Archives of Razi Institute
Volume:76 Issue: 3, Sep 2021

Despite the availability of a vaccine, pertussis is still a worldwide health problem. Outer membrane vesicles (OMVs) in gram-negative bacteria can stimulate the immune system due to several outer membrane proteins and are very good candidates in vaccine development. OMVs obtained from Bordetella pertussis contain several antigens, which are considered immunogenic, and could make them a potential candidate for vaccine production. The current study aimed to compare the current OMV extraction method (with ultracentrifuge) and a modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors. Vaccinal strain BP134 grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared using two different methods. They were then stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method, and then the protein profile was evaluated by SDS-PAGE. The presence of immunogenic antigens was detected by Western blotting. The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and had a size between 40 and 200 nm. The total protein yields of the OMV isolated using the current and modified methods were 800 and 600 µg/ml, respectively. Evaluating the protein profile of extracted OMVs showed the presence of different proteins. Finally, the presence of PTX, PRN, and FHA was observed in OMVs extracted from both methods. Comparison of the two OMV extraction methods showed that the obtained vesicles have a suitable and similar shape and size as well as the expression of three important pathogenic factors as immunogens. Despite the relatively low reduction in protein yield as the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from B. pertussis, especially in developing countries. It should be noted that further experiments including immunogenicity determination of OMVs obtained as vaccine candidates in animal models are required.

Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies.

Fowlpox (FP) is a viral disease that is widely distributed throughout the world. The disease has an economic impact on the poultry industry, and its prevalence has even been reported in vaccinated flocks. The present study used flow cytometry to evaluate the CD4+ and CD8+ T-cell immune response of chicks induced by FP vaccine. 120 specific pathogen-free (SPF) 21-day-old chicks were randomly divided into three groups of 40. One group was used as negative control with PBS inoculation, the other two groups were inoculated with the local fowlpox vaccine produced by Razi Institute and commercial FP vaccines, and they were kept for five weeks. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll–Hypaque density gradients and the percentages of CD3+, CD3+, CD4+, and CD3+CD8+ T lymphocytes were analyzed with flow cytometry. Seven days post-immunization, a maximum (90-100%) swelling formation (“take”) on the vaccination site was observed. The ratios of CD4+ to CD8+ T-lymphocytes in both vaccinated groups were significantly higher (p < 0.05) than the control group inoculated with PBS. The percentages of CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes were increased in chickens vaccinated with commercial and local FP vaccines. There were no significant differences between the groups receiving commercial and local fowl pox vaccines. The present study showed that protective immunity could be associated with increased cellular immune responses, which has been interpreted as enhancing T-cell proliferation and increasing CD4+ to CD8+ ratios through vaccination with the FP vaccine. This study further suggests that the induction of enhanced immune responses is due mainly to the Th1-type response.

Ducks play an important role in the transmission of avian influenza to poultry farms. Because of the importance of vaccination in reducing virus shedding, this study evaluated avian influenza-killed vaccine H9N2 on tissue distribution and shedding of avian influenza virus H9N2 in ducklings. One hundred-day-old ducklings were purchased and, after bleeding from 20 birds, were kept in four separate rooms under standard conditions. Groups 1 and 2 were vaccinated at 9 days, and groups 2 and 3 were challenged with 0.1 ml of allantoic fluid containing 105 EID50 (A/chicken/Iran/Aid/2013(H9)) virus intranasally at 30 days. Group 4 chicks were kept as the control group. Chicks were observed two times daily. On days 1, 3, 5, and 8 after inoculation, 3 chicks were randomly selected from each group and cloaca and trachea swabs samples were collected from each bird. Then the ducklings were euthanized and trachea, lung, spleen, intestine, liver, and brain tissue samples were collected for molecular detection. The virus was detected in the tissues and tracheal and cloacal swabs by polymerase chain reaction (PCR), and anti-AIV titres were measured by HI test. The results showed no clinical signs in the challenged groups. In the vaccinated challenged group, virus was detected only in cloacal swabs, but in the unvaccinated challenged group, virus was detected more in tracheal swabs than in cloacal swabs. In challenged-unvaccinated chicks, virus was detected in the trachea and lungs, and in challenged-vaccinated birds, virus was detected in the intestines. In conclusion, vaccinating ducks against the AI H9N2 virus reduced shedding and tissue distribution of AI viruses in challenged ducks.

Brucellosis is an anthropozoonotic disease. Infection of livestock with Brucella is endemic in most parts of Iran. Sistan-Baluchestan is bordered on the east by the countries of Afghanistan and Pakistan. The high prevalence of brucellosis in livestock in the eastern neighboring countries results in transmission of the disease to this province. The present research aimed to determine the prevalence of brucellosis in small ruminants in the Sistan region of Iran and to compare serological and molecular tests for the detection of brucellosis. Blood samples were taken from 150 randomly selected sheep and goats, and sera were separated. All sera were analyzed by serological (Wright and 2-ME) and molecular (Polymerase Chain Reaction (PCR)) tests. Serological tests were carried out according to the instructions of the Iranian Veterinary Organization The degree of agreement between serological tests and PCR was determined by kappa value. In this study, 17 cases (11.3%) were identified as positive by the PCR method. Wright and 2-ME tests had the highest agreement with PCR in titers ≥2/80 and ≥2/40, respectively. The results of this study show that the brucellosis in sheep and goats has a greater prevalence in the Sistan region than in most other parts of Iran, and this is important in terms of public health. It is suggested that brucellosis vaccination coverage in livestock be increased in this area and that the people in Sistan region must be notified about methods for preventing brucellosis. Also, further studies to compare conventional serologic tests with the gold standard test are recommended.

