mohammadmehdi ranjbar

The aim of this study was to using purified chicken antibody (IgY) for developing solid phase competitive (SPC) enzyme-linked immunosorbent assay (ELISA) to detect the foot-and-mouth disease virus (FMDV) A serotype. After immunization of chickens, polyclonal immunoglobulin (IgY) antibodies were extracted and purified from egg yolk and yield was about 5.00 mg mL-1 of yolk as well as near 0.40 mg mL-1 of specific IgY antibody against FMDV serotype A. Also, optimized sucrose density gradient method produced 228 µg mL-1 whole virus which is much higher than that of the conventional method of sucrose density gradient method. The optimum concentration of purified capture IgY and bind type A antigen were 0.50 µg and 0.10 µg per well, respectively. The OD values < 0.70 were considered positive, and values ≥ 0.70 were negative for in-house kit base on standard controls. Statistical analysis base on 80 serum samples showed the 96.66% sensitivity, 100% specificity, 100% positive predictive value, 90.90% negative predictive value, 97.50% accuracy, and 98.33% reliability for serum samples for two commercial and in-house kits. The SPCE developed based on IgY antibody is a suitable alternative for the detection of antibodies after vaccination against type A FMDV with high sensitivity and specificity. The present research demonstrated the possibility of commercial development of the SPCE kit using IgY antibodies for the detection of FMDV antibodies in serum samples with adequate sensitivity and accuracy.

Virus of caprine arthritis encephalitis belongs to the Retrovirdae family, which can cause arthritis, mastitis and abortion in adult goats, and encephalitis in kids especially in dairy breeds. Therfore, income of farmers are affected .since, in the recent years many importation of small ruminant animals especially dairy goats breeds to our country have been done . This study was conducted to evaluate infection of CAEV in imported and indigenous dairy goat breeds which were kept together in one herd.

In this study, blood samples were taken from 249 dairy goats in one of the industrial breeding farm. Buffy coat was separated from the samples using centrifugation and DNA was extracted from them. Nested polymerase chain reaction (Nested PCR) method was performed with specific primers in order to detect the gag gene.

The gag gene was detected in21 blood samples (8.3%) out of 249  samples and result of CAEV  Infection rate was 11 (13.8%), 7 (8.1%) and 3 (3.6%) for Alpine, Saanen and Indigenous breeds, respectively. Percent of infection based on old was 15.8%, in < two years goats, 8% in 2 3 years and 7.7% > three years old.

Unfortunately, the imported and local breed of dairy goats were infected with CAEV, therefore, phylogeny studies are required to for identification of prevalence of the disease in herds of the country.

 Despite the success of "direct-acting antivirals" in treating Hepatitis C Virus (HCV) infection, invention of a preventive HCV vaccine is crucial for global elimination of the virus. Recent data indicated the importance of the induction of Pan-genomic neutralizing Antibodies (PnAbs) against heterogenic HCV Envelope 2(E2), the cellular receptor binding antigen, by any HCV vaccine candidate. To overcome HCVE2 heterogeneity, "generation of consensus HCVE2 sequences" is proposed. However, Consensus Sequence (CS) generating algorithms such as "Threshold" and "Majority" have certain limitations including "Threshold-rigidity" which leads to induction of undefined residues and insensitivity of the "Majority" towards the "evolutionary cost of residual substitutions".

 Herein, first a modification to the "Majority" algorithm was introduced by incorporating BLOSUM matrices. Secondly, the HCVE2 sequences generated by the "Fitness" algorithm (using 1698 sequences from genotypes 1, 2, and 3) was compared with those generated by the "Majority" and "Threshold" algorithms using several in silico tools.

 Results indicated that only "Fitness" provided completely defined, gapless HCVE2s for all genotypes/subtypes, while considered the evolutionary cost of amino acid replacements (main "Majority/Threshold" limitations) by substitution of several residues within the generated consensuses. Moreover, "Fitness-generated HCVE2 CSs" were superior for antigenic/immunogenic characteristics as an antigen, while their positions within the phylogenetic trees were still preserved.

