Archives of Razi Institute
Volume:74 Issue: 4, Autumn 2019

Coxiella burnetiiis an obligate and gram-negative bacteria causing query fever (Q fever) disease, despite the importance of Q fever, there is no universal vaccine against this disease. Therefore, application of the recombinant subunit vaccines which use Com1 and OmpH as immunogenic proteins can be useful in this regard. To perform the current project, Com1 and OmpH genes were amplified by polymerase chain reaction (PCR) method, then, the PCR products were purified by DNA precipitation technique. In order to clone, first, both genes along with the pET-22b(+) vector were digested by NcoI and XhoI enzymes and then, Com1 and OmpH genes were ligated in linear vectors by T4 DNA ligase. The recombinant vectors were transformed in BL21 (DE3) strain of Escherichia coli and expression was induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside. Expression of Com1 and OmpH was investigated using 12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Finally, both proteins were purified by Ni-NTA columns and consequently confirmed by western blotting. The results of assessing 1% agarose gel showed that PCR amplification, DNA precipitation, and digestion of both genes were successfully performed.Theresults of colony PCRs and sequencing revealed that Com1 and OmpH were correctly cloned in pET-22b(+) vector. Finally, the results of expression, purification, and western blotting of both proteins showed thatBL21 (DE3) strain of Escherichia colicould be able to express Com1 and OmpH proteins. Based on the collected data, it seems that Escherichia coli as an affordable and simple host can be applied to express Com1 and OmpH genes. It should be mentioned that products of the present project can be examined as recombinant subunit vaccines against Q fever.

Gamma Coronaviruses (GCoVs) are distributed worldwide, affecting a wide range of bird species, the beluga whale, and bottlenose dolphins. Because of the limited proofreading capability in the viral encoded polymerase, they emerge genetically diverse. There has been no molecular surveillance data to describe the epidemiology of GCOVs in avian species. The present study was conducted to detect GCOVs in Tehran birds’ parks, 2015. Cloacal swabs (267 samples) from eight  different bird species ((Chickens (Gallus gallus), Pheasant (Phasianus colchicus), Turkey (Meleagris gallopavo), Partridge (Perdix perdix), Quail (Coturnix coturnix), Duck (Anas platyrhynchos), Goose (Anserini),and  Guinea fowl (Numididae)) were collected, the viral RNA was extracted, the RT-PCR was performed using QIAGEN one step RT-PCR kit and the primers targeting “3'-UTR” and “Nucleocapsid” genes. The detection rate was approximately 8.99%. GCOVs were detected in the chicken, quail, pheasant, turkey, and the partridge with different prevalence rates. Phylogenetic tree based on partial nucleotide sequences of the N gene clustered the samples into two groups. It is the first report of GCOVs in non-commercial birds in Iran.  According to our results, GCOVs are circulating in different avian species, and further studies are needed to isolate these viruses and evaluate their pathogenesis.

Un-methylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) has been considered as a powerful vaccine adjuvant and recognition of CpG-ODN by chicken leukocytes promotes their ability to fight against infections. In our study, efficacy of different routes of CpG-ODN application as an adjuvant on immune responses (antibody titer together with leukogram) following vaccination against Newcastle disease (ND) has been evaluated in broiler chickens (Ross-308). The results indicated that routes of CpG-ODN administration influence immune responses and comparison effectiveness of CpG-OND delivery routes showed that group vaccinated by eye-drop application had the highest antibody titer than that of the group injected intramuscularly (im) and the difference was significant (p = 0.04) on day 35 of age. Antibody titer of the group treated with Clone 30 plus CpG-ODN via eye-drop route was higher than that of the group vaccinated with clone 30 alone on days 28 and 35 of age and the difference was significant (p = 0.04). Co-administration of both vaccine and CpG improved outcome of leukogram of the chickens on days 21 to 42 of age and among the treated groups, WBC of the group received both vaccine and CpG by eye-drop route significantly (p < 0.05) differed from that of the group vaccinated with clone 30 alone on days 28 and 35 but not on day 42 of age. Average final body weight of the control group did not significantly differ from those of the treated groups at end of the experiment. In conclusion, co-administration of ND vaccine plus CpG-ODN via eye-drop route improves immune responses.