More than a decade ago, a novel coronavirus that infects humans, bats, and certain other mammals termed severe acute respiratory syndrome coronavirus (SARS-CoV) caused an epidemic with ~ 10% case fatality, creating global panic and economic damage. Recently, another strain of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused an infectious disease (COVID-19) in humans which was detected for the first time in Wuhan, China. Presently, there is no specific therapy available for the treatment of COVID-19. However, social distancing, patient isolation, and supportive medical care make up the current management for this current infectious disease pandemic. The present in silico study evaluated the binding affinities of some natural products (resveratrol, xylopic acid, ellagic acid, kaempferol, and quercetin) to human angiotensin-converting enzyme 2 and coronavirus (SARS-CoV-2) main protease compared to chloroquine, an inhibitor known to prevent cellular entry and replication of the coronavirus. The respective binding energies of the selected natural compounds and chloroquine towards the proteins were computed using PyRx virtual screening tool. The pharmacodynamic and pharmacokinetic attributes of the selected compounds were predicted using admetSAR. Molecular docking analysis showed that the natural compounds had better scores towards the selected protein compared to chloroquine with polar amino acid residues present at the binding sites. The predicted ADMET properties revealed the lower acute oral toxicity of the natural products compared to chloroquine. The study provides evidence suggesting that the relatively less toxic compounds from the natural sources could be repositioned as anti-viral agents to prevent the entry and replication of SARS-CoV-2.

The World Health Organization has strict rules and recommendations on the selection and use of cell substrates in laboratories. Given the widespread use of safe and secure cell substrates in the production and quality control of viral vaccines and also the high demand for vaccines against viral diseases, obligating the selection of a suitable cell substrate for cultivation and production of biological products. Animal cell lines play a valuable role in the preparation and propagation of viral seeds; thus, the current study used the BHK-21 cell line among others for viral checking with the aim of replacing the BHK-21 C5 cell line with the RK13 cell line to investigate the cytopathic effects of the rubella virus. To this end, attempts were made to determine the characteristics of the BHK-21 C5 cell line including cell growth characteristics and sterility tests to validate its safety and security. Then, by culturing the cells in a 96-well microplate, titration of the rubella virus was subsequently performed by preparing serial dilutions of the virus from 10-1 to 10-5 and inoculated to cell lines in order to compare the sensitivity of BHK-21 C5 and RK13 cell lines to rubella virus. Data analysis according to the results of the tests by ahead default, p-value < 0/05 was equal to p-value = 0.01 based on SPSS analysis with the paired-sample t-test. In addition, the box-plot diagram indicated a significant difference between these cell lines. Based on the results, the BHK-21 C5 cell line seems to be more sensitive to the rubella virus than others. Therefore, it can be used for production and quality control of the vaccine and in research and diagnosis of rubella.

As notifiable diseases, lumpy skin disease (LSD), sheep pox (SPP), and goat pox (GTP) are associated with a profound effect on cattle, sheep, and goat farming industries. Development of the ELISA method could effectively facilitate serodiagnosis of the infected animals. This study aimed to develop an ELISA system based on the recombinant full-length and truncated P32 protein (Tr.P32) of goat pox virus. The P32 protein was expressed in Rosetta strain of E. coli using pET24a+ vector and evaluated by SDS-PAGE and Western blotting. Then, Tr.P32 was purified by Ni-NTA affinity chromatography under denaturing conditions and used to develop a capripoxvirus-specific ELISA. Checkerboard titration and receiver-operating characteristic (ROC) analysis were used to optimize the ELISA system and determine diagnostic specificity and sensitivity, respectively. The diagnostic potential of the developed ELISA was evaluated using positive and negative control sera collected from goat, sheep, and cattle. Results showed that the expression level of full-length P32 recombinant protein was negligible, while Tr.P32, a ~ 31 kDa recombinant protein, was expressed up to 0.270-0.300 mg/200 mL of culture media. The results of checkerboard titration revealed that 675 ng/well of Tr.P32 antigen and 1:10 dilution of control sera (anti GTPV HIS and healthy goat sera) caused maximum difference in absorbance between positive and negative goat sera. The recombinant Tr.P32 showed good reactions with antibodies against GTP virus (GTPV), SPP virus (SPPV), and LSD virus (LSDV), whereas no cross-reactions with anti-Orf virus antibodies were detected. By comparing with the neutralization index (NI), cut off, diagnostic sensitivity and specificity of the developed indirect-ELISA were estimated, 0.397, 94% and 96.6%, respectively. These findings indicate that the ELISA system based on Tr.P32 protein could potentially be used in sero-surveillance of all capripoxviruses; however, further investigations are required.

