase chain reaction (PCR) products were obtained for coa-positive isolates. A single band was detected in coa PCR with a size ranges from 370 to 830 bp in most isolates (n=77, 96.25%). For three isolates (3.75%), two amplification products were obtained. A PCR product of an estimated size of 590 bp was most frequent, as obtained for 48 (60.00%) isolates. Whereas, 370 and 830 bp PCR products were the least presented, for two (2.50%) and one (1.25%) isolate, respectively. Subsequently, for restriction fragment length polymorphism (RFLP), typing of coa gene and AluI restriction enzyme were used for the digestion of the products. AluI for most of PCR products generated a unique pattern; however, four PCR products (the sizes ranged 750, 670, 590, and 510 bp) generated three or more patterns. Based on AluI RFLP of coa gene, the isolates were classified into 23 groups. Two groups of isolates were dominant, making 45% of the total. According to the findings, one or two types of coa RFLP were dominant among samples that were infected with more S. aureus isolates belonging to different coa RFLP types.

Colibacillosis is known as a fatal bacterial disease resulting in a high level of commercial loss worldwide. This study amid to elucidate the sequence, genetic characteristics, and phylogeny of the bor gene in Escherichia coli (E. coli) strain c1378 (O78:K80) isolated from avian colibacillosis in Iran and develop a rapid and optimal polymerase chain reaction (PCR) molecular-based technique with specific primers to detect this gene in E. coli. A virulent avian E. coli (i.e., laboratory designation E. coli strain c1378) isolated from a chicken with systemic colibacillosis from a broiler farm in Tehran, Iran, in 2004 was used as a source of the bor gene. After DNA extraction, PCR method was used to amplify the bor gene. A 658 bp fragment of the bor gene was amplified, sequenced, blasted, and phylogenetically studied. The most similar sequences to the bor gene in E. coli strain c1378 were E. coli APEC O78, Enterobacteria phage HK630, and Escherichia coli BW2952, respectively. There was a high similarity between the bor gene in E. coli bacteria with their phage and plasmid. Moreover, a high similarity was observed between the bor and iss genes (approximately 92%) showing that they were homologous genes. In addition, the similarity analysis of different bacterial species, as well as their plasmid and bacteriophage, to the bor gene indicated that the highest similarity to O78:K80 was related to Paracoccidioides brasiliensis, Bacillus thuringiensis CT43 plasmid pBMB0558, and Salmonella enterica subsp. enterica serovar Kentucky strain CVM29188 plasmid, respectively. Altogether, the results of the present study confirmed the presence of the bor gene in the studied isolates and clarified its sequence, phylogenetic relationship, and similarities of E. coli strain c1378 (O78:K80) isolated from avian colibacillosis.

Enzootic bovine leukosis (EBL) is known as bovine lymphosarcoma and normally affects the old cattle. EBL is caused by bovine leukemia virus (BLV), which is generally spread all around the world. This virus is transmitted via bovine blood products within and between cattle herds. Glycoprotein GP51 in the blood is responsible for cattle immune responses to BLV. This virus has been previously detected in cattle and even in the calf. However, to the best of my knowledge, this is the first short communication reporting the detection of EBL in calves in Iran. The samples of the present study were obtained from two calves of different mothers within the same dairy farm of 2000 cattle, located near Tehran, Iran. General clinical signs of the two calves, such as heart rate, respiratory rate, and body temperature were recorded. The clinical observations were confirmed by hematological tests, histopathological examination, enzyme-linked immunosorbent assay, polymerase chain reaction, and phylogenetic analyses. The originality of the detected virus was assessed by blasting against the other strains of BLV available on the NCBI web page. Regarding the clinical symptoms, bulging eyes were noticeable. Moreover, atypical and malignant lymphocytes were detected in the circulatory system and periorbital tissue. It should be noted that the presence and expression of GP51 in both calves and cattle was similar to the previously detected ones in Korea and Japan. The latter result confirms the originality of retrovirus structural subunit GP51.  Similar observations were reported in a six-month follow-up.