Rural poultry farming is common in the Northern provinces. Similar to commercial poultry, rural poultry is susceptible to most infectious diseases. In addition, by increasing the density of poultry farming, the probability of disease incidences has been increased. Newcastle disease is the most highly infectious disease which is endemic in Iran and causes outbreaks among commercial and rural poultry every year. The present study aimed to investigate the prevalence and virus circulation of Newcastle disease among rural poultry in Northern provinces of Iran. In the current study, 70 villages in 3 provinces (20, 30, and 20 villages in Mazandaran, Golestan, and Gilan, respectively) and a total of 1,374 birds (600, 400, and 374 birds in Mazandaran, Golestan, and Gilan, respectively) were sampled. Each village was regarded as an epidemiological unit. In the present study, birds of 67 (96%) villages were positive (presence of antibodies against Newcastle disease virus), including 28 (93.3%), 19 (95%), and 20 (100%) villages in Golestan, Mazandaran, and Gilan, respectively. Moreover, out of 1,374 birds, 616 (45%) of them were seropositive against Newcastle disease virus with 242 (41%), 159 (39.8%), and 211 (56%) samples in Mazandaran, Golestan, and Gilan, respectively. According to the results of the current study, the seroprevalence rate was reported to be high in both villages and birds. Such a high seroprevalence rate was indicative of the continuous exposure of the rural poultry to Newcastle virus and high virus circulation rate in the mentioned provinces which could result in the dissemination of the disease to commercial farms. Consequently, the implementation of proper control and care programs (e.g., vaccination of native poultry) can facilitate the reduction of Newcastle disease prevalence.

The potentially pathogenic Non-Tuberculosis Mycobacteria (NTM) are emerging nowadays which result in pulmonary and non-pulmonary infections in human. This group of bacteria consists of at least 200 different species. While the pulmonary disease is the most common form of NTM infections, NTM can cause diffused infections as well as extrapulmonary infections in every organ, such as bone marrow, skin, eye, and brain. The NTM cause tuberculosis-like infections, therefore, correct identification of these Mycobacteria is necessary to avoid faulty treatment. Different species of NTM isolates were identified from clinical specimens using phenotypic methods and Line Probe Assay. Minimum Inhibitory Concentration for selected antibiotics was obtained by the broth micro-dilution method. Totally, 42 NTM isolates were identified in this study. Moreover, the frequency of NTM between all positive mycobacterium cultures was estimated at 12%. The most common Rapidly Growing Mycobacteria included Mycolicibacterium fortuitum (30.9%), Mycobacterium abscessus (7.1%), and Mycobacterium chelonae (2.3%), whereas Mycobacterium simiae (40.4%), Mycobacterium kansasii (16.6%), and Mycobacterium avium complex (2.3%) were the most recurring among the Slowly Growing Mycobacteria. Amikacin, clarithromycin, and ciprofloxacin were the most effective antibiotics against isolated NTM. The NTM isolates are frequently being separated from Iranian patients, and are mostly resistant to the wide spectrum of antibiotics. Correct identification and determination of antibiotic susceptibility can be helpful in the healing process of the patients who suffer from non-tuberculosis mycobacterial infections.