After the emergence of the highly pathogenic avian influenza viruses (HPAIV) subtypes H5N6 in 2013 and H5N8 in 2014, a surveillance study using molecular epidemiology approaches was carried out during 2014 – 2019 in Iran to discover any potential introduction or outbreak of HPAIV in wild bird populations. All sick and dead wild birds found in nature, or in cases of an outbreak, a collection of representative samples was tested using the specific molecular methods for HPAIV H5 subtypes. Additionally, wild bird species in wetlands, several zoos, zoological gardens, or rehabilitation centers were tested for HPAIV. During the active surveillance plan, several individual and outbreak cases of HPAIV and orthoavulaviruses were identified. In general, more than 900 fecal materials, cloacal and oropharyngeal swabs, and/or tissue samples were collected from apparently healthy live birds representing several different species and families. In addition, tissue and swab samples were collected and investigated from any reported wild birds’ mortality cases in different parts of Iran in the framework of this study. No positive bird was found among apparently healthy live birds; however, the highly pathogenic influenza viruses of H5N1, H5N2, H5N6, and H5N8 were found in individual dead birds or mass die-off cases.

Staphylococcus aureus is a well-known commensal and pathogen agent of many wild and domestic animals. A wide variety of infections can be caused by S. aureus, from suppurative skin infections to life-threatening septicemia. This study was conducted to determine the prophage typing and the pattern of antibiotic resistance of S. aureus isolated from broiler poultry before they have been slaughtered. In this study, 200 nasal and cloacal swab samples from 20 different flocks were collected for bacterial isolation. Staphylococci were identified using biochemical and molecular methods before being examined for mecA gene detections in all samples resistant to oxacillin and cefotaxime. The highest value of antibiotic resistance was observed against ciprofloxacin (94%), and the maximum value of susceptibility was to gentamicin (85%). Twenty-eight (27%) samples were resistant to oxacillin. In methicillin-resistance Staphylococcus aureus (MRSA) isolates, 5 prophage types were observed, where the SGB prophage with a frequency of 75% was identified as a dominant prophage; in isolates of S. aureus susceptible to methicillin, 8 prophage types were observed, where SGFa prophage with a frequency about 82% was the dominant prophage. The high prevalence of MRSA isolates can indicate the risk of transmission of these bacteria to the food cycle. Furthermore, existence of various prophages in these isolates can be considered a threat to public health in producing pathogenicity factors in this bacterium while also empowering other bacterial pathogenicity, even other bacterial genera.

Multidrug-resistant (MDR) Salmonella serovars are considered a significant threat to veterinary and public health. Developing new antimicrobial compounds that can treat the infection caused by these notorious pathogens is a big challenge. Bacteriophages can be adsorbed on and inhibit the growth of bacteria, providing optimal and promising alternatives to chemical antimicrobial compounds against foodborne pathogens due to their abundance in nature and high host specificity. The objective of the current study was to isolate and characterize new phages from poultry farms and sewage and to evaluate their efficacy against S. Enteritidis isolates. The study reports three lytic phages designated as ϕSET1, ϕSET2, and ϕSET3 isolated from poultry carcasses and sewage samples in Qalubiya governorate Egypt. The effectiveness of phages was evaluated against multidrug-resistant S. Enteritidis strains. Electron microscopy showed that these phages belong to the Siphoviridae family. Phages were tested against 13 bacterial strains to determine their host range. They could infect four S. Enteritidis and one S. Typhimurium; however, they did not infect other tested bacterial species, indicating their narrow infectivity. The bacteriophage's single-step growth curves revealed a latent period of 20 min for ϕSET1 and 30 min for ϕSET2 and ϕSET3. The isolated Salmonella phages prevented the growth of S. Enteritidis for up to 18 hrs. The findings revealed that Salmonella phages could be used as alternative natural antibacterial compounds to combat infection with MDR S. Enteritidis in the poultry industry and represent a step forward to using large panels of phages for eliminating Salmonella from the food chain.

Bifidobacteriaceae family are gut microbiota that exhibit probiotic or health promoting effects on the host. Several studies have suggested that gut microbiota are quantitatively and qualitatively altered in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). The present study aimed to assess the members of Bifidobacteriaceae family in fecal samples of patients with CKD and ESRD and compare them with non-CKD/ESRD patients to find any changes in their counts and diversions in these patients. Twenty fresh fecal samples from patients with CKD/ESRD and twenty from non-CKD/ESRD patients were examined. Whole DNA was extracted from fecal samples and the gut microbiota composition was analyzed by next generation sequencing (NGS). A total of 651 strains were identified from 40 fecal samples, 8 (1.23%) strains of which were identified as family Bifidobacteriaceae. The most abundant species in both control and disease groups were Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum, and the least abundant species in the disease group was Bifidobacterium animalis subsp. lactis. There was no significant difference in the abundance of various species between the disease and control groups (p < 0.05). This study confirms that the members of the Bifidobacteriaceae family are not altered in patients with CKD/ESRD.