 "Fitness" algorithm is capable of generating superior/optimum HCVE2 CSs for inclusion in a pan-genomic HCV vaccine and can be similarly used in CS generation for other highly variable antigens from other heterogenic pathogens.

Foot-and-mouth disease is a highly contagious and sometimes fatal viral disease affecting livestock. Like other viruses, this virus continuously evolves and mutates, posing a significant challenge to vaccination due to the vast diversity of serotypes. There is no cross-protection among the serotypes, and two strains within a specific serotype may have nucleotide sequences that differ by up to 30% for a particular gene. This indicates that foot-and-mouth disease vaccines must be highly specific to the strain involved. Given the importance of the foot-and-mouth disease vaccine and its role in health and food security, using new peptide candidates for immunization could be beneficial.

The aim of this study was to investigate the immunogenic effects of specific peptides produced against the FMD virus.

After conducting bioinformatics studies, two sequences of 14- and 20-amino acid peptides from the peptide sequences of the immunogenic regions of the VP1 protein of foot-and-mouth virus O2016 were designed and synthesized. The peptide formulation (conjugated to BSA) was then prepared with aluminum phosphate adjuvant, and injections were administered to groups of nine rabbits at two-week intervals for a total of five times. Subsequently, the amount of antibody production was investigated and analyzed using the indirect peptide ELISA test, comparing it to the negative control samples (non-vaccinated calves) against the peptides attached to the BSA base.

The results showed that both peptides were capable of stimulating the immune system in rabbits, and antibodies against these peptides, as well as against the complete foot-and-mouth disease virus, demonstrated cross-reactivity with each other. Additionally, the highest immunogenicity in the ELISA test was related to the 20 amino acid sequence at a concentration of 300 µg per well in the checkerboard ELISA and 400 µg per dose injected into the rabbits.

These two peptides, with further research, could serve as suitable candidates for evaluating the immune response against foot-and-mouth disease virus in serology tests, examining vaccinated and non-vaccinated animals, and conducting immunization studies.

Traditionally, diphtheria-tetanus-pertussis (DTwP or DTaP) as pediatric vaccines are produced from the corresponding inactivated toxins or whole cell pathogenic bacteria of Corynebacterium diphtheria toxin (Tox), Clostridium tetani toxin (TetX) and Bordetella pertussis. There are major concerns in the classic or acellular DTP (DTaP) vaccine production processes from native live sources of bacteria as it may raise concerns on adverse effects and safety issues, complexity of the purifications for each agent as well as costs. Here, we designated recombinant multi-epitope candidate vaccine by vaccino-informatics study to address mentioned issues and to develop a single trivalent fusion protein as potent recombinant DTP vaccine. To follow these goals, stages of immune-bioinformatics were retrieved by means of proteinaceous toxins sequences, predicting secondary/tertiary structure and transmembrane topology, energy minimization and models validation. Then, conformational and linear B cell epitope prediction by several different servers, mapping consensus linear/discontinuous immunogenic regions and constructing synthetic fusion vaccine candidate in respect to optimal immunogenic, physico-chemical properties and highly expression in prokaryote host were achieved. Finally, reverse translation, codon optimization, addition of cloning tags for pet 28a vector and optimizations of physico-chemical characteristics of synthetic trivalent fusion protein were performed. By different hybrid immune-informatics and structural bioinformatics analysis predicted and experimental epitopes finally, 12 new consensus highly immunogenic linear and discontinuous epitopes in Tox, TetX and PTXa proteins were selected. Peptide sequence of these immunogenic regions were as follow; PTXA (AA34-64, AA184-256 and AA98-116), Tox (AA47-76, AA117-159, AA515-557 and AA245-265) and TetX (AA226-249, AA819-844, AA923-967, AA1009-1067 and AA1225-1315). Moreover, the characteristics of recombinant trivalent fusion construct were; 546 residue length, solvable (Grand average of hydropathicity (GRAVY) was -0.475), estimated half-life was >10 hours in Escherichia coli, pI 5.94 (a little acidic), stable protein (The instability index (II) 35.58) as well as thermal stable (Aliphatic index (AI) 71.67). The putative antigenic epitopes from different organisms in a single protein, as in the current study, possibly will improve the protective effectiveness as novel potent, safe, cheap and broad-spectrum vaccines for better prevention of diphtheria, tetanus and pertussis infections in future.