The venom of animals, including snakes, scorpions, and spiders is a complex combination of proteins, peptides, and other biomolecules as well as some minerals. Among the biomolecules of some animal’s venom, small peptides that lack disulfide bands known as Non-Disulfide Bridge Peptides (NDBPs) potentiate the bradykinin by preventing the conversion of angiotensin 1 to angiotensin 2 using the mechanism of inhibiting the Angiotensin-Converting Enzyme activity and finally reducing the blood pressure in the victims. This feature of the NDBPs of animal’s venom is suggested as the potential of biological drugs. This study aimed to isolate venom components of three species of Iranian medically important scorpions and study the bradykinin potentiating effect of them. The scorpion specimens were collected from the venomous animals and antivenom production department of Razi Vaccine and Serum Research Institute, Karaj, Iran. Moreover, venom extraction was performed by electrical shock (5 volts). The obtained liquid venom of three species specimens was frozen and lyophilized immediately and then preserved in a cool and dried place. The isolation of the venom components for each scorpion was carried out using high-performance liquid chromatography. The obtained ranges of venom fractions (zones) were tested on isolated tissues of guinea-pig ileum and rat uterus using organ bath instrumentation in several replicates. The bioassays resulted in the peptides, including Z1 and Z2 regions in the venom fractionsof the Hottentotta saulcyi, Z2 in Odontobuthus doriae, as well as Z2 and Z3 in Mesobuthus eupeus demonstrated bradykinin potentiating effect. It is concluded that Bradykinin Potentiating Factors were traceable in the venom of all three scorpion species. Therefore, these venoms have the therapeutic potential to exploit biological-based drugs.

Bone healing is still a great challenge in orthopedic surgery and clinical practice. There is a dearth of research investigating the effect of Zeolite/Collagen (ZC) nanocomposite on bone regeneration. In the present study, a critical segmental defect of the rabbit femur was repaired using defects in femurs repaired by ZC nanocomposite, and the effects were examined histologically. In total, 45 rabbits at seven months of age weighing 3.5 kilograms were utilized in this study. After making the bone defects, all animals were randomized into three groups (n=15). In a normal control group (NC), a defect was created, no intervention was made, and the skin incision was sutured. On the other hand, in the ZC group, the nanocomposite of ZC was placed into the created defect. In the hydroxyapatite group (HA), the hydroxyapatite was placed into the created defect. The samples were collected on days 15, 30, and 45 postoperatively and assessed histopathologically. The mean scores of the index of the union were compared and considerable alterations were observed in this regard in the experimental groups (P<0.05). The values of the index of spongiosa demonstrated that on day 15, it was the highest in the ZC group (2.2) and lowest in the HA and NC groups (0.6). Moreover, the values of the index of bone marrow demonstrated no noticeable alteration among the values of the index of bone marrow in the experimental groups (P>0.05). The findings of this study demonstrated that ZC nanocomposite might be considered for reconstruction in bone damages. It seems the ZC nanocomposite bears a crucial capability in the reconstruction of bone damages and might be used as a biological frame in bone damages.

The development of the marine aquaculture industry has led to the generation of significant amounts of fish wastes. Marine farm wastes exert adverse effects on the surrounding area of the cages. On the other hand, wastes of fish and other aquatic animals are regarded as major sources of valuable natural bioactive compounds, including enzymes, proteins, bioactive peptides, oil, amino acids, collagen, gelatin, calcium, biopolymers, and water-soluble minerals. To investigate the potential of marine fish waste, the whole digestive system of yellowfin seabream (Acanthopagrus latus) was extracted for extraction and identification of trypsin enzyme. Fish (179.93±93.67 g; 184±28.17 cm) were caught from the Persian Gulf and stored at -20 °C. Yellowfin seabream were dissected and their whole digestive systems were removed. Samples were thoroughly washed with distilled water and purified through defatting using acetone and ammonium sulfate precipitation. The following issues were assessed: the total and specific activity of trypsin, protein determination, molecular weight, enzyme activity and stability in different pH values and temperatures. The obtained results indicated that specific activity and protein content of trypsin enzyme were 4.4 U and 3.4 mg/ml, respectively. The molecular weight of 23 kDa was reported for trypsin using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. Maximum activity and stability of trypsin were observed at 60°C and 45°C, respectively. Trypsin demonstrated maximum activity and stability at a pH value of 8.0. In general, the results of the current study suggested that trypsin extracted from the digestive system of yellowfin seabream has considerable potential for industrial applications, such as the food industry, owing to its characteristics and stability under alkaline conditions.