Leishmaniasis causes parasitic infections, especially in developing countries. The disease has not yet been controlled because of the absence of an effective vaccine and low-cost treatment. Achillea santolina essential oil (ASEO) might control the disease as it has antimicrobial properties. This study investigated the in vitro antileishmanial activity of ASEO against Leishmania infantum promastigote using the methylthiazole tetrazolium (MTT) and trypan blue colorimetric methods. The standard strain of L. infantum (MCAN/IR/96/LON49) promastigotes was prepared and cultured in a 96-well Novy-MacNeal-Nicolle (NNN) medium. The effects of different concentrations of saline, ASEO, and glucantime (10, 50, 100, 200, 500, and 1000 mg/mL) were examined in 24-, 48-, and 72-hour intervals using the MTT and trypan blue test methods.The use of ASEO reduced viability in all concentrations compared to the control group in times of 48 (p<0.05) and 72 h (p<0.05). Treatment with glucantime and ASEO had similar efficiency with the concentration of 1000 mL/mg in both methods after 72 h. The results showed that viability was significantly lower in the ASEO group with increases in time using both methods (p<0.05). Cohen’s Kappa coefficient showed a significant agreement between the obtained results for the two methods (Kappa=0.856; p<0.001).In sum, the results showed in vitro antileishmanial activity of ASEO, but more clinical studies are needed to confirm the efficiency. ASEO can be used as an agent and/or in combination with synthetic agents for the treatment of leishmaniasis disease.

This study purposed to discover the connection between the central glutamatergic and histaminergic systems on feeding behavior in layer chickens. In the first experiment, chicks obtained intracerebroventricular (ICV) injections of saline (control solution), α-FMH (250 nmol), glutamate (300 nmol), and α-FMH + glutamate. Experiments 2-6 were comparable to the first experiment, apart from the birds being injected with chlorpheniramine (histamine H1 receptor antagonist, 300 nmol), famotidine (histamine H2 receptor antagonist, 82 nmol), and thioperamide (histamine H3 receptor antagonist, 300 nmol) instead of α-FMH. In Experiment five, experimental groups were divided into (A) control solution, (B) MK-801 (N-methyl-D-aspartate receptor antagonist, 15 nmol), (C) histamine (300 nmol) and (D) MK-801 + histamine. Experiments 6-10 and Experiment five were similar apart from the ICV injections of CNQX (AMPA receptor antagonist, 360 nm), UBP-302 (Kainate receptor antagonist, 390 nm), AIDA (mGluR1 antagonist, 2 nmol), LY341495 (mGluR2 antagonist, 150 nmol), and UBP1112 (mGluR3 antagonist, 2 nmol) given instead of MK-801. Afterward, cumulative food intake was recorded at30, 60, and 120 minutes after the injection process. According to the results, ICV injection of glutamate considerably reduced food intake (p<0.05). Co-injection of α-FMH + glutamate and/or chlorpheniramine + glutamate reduced the hypophagic influence of glutamate (p<0.05), whereas thioperamide + glutamate augmented glutamate-induced hypophagia in neonatal chicks (p<0.05). Co-injection of MK-801 + histamine or UBP-302 + histamine reduced the hypophagic influence of the histamine (p<0.05), whereas LY341495 + histamine augmented the hypophagic influence of the histamine (p<0.05). Given the results, it is suggested that the effect of the connection between these systems on the process of food intake regulation is mediated by H1 and H3 histamines as well as NMDA, Kainate, and mGluR2 glutamate receptors in neonatal layer chickens.

The current study evaluated the effects of cryopreservation medium supplementation with folic acid as an antioxidant on post-thawed semen quality in bulk. Semen samples were collected from four proved Iranian Mahabadi bulls and diluted in extender containing 1.5% soybean lecithin. The diluted semen was assigned into six parts and supplemented with different doses of folic acid as follows: FA0 (extender without folic acid), FA0.05, FA0.1, FA0.2, FA0.4, and FA0.8 (extenders containing 0.05, 0.1, 0.2, 0.4, and 0.8 mM folic acid, respectively). Then, the semen samples were cryopreserved in liquid nitrogen. Sperm motility and velocity parameters, membrane integrity, abnormal morphology, viability, and lipid peroxidation were evaluated after thawing. In the results, FA0.05 presented higher (p≤0.05) total motility, progressive motility, membrane integrity, and viability and lower lipid peroxidation compared to other groups. Abnormal morphology was not affected (p>0.05) by treatments. In conclusion, supplementation of cryopreservation medium with 0.05 mM folic acid is a helpful method to conserve the quality of post-thawed semen in bulk.