Foot-and-mouth disease virus (FMDV) causes severe infection in livestock and makes considerable economic impacts. Therefore, a rapid, highly specific, and accurate method for the diagnosis of FMDV infections is required to ensure that appropriate treatment is administered to reduce economic losses. In this study, the diagnostic tests for FMDV detection by enzyme-linked immunosorbent (Ag-ELISA), reverse transcription–PCR (RT-PCR), and real-time RT-PCR (rRT-PCR) were carried out as the World Organization for Animal Health recommended and were based on the VP1 gene. Positive samples were detected by RT-PCR and rRT-PCR (73.16%) and by ELISA test (55.99%). According to the information obtained from the present study molecular methods provide much more reliable and definitive results than Immune assay methods.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the ELISA. Diagnostic tests with high sensitivity is necessary both to distinguish infected vaccinated animals and disease control programs for identifying the carrier animals. To detection of FMD virus, the current strategies are mainly based on capture antibody (sandwich) ELISA test. Applying laying pullets as animal bioreactor for production specific egg yolk antibody (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. In the present study, we aimed to produce a concentrated and purifies IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized by inactivated FMDV serotype virus and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected and then, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using polyethylene glycol 6000–ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography and dialyzed. The purified IgY concentration estimated by Bradford assay, confirmed it presents by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving optimum concentration of antigen/antibody (sera) in sandwich ELISA, checkerboard titration test was setup base on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both Real time PCR (quantitative PCR, qPCR) and with a commercial ELISA kit, used for evaluation of sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit was 100% and 98%, respectively. According to this, the present developed capture ELISA kit based on IgY has high sensitivity and specificity for FMD virus detection and it could be used in future as both commercial detecting and serotyping applications.

Cryptosporidiosis is a parasitic disease caused by the cryptosporidium protozoan in vertebrate animals. In livestock, especially ruminants, infants develop diarrhea syndromes. The infection is widespread throughout the world, as well as in Iran, which is reported from various species. Morphological diagnosis of Cryptosporidium species has many limitations and is of no taxonomic value alone, therefore using molecular methods helps remove these limitations to some extent. The current study is aimed at microscopic, molecular, and antigen detection and isolation of Cryptosporidium Parvum parasite. Firstly, there were 300 specimens collected from different parts of Iran. Next, purification of oocysts from feces was carried out according to the method of Casemore et al., through the flotation technique following which staining was done by means of the modified Ziehl-Neelsen method (Henriksen method), and identification through diagnostic keys. ELISA test was also performed on the specimens for which the results were from 1 positive to 4 positive. Results of our study demonstrated that from the 300 cases tested for Cryptosporidium through the molecular method, 48 cases (16%) were positive. These cases were collected during summer, autumn, and winter, however, more than 50% of positive cases were found among specimens collected in autumn. Furthermore, there were 54 positive cases found by means of PCR test, which indicates 6 cases more than that for ELISA results. Finally, results from PCR detection and ELISA, were subjected to Chi-Square analysis, where no significant difference between the collected data was observed (p=.0587).

Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats.

Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software.

The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification.

Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Cervical cancer is the leading cause of morbidity and cancer mortality in women throughout the world and persistent infection with human papillomavirus (HPV) is the main etiology for the development of this cancer. Peptide vaccines derived from E6 and E7 oncogenes are promising candidates for HPV vaccine production due to their safety, high stability, and ease of production. In the current study, expression strains such as Escherichia coli BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS were applied for poly epitopic E6 expression and the effects of temperature, induction time, and inducer concentration on the expression level of recombinant HPV16-E6 polypeptide protein were examined in the mentioned strains.

