Veterinary Research Forum

Abstract Inflammatory bowel disease (IBD) encompasses a group of chronic inflammatory conditions that primarily impact the gastrointestinal system. While ulcerative colitis and Crohn's disease are the principal manifestations in humans, animals frequently exhibit lymphocytic-plasmacytic enteritis/colitis and eosinophilic enteritis/colitis. Growing evidence suggests a complex interplay among genetic predisposition, gut microbiota imbalance and abnormal immune responses to intestinal microbes in susceptible individuals. This intricate involvement results in remarkably similar clinical presentations across species. Patients often experience symptoms such as diarrhea, vomiting, weight loss and anemia. Extraintestinal manifestations including uveitis, skin rash and arthritis may also occur. Endoscopy and biopsy typically serve as the gold standard for confirming the diagnosis and differentiating it from other gastrointestinal disorders in humans and animals. The treatment approach generally focuses on managing disease activity through immunosuppressive medications such as glucocorticoids, administered at appropriate dosages. However, the precise cause of IBD remains a topic of ongoing research. With the emergence of additional treatment options like herbal compounds and fecal microbiota transplantation, which have demonstrated effectiveness in restoring gut health in IBD patients, there is optimism for novel therapeutic strategies. Ultimately, conclusion is that chronic gastrointestinal conditions like IBD are complex in both human and veterinary medicine. These diseases share numerous common pathophysiological features, yet, diagnostic and treatment challenges continue to exist.

Abstract Today, a combination of immunological and bioinformatics tools has become common for vaccine design, making vaccine production affordable. Considering the importance of recombinant protein purification for vaccine production in a cost-effective way, our study aimed to in silico design a fusion protein vaccine against egg drop syndrome virus (EDSV) with a higher isoelectric point for more affordable purification. The in silico design of fusion protein, including egg white lysozyme and fiber protein as an antigen from egg drop syndrome virus, was performed. In addition to isoelectric point changing, lysozyme probably helps the antigenicity by increasing the size of the antigen. Also, lysozyme can act as a preservative. The physicochemical characteristics, stability, secondary and tertiary structure, epitope prediction, antigenicity, and mRNA structure were analyzed using computational and bioinformatics tools. The results showed that the isoelectric point of the gene construct was 8.87, which can be purified by ion exchange chromatography. Validation of the Ramachandran plot showed that the predicted model was accurate and suitable. The tertiary structure of the fusion protein was modeled as well, and its trimer structure, being required for immunogenicity, was preserved. The antigenicity of the target construct was also suitable. Protein was stable and hydrophilic based on aliphatic index and grand average of hydropathicity, which can be a good candidate for a vaccine. After experimental studies, this fusion protein may be used as a vaccine against EDSV.

Abstract This study evaluated the impact of combining piperine and prednisolone on clinical symptoms and immune responses in Wistar rats with rheumatoid arthritis (RA) induced by Freund's complete adjuvant due to piperine known anti-inflammatory and immunomodulatory properties. The RA rats were randomly divided into five groups (n = 10): The RA rats were treated with phosphate-buffered saline, RA rats treated with piperine (100 mg kg^-1 orally), RA rats treated with prednisolone (10.00 mg kg^-1 orally), and RA rats treated with a combination of piperine and prednisolone (half doses of each orally). Treatment started on day five post-induction when all rats had a clinical score of ≥ 1. Disease symptoms were monitored every other day until day 23 post-induction. Combining the two medications at half doses led to a more significant reduction in disease severity, weight improvement, and histopathological changes compared to using each drug alone at the full doses. The combined treatment group exhibited the most favorable response in C-reactive protein, myeloperoxidase, and nitric oxide biochemical tests compared to the other treatment groups. The combined treatment group showed decreased expression of T-bet and RORɣt genes. However, there was no statistically significant difference in the expression of Foxp3 and GATA3 genes compared to the group receiving prednisolone alone. Overall, combining piperine with prednisolone may prove to be a beneficial approach for managing RA.