Quinolone antimicrobials are widely used in clinical medicine due to their wide spectrum with high tissue penetration and ease of use; but increasing resistance with clinical use appears to be common in some bacterial pathogens, including Escherichia coli (E.coli). The aim of this study was to investigate plasmid-mediated quinolone resistance determinants (PMQR) including, qnrA, qnrB, and qnrS as the emerging mechanisms of quinolone resistance of E.coli isolates from different clinical sites in Karbala province, Iraq. A total of 200 clinical samples were collected from patients suffering from infections such as UTI, gastro enteritis (diarrhea), vaginitis, and wound infections; 30 samples were diagnosed as E.coli clinical strain from both sexes and different ages after identification by biochemical test, VITEK-2 compact system, and by molecular method using 16Sr DNA marker. Antimicrobial susceptibility and minimal inhibition concentration (MIC) testing for nalidixic acid, norfloxacin, ciprofloxacin, levofloxacin, and gatifloxacin was performed using the broth microdilution method. All strains were screened for PMQR genes qnrA, qnrB, and qnrS by the PCR method after DNA extraction from tested clinical isolates of E.coli. The results showed that E. coli is largely isolated from vaginal (40%) and urine (32%) samples, followed by wound infections (24%) and stools (21%).The high occurrence rate of E. coli(33.33%) isolates was observed in participants aged 31-45 years, while a lower occurrence (10%)was recorded in a group of ˃ 60-year-old female participants. Females have a notably increased frequency of E.coli compared to males, with the female to male ratio being 87%:13%. Molecular investigation showed the total percentage of E.coli isolates harboring qnr genes to be 21/30 (70%); this figure is composed of 14/30 isolates harboring qnr in combined or mixed form (46.66%) and 7/30 (23.33%) isolates harboring qnr in single form (3 isolates harboring qnrA alone, 1 isolate harboring qnrB alone, 3 isolates harboring qnrS alone).The prevalence rates of qnrA, qnrB, and qnrS were 40%, 43.33%, and 53.33%, respectively. The results also showed that among E.coli isolates encoding qnr genes A, B, and S, 24%, 12%, and 36% were resistant to nalidixic acid, respectively. Among those isolates carrying qnrA, qnrB, and qnrS genes, 15.8%, 5.3%, and 26.3%, respectively, were resistant to ciprofloxacin. Moreover, Norfloxacin resistance was seen in 20.0%, 5.0%, and 30.0% of E.coli isolates harboring qnr A, B, and S genes, respectively. Levofloxacin resistance was seen in 37.5%, 75.0%, and 37.5% of the isolates carrying the qnrA, qnrB, and qnrS genes, respectively. The lowest resistance rates of qnrA, B, and S-positive E.coli strains were against gatifloxacin (0,0, and 25%, respectively).A high prevalence of qnr genes enhances the increasing resistance rate of E.coli against the quinolone antibiotic under study.

Amphotericin B (AmB) is an effective antifungal agent; however, the application of AmB is associated with a number of drawbacks. Application of nanoparticles (NPs) is known to improve the efficiency of drug delivery to the target tissues, compared to the traditional methods. In this study, a novel method of NPs preparation was developed. The trimethyl chitosan (TMC) was synthesized using low molecular weight chitosan and was used for the preparation of TMC-NPs through ionic gelation method. Afterward, AmB-loaded TMC-NPs (TMC-NPs/AmB) were prepared and their drug delivery potential was testes. The TMC-NPs and TMC-NPs/AmB were characterized for their structure, particle size, Zeta potential, polydispersity index, morphology, loading efficiency, loading capacity, in vitro release profile, release kinetic, and entrapped AmB potency. The cytotoxicity and antifungal activity of TMC-NPs/AmB against Candida albicans biofilm were evaluated. The quaternization of TMC was estimated to be 36.4%. The mean particle size of TMC-NPs and TMC NPs/AmB were 210±15 and 365±10 nm, respectively, with a PDI of 0.30 and 0.4, ZP of +34±0.5 and +28±0.5 mV, respectively. Electron microscopy analysis indicated uniform spherical shapes with smooth surfaces. The TMC-NPs/AmB indicated LE of 76% and LC of 74.04 % with a potency of 110%. The release profile of TMC-NPs/AmB was best explained by the Higuchi model. The initial release after 10 h was obtained at 38%, and the rates of release after 36 and 84 h were determined at 67% and 76% respectively, which was significantly different (P<0.05) from previous time points. The minimum inhibitory concentration (MIC) (50%) of NPs/AmB and AmB were 0.65 and 1.75 μg/mL, and the MIC 80% were determined at 1.95 and 7.75 μg/mL, respectively, demonstrating a significant improvement in antifungal activity. The half-maximal inhibitory concentration for TMC-NPs/AmB and AmB were estimated at 86 and 105 μg/mL, respectively, indicating a significant reduction in cytotoxicity and the adverse effect. This study could successfully introduce a practical method to synthesize TMC-NPs. The encapsulation process was efficient and significantly improved the antifungal activity of AmB. The developed method can be applied to improve the feasibility of oral delivery while reducing the adverse effects associated with traditional methods.

Scorpions are among the most medically important arthropods in Iran, particularly northwestern areas. To date, five scorpion species, i.e. Mesobuthus eupeus, Mesobuthus caucasicus, Androctonus crassicauda, Hottentotta saulcyi, and scorpio maurus, have been identified. The family Buthidae is responsible for most cases of scorpionism in Iran. The Mesobuthus eupeus species belong to this family and is commonly distributed from Turkey to China, including Iran. Among these species, Mesobuthus eupeus is regarded as the most medically important species and responsible for most cases of envenomation in this area. Morphological differences between some species collected in the study area have been reported. The present study, thus, aimed to identify the subspecies of Mesobuthus eupeus in northwestern Iran. Scorpions were captured in the summer months from 37 localities in three northwestern provinces in Iran: West Azerbaijan, East Azerbaijan, and Ardabil. Scorpion collection was carried out using rock rolling and ultraviolet methods. A total of 376 specimens of Mesobutus eupeus (177males and 199 females) were collected and identified as Mesobuthus eupeus (98.4%) and Mesobuthus eupeus philippovitschi (1.6%). Owing to the findings of our study, M.e.philippovitschi has been added to the scorpion fauna of northwestern parts of Iran for the first time. Unlike M.e. eupeus which is widely distributed from plains to mountainous regions, M.e.philippovitschi has limited distribution and is found mostly along the borders with neighboring countries. This subspecies is the most medically important and most prevalent one in the region. The findings of the present study also provide the basis for future consideration of regional antivenom production for this medically important species.