In the present study, the optimized gene of E6 recombinant protein was cloned into pET28a vector under the control of T7 promoter and the resulting plasmid was successfully transformed into Escherichia coli strains BL21 (DE3), BL21 (DE3)-AI, Rosetta, C41 (DE3), and BL21 (DE3)-pLysS. Then, the ability of these strains to express the desired recombinant protein in different conditions was compared.  Two temperatures of 25 ° C and 37 ° C, IPTG concentrations between 0.1 and 1.0 mM, and different incubation times were selected to examine the expression level of recombinant E6 protein in these strains.

The highest level of expression was obtained in Escherichia coli BL21 (DE3) -AI strain inducing at Optical Density (OD) of 0.9, 0.1 mM IPTG, and 16 hours induction at 25 °C.

The results of this study can be used to produce E6 protein as a vaccine candidate in the treatment of HPV-related cancers.

Cervical cancer is one of the leading causes of death worldwide, causing approximately 500,000 new cases and 250,000 cancer deaths each year. Persistent infection with high-risk human papillomaviruses (HPVs), particularly type 16, is the primary cause of cervical cancer development and maintenance among women worldwide. The E6 therapeutic vaccines can induce strong anti-tumor T cell-mediated immune responses, such as cytotoxic T lymphocytes, that play vital roles in current therapeutic vaccine development. In our study, bioinformatics approaches and in silico analyzes, such as protein sequence retrieval, identification of conserved regions, drawing of pedigrees, prediction of T-cell epitopes, calculation of population coverage of predicted epitopes, and molecular docking, were used to predict the major histocompatibility complex (MHC) Class I and Class II T cell epitopes of HPV16 E6. Taking into account the scores from different steps, six CD8+ T cells and three CD4+ epitopes were selected. The fusion of the selected epitopes created a universal potential vaccine with a population coverage of 86.41%. The population coverage was obtained by evaluating the potential of these epitopes to elicit innate and acquired immunity. These theoretically confirmed peptides could be employed in a poly-epitope construct as a candidate vaccine for further analyses. Also, these results provide new insights into therapeutic vaccine development.

Unearthing the immune defects associated genes and genetic variations may lead to locating novel targetable elements and introducing the underlying mechanisms and pathways of the desired condition. The major histocompatibility complex (MHC) genes are essential for protein antigens presentation and inducing immune response as well as are associated with production and phenotypic traits. The MHC class II genes of cattle and buffaloes, Bovine Leukocyte Antigen (BoLA) or Buffalo Leukocyte Antigen (BuLA), are located on the short arm of chromosome 23. It has been demonstrated that the second exon of BoLA-DRB3 (BoLA-DRB3.2) is highly polymorphic, having more than one hundred identified alleles, that each of them can form special binding pockets for corresponding antigenic peptides. Concerning the populations of cows, unique native and hybrid, and buffaloes are distinctly divided into different regions of Iran, analysis and interpretation of the polymorphisms’ expression status of this locus can be implemented to find better breeding strategies like selecting highly resistant animals to infection diseases, in herds. It also can help to develop effective and novel vaccines regarding allele frequencies in populations; different allelic variants of MHC class II binding to different peptides. Immunogenic evaluation of animals’ genome/genes characteristics has always been used as a model for the study of similar genes in humans.

After SARS and MERS outbreaks, COVID-19 is the third coronavirus epidemic that soon turned into a pandemic. This virus causes acute respiratory syndrome in infected people. The mortality rate of SARS-CoV-2 infection will probably rise unless efficient treatments or vaccines are developed. The global funding and medical communities have started performing more than five hundred clinical examinations on a broad spectrum of repurposed drugs to acquire effective treatments. Besides, other novel treatment approaches have also recently emerged, including cellular host-directed therapies. They counteract the unwanted responses of the host immune system that led to the severe pathogenesis of SARS-CoV-2. This brief review focuses on Mesenchymal stem cell (MSC) principles in treating the COVID-19. The US clinical trials database and the world health organization database for clinical trials have reported 82 clinical trials (altogether) exploring the effects of MSCs in COVID-19 treatment. MSCs also had better be tried for treating other pathogens worldwide. MSC treatment may have the potential to end the high mortality rate of COVID-19. Besides, it also limits the long-term inability of survivors.