Zoonotic cutaneous leishmaniasis t caused by Leishmania major is spread in focal areas of more than 90 countries in the tropics, subtropics, and southern Europe. In the absence of any effective vaccine, the only means to treat and control leishmaniasis is conventional medication. Glucantime is the first choice of anti-leishmanialdrug, has serious side effects like high toxicity, exorbitant cost, problems with the administration and development of resistance. Curcumin is the active component from the rhizome of herb Curcuma longa, possessing many pharmacological and biological activities with antiprotozoal and anti-proliferative effects which make it a good alternative to existing therapy. Antimicrobial peptides like CM11, a small peptide consisting of 11 amino acids, are also novel potential drugs against at least wide spectrum of microbial organisms. The aim of this study was to evaluate the effect of curcumin alone and in combination with CM11 on promastigote form of L. major (MRHO / IR / 75 / ER) for 12h and 24h in vitro. The results of Giemsa staining showed that the morphology of the flagellum and cell shape increased changed with increasing concentration of curcumin (5 µM, 10 μM, 20 μM, 40 μM and 80 μM). MTT and Trypan blue results demonstrated that the promastigotes were susceptible against curcumin in dose and time dependent manner, while CM11 alone at concentration of 8 µM as well as in combination with 10 and 20 µM curcumin had no significant effect on promastigotes. Our results revealed that curcumin can provide a new curative candidate against cutaneous leishmaniasis.

Snails are creatures present in various ecosystems that, in addition to being present in human surroundings, some of them are also important in veterinary medicine and medicine as the intermediate hosts of Digenean trematodes. The present study was conducted to identify and determine the geographical distribution of freshwater snails and investigate the relationship of variables, such as season and geographical region, with snail species and dispersion in Lorestan in the west of Iran. A total of 4400 samples of freshwater snails were collected using the multistage sampling method (i.e., stratified, cluster, and randomized) from 110 points in five geographical regions in four seasons and then identified based on their morphological characteristics by diagnostic keys. The ArcGIS software (version 10.3) was used to evaluate the spatial distribution of the freshwater snails. In this study, seven species of freshwater snails were identified in six families belonging to six genera, namely Melanopsis doriae (6.30% of the variation in species), Theodoxus doriae (5.55%), Bithynia tentaculata (43.22%, the dominant species), Physa acuta (24.98%), Lymnaea truncatula (9.75%), Gyraulus euphraticus (8.18%), and Lymnaea gedrosiana (2.02%). The geographic distribution of freshwater snails was recorded across five regions in 22 points per region for every season. The spatial distribution maps showed that the distribution of freshwater snails varies according to region and season (P<0.001). The obtained results revealed the effects of season and geographical region on the distribution and population density of snails in the province. These data can be used for the implementation of control programs against parasitic diseases in the region, including trematodes.

The aim of this study was to identify the cell surface cluster of differentiation (CD) markers of the cell lines infected by Theileria annulata schizont. The CD molecules are very useful for the characterization of cells and different subpopulations of leukocytes. They are usually recognized by specific antibodies using flow cytometry and immunohistochemistry. In the current study, we applied reverse transcriptase-polymerase chain reaction (RT-PCR) to define the profile of cell surface markers in a cell line infected by an attenuated S15 vaccine strain of T. annulata schizont and a new laboratory-established cell line infected by a non-attenuated form. In order to determine the specific markers that can be used for excluding the non-attenuated cell lines, the characterization of the surface proteins profile of the S15 vaccine cell line is important. The RT-PCR was carried out by specifically designed primers using a panel of seven bovine CD markers, as well as beta-actin as an internal control house-keeping gene. We showed that both of the examined cell lines had a consistent expression of CD4, CD5, CD11a, CD14, CD43, and CD45 markers. However, the specific finding in this study was the expression of B-cell markers CD79a and CD5 by the newly-transformed cell line. On the other hand, CD5 as a marker for B-cell subset was expressed by S15 vaccine strain. In conclusion, we consider CD79a surface protein as a new marker for the cell lines infected by non-attenuated T. annulata schizont, while the cell lines infected by the vaccine strain do not express this marker. In addition, the identification of CD marker expression based on the RT-PCR assay might be a suitable and appropriate alternative technique for flow cytometry.