Echis carinatus (E. carinatus) is known for its hematological and nephrotoxic properties in the envenomed patients. Based on the limited data upon the cardiovascular changes associated with this dangerous venomous snake in Iran, the current study purposed to evaluate the venom-induced hemodynamic manifestations in rats. Venom (120 µg/kg) was administered intravenously within one minute through the left femoral vein, and the hemodynamic parameters were continuously recorded using a pressure transducer (MLT844, ADInstruments, Australia). The venom caused prominent hypotension leading to death a few minutes after a transient uprise in blood pressure. It also induced a decrease in heart and pulmonary rates, yet it had no arrhythmogenic properties. Additionally, pre-treatment with the pepsin-derived Iranian polyvalent antivenom (30 µl/Kg) completely neutralized the hemodynamic responses but had no effect when instilled two minutes after venom injection. Heparin (300 IU/kg) and epinephrine (1.5 µg/kg) prevented dramatic hypotension when used 10 minutes before venom instillation; however, atropine (1 mg/kg), dexamethasone (1 mg/kg), and ketorolac (10 mg/ml) had no effects. All treated rats were killed post-injection. Histologically, the lung was the most vulnerable organ with mononuclear infiltration, microcystic formation, and significant capillary congestion. Prominent renal pathological deterioration also occurred, including mesangial cell infiltration and diffuse bleeding, leading to acute tubular necrosis. Modest portal inflammation and vascular congestion were observed in the hepatic tissue of the envenomed rats. The crude venom of Iranian Echis carinatus caused hypotension leading to bradycardia, a decrease in pulmonary rate, and death without significant histological changes to the heart.

Conventional cancer treatments are costly and have different serious side effects for patients. Natural herbal treatments are widely accepted among people because of their minimal side effects, although there is little scientific knowledge about them. One of these remedies utilizes the root of Biebersteinia multifidi that has been used for years in Iran to treat different chronic genital diseases. The current study examined the effects of methanolic and ethanolic extracts of B. multifida (induction of necrosis and apoptosis) on breast cancer (MCF-7), ovarian cancer (A2780), and human cervix cancer (HeLa) cell lines in comparison with normal breast cells. These effects were determined to be morphological alterations in cell light microscopy, by flow cytometry (staining with annexin V and propidium iodide), and by measuring live cells and inhibition concentrations by MTT assay. IC50 of B. multifida on the MCF-7 cell line (methanolic extract) was 400 µg/ml and for A2780 was 250 µg/ml. The IC50 amount of B. multifida on the MCF-7 cell line (ethanolic extract) was 750 µg/ml and 1500 for A2780. Results demonstrated that apoptosis and necrosis occurred in MCF-7 and A2780 following the addition of ethanolic and methanolic extracts of B. multifida to the medium. These findings confirmed the anti-cancer effects of mehthanolic extracts of Biebersteinia multifida root and its safety for normal cells; thus, it can be applied in cancer therapy as a novel medication.

The current study was conducted to evaluate the effect of dietary fish oil on the semen quality and fertility potential of Zandi rams. For this purpose, a total of 15 Iranian Zandi rams were randomly assigned into three equal groups. The first group was a negative control and were fed without oil supplement. The second group was a positive control, and their diet contained palm oil, and the last group had a diet containing fish oil. All the diets were isocaloric and isonitrogenous. The rams were fed for 70 days, and the semen smaples were collected every 10 days. In experiment I, the evaluated parameters included semen volume, sperm concentration, motility, membrane integrity, and viability. In experiment II, 210 Iranian Zandi rams received CIDR for 12 days and 400 IU of equine chorionic gonadotropin at the time of CIDR removal. Then, they were assigned into three equal groups and artificially inseminated with semen samples. According to the obtained results, the supplementation of ram diet with fish oil as a source of omega-3 fatty acids improved the semen volume, sperm concentration, total motility, progressive motility, viability, membrane integrity, pregnancy rate, parturition rate, and lambing rate of the rams (P≤0.05). In conclusion, fish oil as a dietary supplement for rams could be an effective strategy to improve the semen quality of rams for artificial insemination and other goals.

For the first time ever, this research studied the response of stroke volume in animals to various modes of motor activity after a traumatic brain injury. The study showed a pronounced decrease in stroke volume (SV) the first day after modeling an open craniocerebral injury in rats of all age groups. At the same time, the smallest SV response to brain injury was observed in immature animals. After modeling a traumatic brain injury, it was found that the implementation of systematic dynamic exercises by animals of pre-senile agedoes not contribute to increase SV; a significant increase in SV after a traumatic brain injury is observed only in immature animals. It was revealed that limiting physical activity and the performance of isometric exercises after a traumatic brain injury restrain a natural increase in SV in immature rat pups and decrease SV in sexually mature and pre-senile animals.