Calf scours (diarrhea) in unweaned calves play a major role in economic losses of animal farming industry worldwide. The present study was conducted to investigate and interpret the presence of BRV, BVDV, and Escherichia coli K99 by molecular and serological approaches simultaneously.

A total of 73 E. coli-negative diarrheic fecal samples were collected from one-week to less than one-month-old calves of Holstein dairy cattle herds of some provinces of Iran during autumn and winter. The samples were directed to antigen detection by ELISA (Enzyme Linked ImmunoSorbent Assay), RNA extraction by semi-manual approach, and cDNA synthesis for PCR amplification.

Out of 73 calves’ diarrheic  fecal samples, 28 (38.3%) and 1 (1.36%) were positive for BRV and BVDR by ELISA, respectively. However, 31 (42.4%) samples were positive for BRV and non for BVDV by RT-PCR. The Kappa coefficient showed significant differences in BRV and BVDR detection between ELISA and RT-PCR methods. The distribution of the BRV-positive samples among bovine diarrheic calves was 80, 52.6, and 50% in Eslamshahr, Qazvin, and Hamedan, respectively.

ELISA and RT-PCR indicated high prevalence rate of BRV in autumn and winter, respectively. The present study results showed that positive cases detected by RT-PCR were more than those detected by ELISA. Further studies are needed to achieve a comprehensive preventive and therapeutic strategy to address  diarrhea bovine pathogens.

Crimean–Congo hemorrhagic fever (CCHF) is a fatal disease caused by Nairovirus classified within the Bunyaviridae family. The virus is transmitted to humans through the bites of infected ticks or direct contact with viremic animals or humans. The current study aimed to detect the virus genome in ticks from Khorasan Razavi Province.

One hundred hard ticks were collected randomly from 100 sheep in four different areas of the province. Collected ticks were kept alive and identified. All the ticks were analyzed for the presence of CCHF virus genome using reverse transcriptase polymerase chain reactions (RT-PCR).

The identified ticks were belonging to Hyalomma marginatum (16% female and 6% male), Rhipicephalus turanicus (52% female and 25% male), and Dermacentor raskemensis (1%). The CCHF virus genome was found in Hyalomma marginatum (5% male from Taibad and Sabzevar region and 1% female from Taibad). Genetic analysis of the virus genome isolated from two regions (Sabzevar and Taibad) showed 100% identity.

This study indicated that CCHF should be regarded as a risk-borne infection in this province. Therefore, special health management is needed to control this disease.

Humans have always been encountered to big infectious diseases outbreak throughout the history. In December 2019, novel coronavirus (COVID‑19) was first noticed as an agent causing insidious pneumonia in Wuhan, China. COVID‑19 was spread rapidly from Wuhan to the rest of the world. Until late June 2020, it infected more than 10,000,000 people and caused more than 500,000 deaths in almost all of countries in the world, creating a global crisis worse than all previous epidemics and pandemics. In the current review, we gathered and summarized the results of various studies on characteristics, diagnosis, treatment, and prevention of this pandemic crisis.

SARS-CoV-2, as a major threat to human health and economy, has brought in uncertain consequences in the early decade of the 21st century. Since no antiviral therapy or effective vaccine against SARS-CoV-2 is currently available, deciphering the possible mechanisms by which the host responds to the virus seems critical, as it may affect the scientific community around the world toward the development of novel therapeutics. Here, we identified the key regulatory molecules modulating the host response to SARS-CoV-2 that affected the transcriptional profiles of respiratory infections in vitro.

We used the data recently published on the effect of SARS-CoV-2 on two lung cell lines. We selected the shared differentially expressed genes (DEGs) between the two cell lines. To find the key regulatory molecules, we used transcription factors-miRNA-gene interaction databases and analyzed the data using the FANMOD software to detect the crucial regulatory motifs. Cytoscape was then applied to construct the network. We used the KEGG pathway and Gene Ontology (GO) enrichment analysis to predict the probable intermediating biochemical pathways and biological processes.