The current research aimed to quantify melittin (MEL) in Iranian honey bee (Apis mellifera meda) venom. To this end, a liquid chromatography-electrospray ionization-ion trap tandem mass spectrometry (LC-ESI-IT-MS/MS) approach was employed. Melittin is the main toxic peptide of honey bee venom with various biological and pharmacological activities. It was extracted with pure water from the bee venom samples. The analyses were performed on XBridge BEH300 C4 column using a gradient method with the mobile phase consisting of ultrapure water and acetonitrile (containing 0.1% formic acid). Signals of the melittin were recorded with the selected reaction monitoring (SRM) mode, which is a quantitative approach capable of quantifying analyte peptides with high sensitivity and. The mass spectrum of MEL was obtained in the positive ion mode and the quantification analysis was performed using precursor to product ion transition of m/z 570.2/669.9. This method demonstrated good linearity (R2˃0.997) in the range of 1-100 µg mL-1, with a limit of quantification (LOQ) of 1.0 µg mL-1. The content of MEL in Iranian honey bee venom accounts for 43–55% of total dry weight. This method can be used to evaluate the quality and authenticity of bee venom samples for different therapeutic applications of MEL.

Avian influenza (AI) is an acute infectious disease with worldwide significance causing extensive economic losses in the poultry industry. Avian influenza viruses (AIVs) belong to the family Orthomyxoviridae and categorized in the genus influenza virus A. These viruses have been isolated from more than 100 species of free-living birds. Migratory birds are considered as reservoirs for AIVs and are the major agents responsible for global outbreaks. The Passeriformes are found in most parts of the world and cover a variety of habitats from rural to urban areas. House sparrows are members of the family Passeridae and due to their free flying, are strongly associated with seabirds, indigenous, and industrial poultry. The aim of this study was to determine the role of house sparrows in AIV (H9N2) circulation in the Ahvaz region. The intestinal and tracheal samples were taken from 200 sparrows around Ahvaz during 2017. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using specific primers in order to detect M and H9 genes of AIVs. The positive specimens in the PCR for the M gene were inoculated into 9-11-day-old embryonated chicken eggs via the allantoic fluid. The results showed that 11 out of 200 samples were positive for the two genes of M and H9. According to the findings of the present study, house sparrows are infected with H9N2 and pose a threat to commercial poultry. These birds may play a significant role in the transmission of AIV between wildlife and domestic animals. Therefore, this issue is important to be considered in preventive measurements.

In order to describe the proportion and pattern of culling in commercial goatherds, this survey was carried out in an industrialized goatherd in Torbat-e-Jam, Iran, over a period of 18 years from 1996 to 2013. In total, the data of 3945 goats were used in this study. Finally, out of all samples, 499 (12%) goats were culled. The involuntary culling was performed mainly due to shortage disorders (3.8%), viral disorders (3.3%), microbial diseases (2.8%), and other disorders (2.1%). Sheep pox was the most important reason (64%) for culling due to viral disorders. Tick paralysis was the most common parasitic disease that contributed to culling and responsible for 88% of parasitic disorders. On the other hand, enterotoxemia accounted for 55% of microbial disorders is considered the most common cause of culling. The high proportion of culling due to shortage disorders, especially nutritional deficiencies should be considered the most important cause of culling. It requires precautionary measures and planning in order to reduce the aforementioned rate.