Obesity triggers the development of adipokines such as leptin, resistin, and visfatin, which have been associated with the development of diabetic nephropathy and other vascular disorders. The main purpose of the current investigation was to identify the physiological impact of visfatin on immunological response and its inflammatory effects on nephropathy. Fifty Iraqi patients with chronic kidney disease (CKD) at various stages, as described by the National Kidney Foundation (NKF) and ranging in age from 48.367.56 to 53.68 8.46 years on average were considered. Prior to the start of the investigation, informed consent was obtained from all participants, and the ethics committee approved the study. Patients were classified into two groups: Group (A) comprised patients with a GFR higher than 60 mL/minute, and Group (B) comprised patients with a GFR of less than 60 mL/min. There was no considerable variance between the groups as regards visfatin, but a highly significant correlation between serum visfatin and CRP was observed. The results of the current investigation indicated that serum visfatin levels are significantly correlated with CRP in CKD patients; it is also correlated with deterioration of kidney function. Moreover, higher visfatin levels were accompanied by increased serum triglyceride and cholesterol levels. These findings would suggest that visfatin may perform an essential function in uremia-related inflammation and may serve as a potential target for treatment and prevention of renal associated complications. Future studies may delineate whether visfatin is a marker of disease activity and severity as well as a predictor of outcome in CKD.

Testes have several primary functions, such as male gametes production (spermatozoa) and secretion of several endocrine factors, including the production of steroid and protein hormones which facilitate elements in the healthy reproductive function of mammals. The potential of an animal functional endocrine reservoir could be an interesting finding in animal reproduction management. Therefore, the current study aimed to analyze the functional endocrine reserves of testes in 12- and 18-month-old boars of four different boar breeds, namely Large White, Landrace, Duroc, and Tempo, as the animal model in this research (n=10). To determine the functional endocrine reserves of the testes at 12 and 18 months of age, boars received injections of  human chorionic gonadotropin (hCG) 3 times every 72 hours. Testosterone was measured in blood samples (10 mL) taken from the jugular vein before the administration of hCG as well as 2, 12, 24, 48, and 72 hours after administration of each of three hCG injections. The index of endocrine activity of the testes was determined by the formula: Ita = Т1 –Т0 / Т0, where Ita is the index of endocrine activity of the testes, T0 is the testosterone concentration before the administration of hCG, and T1 is the maximum testosterone concentration after the third administration of hCG. The effects of the hCG injection on boar testes at different age periods indicated that the testes of Large White and Duroc boars have higher potential endocrine reserves compared with the Landrace and Tempo breeds.

In China, Japan, and Korea, Panax ginseng has been used in traditional medicine for thousands of years. Panax is a plant used as a general tonic or adaptogen for chronically ill patients. The current study evaluated the cytotoxicity of Panax ginseng extract (PGE). Different cell lines (HCT-116, LNCaP, and normal cell line VERO) were treated with different inhibitory agentsat different concentrations (1000, 500, 250, 125, 62.5, and 31.25 µg/ml) as follows: G1 (Methanol Panax ginseng extract, PGE), G2 (Doxorubicin, DOX), and G3 (Methanol Panax ginseng extract +DOX, PDD). Each inhibitory agent group was used to treat the cancerous cell lines HCT-116, LNCaP, and normal cell line (VERO) to obtain IC50% by MTT assay. The inhibitory ability of the 1000 μg/ml PGE was significantly increased in all the three-cell lines compared with other concentrations. The recorded data revealed that the inhibition ability of PGE and Doxorubicin towards the HCT-116 cell line significantly increased compared with the other cell lines. The interaction between different PGE concentrations and cell lines showed that the 1000 μg/ml PEG had the highest inhibitory effects on HCT-116 compared with other combinations. The interaction between different DOX concentrations and different types of cell lines showed that the 1000 μg/ml DOX had the highest inhibitory effects on LNCap compared with other combinations. The PGD inhibition ability reflected a significantly higher difference toward the HCT-116 cell line as compared with other cell lines. IC50% is the concentrations (µg/ml) to kill 50% of cell line. It was calculated by MTT assay for three cell lines: HCT-116, LNCaP, and VERO. The rate of effectiveness of the inhibitory factors (PGE, DOX, and PGD) showed highly significant differences toward the cell line HCT-116 compared to the other cell lines. This indicates the safety of the PGE compound and its low toxicity toward normal cells, quite the opposite of cancer cells as compared to the common drug DOX and combined PGD (PGE+DOX). PGD combined with DOX (PGE + DOX) showed antagonistic results toward the HCT116, LNCaP, and VERO cell lines, while UDE combined with DOX (UDE+DOX) showed synergistic activity.