Our data demonstrated that four triangle-shaped (3edge) feed-forward loop motifs (FFLs) played significant roles, and the integrated FFLs subnetwork was constructed. STAT1, IRF9, IRF7, and PRK12 were the genes shared among them. The most important biological processes relating to the effect of the new virus were linked to response to cytokine, innate immune response, and adaptive immune response. Besides, significantly enriched pathways associated with other different viral infections included the nuclear factor κ-light-chain-enhancer of activated B cells (NF-kappa B) signaling pathway, the nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling pathway, and the Jak-STAT signaling pathway.

Most of the pathways were related to the cytokines storm that may contribute to different levels of lung injury. These regulatory motifs shed light on the transcriptional signature of the respiratory cells and may be responsible for the development of COVID-19 or can also be used as a potential target for further drug therapies or vaccines.

This study was conducted to evaluate prevalence of BVD and the strategies which were applied for prevention of BVD over the past 50 years in some provinces of Iran. Blood samples of 500 Iranian Holstein race were randomly collected from three dairy farms located in Tehran, Isfahan and Qazvin provinces. BVD control protocols of these farms were recorded. ELISA technique was used to measure the antibody and antigen titers for BVD. RT-PCR technique was performed to investigate the presence and the type of virus in all samples. The prevalence of Ab-/ Ag-, Ab+/ Ag-, Ab-/ Ag+ and Ab-/ Ag+ were 10.2%, 78.8%, 7.2% and 3.8%, respectively. Furthermore, approximately 4.2% of the animals were PI and the prevalence of antibody and antigen titers had not significant difference in three provinces. All positive samples were BVDV type 1. Type 2 was not observed in this study. The results of the study indicated that the efficiency of the used protocols to control BVD diseases is not successful. So, to control the BVD, applying large management policies at the national level is fundamental beside the biosecurity strategies.

Toxoplasma gondii is a widely-distributed parasite all over the world whose attribut-ed severe afflicting complications in human necessitate the development of serodiagnostic tests and vaccines for it. Immunological responses to monovalent vaccines and the application of diagnostic reagents including single antigens are not optimally effective. Bioinformatics approaches were used to introduce these epitopes, predict their immunogenicity and preliminarily evaluate their potential as an effective DNA vaccine and for serodiagnostic goals.

A 3D structure of proteins was predicted by I-TASSER server, and linear and conformational B cell and T cell epitopes were predicted using the online servers. Then, the predicted epitopes were constructed and called Toxoeb, and their expression in the prokaryotic and eukaryotic cells was demonstrated using SDS-PAGE. In the next step, Western blotting with pooled sera of mice infected with T. gondii was done.

The current in silico analysis revealed that the B cell epitopes with high immunogenicity for GRA4 protein were located in the residues 34-71, and 230-266, for GRA14 in 308-387, for SAG1 in 182-195, 261-278, and for GRA7 in residues 101-120, 160-176. The T cell epitopes were selected in overlapping regions with the B cell epitopes. The immunogenic region for GRA4 are in the residues 245-253, 50-58, and 40-54, for GRA14 in 307-315, 351-359, and 308-322, for SAG1 261-269, and 259-267, and for GRA7 in the residues 103-112, and 167-175. The results of the western blotting showed that the expressed protein had immunogenicity.

Our constructed multi-epitope of T. gondii could be considered as a candidate for diagnostic and vaccination purposes.

For fertilization process, sperm cell-surface protein called "IZUMO” requiredas a ligand for IZUMO1R/JUNO and CD9 receptor on egg. The IZUMO1:IZUMO1R/JUNO interaction is a crucial adhesion step between sperm and oocytein plasma membrane binding and fusion, but also is not sufficient for cell fusion and others receptors are play roles in this interaction. Here, we found G protein-coupled receptor 15 (GPR15) as another partner of sperm-egg interaction which is located on egg.

Initially,system biology methods applied for known and predicted protein-protein network interactions of IZUMO1. Also, primary structure of IZUMO1 and its IZUMO1R:JUNO receptors retrieved from reference databases, and their 3-dimensional structure (3D) were modeled by both Threading and homology modeling. Then, modeled structure were energetically minimized and validated by Rammapageplots and eventually prepared for molecular docking (hydrogen and atomic charges) by Chimera UCSF 1.10. Also, the docking studies were performed by HEX version 8. Finally, binding energy, pose of interactions,RMSD, hydrogen and electrostatics bonds were analyzed.

System biology analysis showed that beside IZUMO1R/JUNO and CD9 ,GPR15 maybe is another functional partner for IZUMO1 by score of 0.705 (text mining approach). Also, Rammachandran plot of modeled structures represent high quality of modeling procedure and so modeled structure used for docking. Molecular docking analysis showed GPR15 could interact with IZUMO by SER176,THR91, ARG236 and SER240, ASN239and ASN151hydrogens. The strongest hydrogen bond is between thoronine 91 (THR91) and serine 240 (SER240). On the other hand, amino acids Threonine 91 and ASN239 are the most important amino acids for binding to hydrogen bonding two proteins to each other and react simultaneously with two amino acids. Also, 21 amino acids from IZUMO1 and GRP15 play a role in the hydrophobic bond between two proteins.

The results of this bioinformatics study can help to find out more about fertility and Sperm fertilization. Further studies on the role of GPR15 in the binding of sperm and oocyte should also be made.

Crytosporidiosis is a parasitic disease caused by the cryptosporidium protozoan in the vertebrate animal. This parasite in livestock special ruminants causes diarrhea syndromes in infants. The main sources of the parasite spread are infectious feces and the host mouth. The purpose of this project was to isolate and identify the cryptosporidium species by morphological indices in diarrheic calves in order to detect and control the disease. According to the method of Casemore et al., purification of oocysts from feces using flotation technique was carried out. Staining with modified Zeil nelson method (Henrisksen method) and identification with diagnostic keys was done. Cryptosporidium parvum alone causes clinical disease in young calves; the latent infection time is 3 to 6 days. Cryptosporidium muris, C. Andersoni and C. ryanae have been reported in weaned calves, and C. Andersoni has been also observed in adult cattle. The total of 150 cattle fecal samples were randomly collected from Alborz province. 60 out of 150 (40%) calf diarrhea positive samples were detected by modified Zeil nelson method staining. In all fecal samples from calves without diarrhea, the oocyst was not observed. In calves below one month, especially in the first two weeks, 40% of the diarrheic samples were infected. This protozoon is a zoonotic and opportunistic parasite and, in Iran, it has been reported from various species like poultry, cattle, sheep and goats and it may cause significant economic losses which need much more attentions.

Acinetobacter baumannii is a Gram-negative bacterium that has recently been identified as a leading nosocomial pathogen. Infections by this pathogen result in significant mortality due to antibiotic resistance. An effective vaccine would help alleviate the burden of disease incurred by this pathogen; however, there are currently no licensed vaccines offering protection against Acinetobacter baumannii infection. In this study, considering the fact that outer membrane protein A is one of the most promising vaccine candidates, we predicted T cell and B cell epitopes on this protein using sequence-based epitope prediction tools and determined whether or not mice immunized with these peptides induce an immune response. We selected consensus epitopes including five peptides in different tools with the highest score. 48 female C5BL/6 SPF injected subcutaneously with the peptides (peptide1 to peptide 5 separately) in 100 μL of the solution and sham groups received adjuvant and PBS alone on the same schedule: on day 0 (primary dose) and two booster doses were administered on days 14 and 28. At the end of time, animals euthanized by Isoflurane, and collected sera for assessment of specific antibodies against each peptide by ELISA (Enzyme-linked immunosorbent assay). Immunization of mice showed one of the novel synthetic peptides (peptide 1 (24-50 amino acids)) elicited immune responses. We conclude to combine theoretical methods of epitope prediction and evaluating the potential of immunogenicity for developing vaccines is important.