Kidneys comprise the paired organ essentially responsible for excreting nitrogenous wastes, excessive water, inorganic salts, and toxic substances produced during the process of body metabolism. The maintenance of osmotic regulation and homeostatic fluid balance of the body is also performed by the kidneys. The current study investigated the histological features in quail birds of both genders at different age stages. A total of thirty-six adult male and female Japanese quail were allocated randomly into four different age groups of 30, 90, 180, and 270 days. They were fed high-protein food and water for two weeks, after which the kidneys of the quail were obtained and histological changes between males and females were evaluated. Data analysis was performed using SPSS-22. The results showed a 24% increase in Bowman's space in male quail compared with females after 30 days. Moreover, a 10% increase in Bowman’s space was recorded at 270 days in male quail compared with females. The results showed a 12% increase in glomerular diameter in females compared with males. The data also showed a 12% increase in the diameter of Bowman’s capsule in females compared to males. The outer diameter of the thin tubule in the loop of Henle in females increased by 4% compared to males. A 12%increase was noted in the outer diameter of the thickened tubule and the collecting tubes in male quail compared to the females at 30 days of age. Increases in the outer diameter of the proximal tubule of 6%, 16%, and 2% in female quail compared to males were recorded at 30, 180, and 270 days, respectively. Finally, the outer diameter of the distal tubule in males increased by 4% compared to females at 30 days and by 2% at 270 days. The current study described in detail the effect of a high-protein diet on the histology of different genders in quail kidney.

Improving the genetic potential of animals using genomic technologies is an effective way to solve problems in domestic breeding of dairy breeds. The aim of the current study is to analyze the genetic diversity of Holstein cattle bred in the Tyumen Region using microsatellite markers. The blood samples of 241 Holstein cow were collected for genomic analysis. Genetic identification was performed using COrDIS kits. The resulting data indicated the presence of genetic diversity in the studied sample of dairy cattle bred at the Tyumen Region for a number of indicators. The number of alleles at 15 microsatellite loci in the tested breeds varied from 5 to 14. Eighteen unique allelic variants can be breed-specific markers for genetic identification of dairy cattle. The recorded data showed a high level of animal genetic variability. A high level of polymorphism (Ае ³3.32) was detected in 47% of the loci under research. High values of the observed heterozygosity up to 0.91 at the Eth3 locus and of the polymorphism up to 0.77 at the Tgla53 locus were detected. In the results, 7% erroneous entries in the progeny pedigree was detected. The results of the current study contribute to the knowledge of genetic diversity of dairy cattle in the Tyumen region, Russia, and also paved the way to seek new relationships between allelic variants of microsatellite loci with the productive traits of animals.

Clostridium perfringens is implicated in the etiology of some diseases including fatal enterotoxaemia. Determining dominant toxin types of this microorganism can be helpful in epidemiologic surveys and the formulation of more proper vaccines. To understand the pathogenicity of this bacterium, it seems necessary to describe the toxin and virulence genes content of strains involved in enterotoxaemia and other associated diseases. The current study aimed to isolate and type the toxins of C. perfringens in sheep with suspected enterotoxaemia in Fars province by culture-PCR and ELISA methods and to compare them to isolates of a healthy group. Samples of intestinal contents were collected from enterotoxaemia cases and a healthy group of sheep. The presence of alpha, beta, and epsilon toxins were evaluated by ELISA method. After culture and isolation of C. perfringens, toxin typing and screening of isolates for the presence of beta-2 and enterotoxin were performed by PCR method. C. perfringens was isolated from 102 of 167 suspected enterotoxaemia cases of sheep and from 22 of 50 healthy sheep. The PCR results showed that type A was the most prevalent toxin type in both groups, but according to ELISA type D was the dominant toxin type in the clinical group. The enterotoxin gene was detected in 10% of all isolates from healthy and suspected group isolates of types A and D. The beta-2 gene was identified in 35% and 63.6% of enterotoxaemia-associated isolates and isolates not associated with disease, respectively. In conclusion, Type D of C. perfringens was the dominant causative organism of fatal enterotoxaemia in sheep in Fars province.

An outbreak of possible acute poisoning by Sinapis arvensis occurred in a flock of 50 fat-tailed sheep located in the Semnan province of Iran. Sinapis arvensis is an annual or winter annual plant of the genus Sinapis in the family Brassicaceae, commonly known as field mustard, wild mustard or charlock. The poisonous constituents are volatile oil of mustard, the alkaloid sinapin, and the alkaloidal glucoside sinalbin. The flock was grazing in land containing high amounts of wild mustard (Sinapis arvensis) in late spring. Seven sheep (aged between 1 and 5 years) died within approximately 3 days. The affected animals displayed signs of depression, reluctance to move, tachycardia, tachypnea, mucoid and hemorrhagic nasal discharges, pale conjunctiva, ataxia, abdominal pain, bruxism, and anorexia. Rectal temperature in these animals was normal to high (39-41.5 °C). Ruminal movements were reduced (1-2/min). Serum biochemical levels in affected sheep showed marked increases of blood urea nitrogen (BUN), Gamma glutamyl transferase (GGT), magnesium (Mg), and phosphorous (P) and a marked decrease in calcium (Ca). In urinalysis, marked hemoglobinuria and proteinuria were observed. Necropsy findings included congestion in lungs and hemorrhage on the epicardial and endocardial heart surfaces, on the surface and medulla of the kidneys, and abomasal mucosa. The liver was also congested with a nutmeg pattern. Rumen contents included digested materials and large quantities of seeds and stems of wild mustard. For the first time, our findings confirmed wild mustard toxicosis in sheep in Iran.