The cattle tick Rhipicephalus spp. causes significant economic losses due to diseases in animals and human. Bm86 is a midgut protein and vaccine candidate, which its sequences among the isolates of Ripsephalus spp are geographically separated, variable, and are the main reason for reducing effectiveness, and subsequently, the failure of the recombinant vaccines.

In this bioinformatics study, the sequences of R. microplus and R. annulatus were retrieved, aligned, and edited. Then, the variation plot and phylogenetic tree were constructed. Afterwards, grouping and taxa marking for designing evolutionary vaccine, COBRA antigen, center of tree and ancestral were done. Also, over COBRA vaccine sequences, modeling analysis and superimpose test were done.

In both R. microplus and R. annulatus, the most variable region were residues 177-181, 270-276, and 351-352, respectively. 6 sequences were selected as appropriate sequences for design of evolutionary vaccine, and 12 for the realignment of and achieving sequences for design of COBRA antigen. On the other hand, R. annulatus sequences were in sister branches and more similar to each other compared to Bm86 protein sequences in R. microplus except ADQ19687. The sequences selected for vaccine design based on the center of tree and ancestral, were AJE29931, AJE29932, and ATW75472, and ATW5476, ADM86722, ACZ55133 sequences, respectively.

Anti-tick COBRA-based vaccines of Bm86 could be more cost-effective and better alternative with broader spectrum, compared to the commonly used recombinant vaccines.

Accumulating evidence indicates that toll-like receptor 4 (TLR4) plays a critical role in promoting adaptive immune responses and are definitively involved in the expansion and maintenance of the neuropathic pain. However, the application of docking in virtual-screening in silico methods to drug discovery has some challenge but it allows us to make the directed and meaningful design of drugs for a target protein, which can be shortening and low costing the evolution and discovery of very promising lead new drugs. Nevertheless, in parallel with virtual screening methods, attendant developments in cell culture and in vivo studies must be achieved. In the present paper, we aimed to discover new drugs that have the ability to bind and inhibit TLR4 functions. So, after using the Pathway studio to investigate the biological pathways and protein interaction maps between TLR4 and neuropathy, we reported the application of the affinity-based approach of different pharmaceuticals; these agents contained all of the approved drugs; which could bind to Toll-like receptor 4 in blind high-throughput in silico screening. Our results demonstrated that among the primary list of 1945 retrieved compounds, 39 approved compounds could be the right candidate to perform a biological test in different in vivo and in vitro conditions and as a lead for further neurophysiological and neuropathological studies and treatment of neuropathic pain.

Rhipicephalus (Boophilus) annulatus is an important tick, which can transmit parasites and bacteria to cattle. It can also cause hides damage, growth reduction and sometimes paralysis. So, the tick infestation can be regarded as a major problem in the livestock industry. Different control methods including the chemical ones are used to fight ticks, but because of developing resistance to chemical treatments, researchers try to find some immunogenic proteins for vaccine production. Investigating these tick proteins could be an important step in the identification of biological molecules for the purpose of developing control strategies. The aims of present study were to evaluate and analyze Boophilus annulatus larval extract proteins by 2- dimensional gel electrophoresis patterns (proteomic profiling) and identification of some its immunogenic proteins by two dimensional immunoblotting and MALDI TOF/TOF mass spectrometry referred to as Mazandaran strains. The steps followed here were: tick preparation and culture, 1-D electrophoresis, and then 2-D electrophoresis, Western blotting and Mass spectrometry and then Mass data were analyzed by Mascot software. Analysis of the produced 2D image identified approximately 80 protein spots with different Molecular weight and PI by Coomassi blue staining. Based on immunogenicity (through Western blotting) and high concentration, 10 protein spots (between 14 and 97 kDa) candidates for MALDI TOF and MALDI TOF - TOF MS. Among the 10 proteins spots, Vitellogenin, Vitellogenin-2 precursor, tropomyosin, hypotethical protein ISCW001652 were identified proteins with immunogenic properties. Also, quantification analysis showed some proteins had more quantity in soluble larvae protein extract and some such as vitellogenin had some isoforms. The results of this study could be a preliminary step towards selecting proteins candidated to develop vaccines against ticks.