Abstract Cell-surface proteins of Clostridium chauvoei were purified using a simple method. Bacterial cultures were centrifuged and agitated vigorously in phosphate buffered saline with or without further glycine treatment and ammonium sulfate precipitation. Rabbits were immunized subcutaneously with a blackleg disease vaccine twice with a two-week interval. Immunized sera were collected one week after the second injection. Enzyme-linked immunosorbent assay (ELISA) was performed using the proteins purified by the second method as the coating antigen. Bradford assay results showed a higher protein concentration in the second than the first method. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis showed multiple bands for the cell-surface proteins of C. chauvoei in the first method and a sharp band equivalent to flagellin protein in the second method. The ELISA results indicated that the purified proteins were capable of detecting antibodies against Blackleg disease vaccine. The purified protein would be an alternative antigen for indirect ELISA in order to monitor the immune response in vaccinated farm animals.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. The receptor binding domain (RBD), located at the spike protein of SARS-CoV-2, contains most of the neutralizing epitopes during viral infection and is an ideal antigen for vaccine development. In this study, bioinformatic analysis of the amino acid sequence data of SARS-CoV-2 RBD protein for the better understanding of molecular characteristics was performed. The SARS-CoV-2 RBD gene was inserted into pET-28a vector, and efficiently expressed in E. coli system. Then, the recombinant proteins (RBD monomer and RBD dimer protein) were purified as antigen for animal immunization. Furthermore, the results showed that the recombinant proteins (RBD monomer and RBD dimer protein) had adequate immunogenicity to stimulate specific antibodies against the corresponding protein in immunized mice. Taken together, the results of this study revealed that RBD protein had a high immunogenicity. This study might have implications for future development of SARS-CoV-2 detection.

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the causative agent of the emerging zoonotic respiratory disease. One of the most important prerequisites for combating emerging diseases is the development of vaccines within a short period of time. In this study, antigen-irradiated, inactivated SARS-CoV-2 viruses and the disaccharide trehalose were used to enhance immune responses in the Syrian hamster. The SARS-CoV-2 virus was isolated from tracheal swabs, confirmed by Real-Time Polymerase Chain Reaction (RT-PCR), and propagated on Vero cells. For inactivation, it was irradiated with 14.00 kGy gamma radiation. Evaluation of the antigenic properties of the spike protein subunit S1 showed that the antigens were intact after gamma irradiation. The gamma-irradiated and formalin-treated viruses were used to immunize hamsters in four vaccine formulations. Neutralizing antibodies increased significantly in all vaccinated groups three weeks after the second and third vaccinations. The concentration of secretory immunoglobulin A in the irradiated vaccine plus trehalose increased significantly in nasal lavage and nasopharyngeal-associated lymphoid tissue fluids three weeks after the second and third vaccinations. The lymphocyte proliferation test in the spleen showed a significant increase in all vaccinated hamsters, but the increase was greater in irradiated vaccine plus trehalose and irradiated vaccine plus alum. We can recommend the irradiated inactivated vaccine SARS-CoV-2 plus trehalose (intra-nasal) and another irradiated inactivated vaccine SARS-CoV-2 plus alum (subcutaneous) as safe vaccines against coronavirus disease of 2019 (COVID-19), which can stimulate mucosal, humeral, and cellular immunities. However, the protectivity of the vaccine against COVID-19 in vaccinated hamsters must be investigated in a challenge test to assess the potency and efficiency of vaccine.

Abstract The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in Escherichia coli, including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from E. coli culture and their immunogenicities were evaluated in vivo by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.

Abstract Coccidiosis is the leading parasitic disease in poultry. One of the most critical Eimeria species, Eimeria tenella, lives inside the cecal epithelial cells and induces bloody coccidiosis. The present study evaluated the effect of radiation-attenuated E. tenella oocytes mixed with inulin adjuvant on broiler chicken. Initially, the effect of irradiation on oocyst attenuation was confirmed. Then, one-day-old broilers (n = 90) were divided into nine groups on seven days of age as follow: Group 1 (400 attenuated oocysts + 1.00 mg of adjuvant), group 2 (400 attenuated oocysts + 0.50 mg adjuvant), group 3 (200 attenuated oocysts + 1.00 mg of adjuvant), group 4 (200 attenuated oocysts + 0.50 mg adjuvant), group 5 (1.00 mg adjuvant), group 6 (400 attenuated oocysts), group 7 (commercial vaccine), group 8 (negative control) and group 9 (blank). On day 21, we performed a challenge with E. tenella oocytes and investigated oocyst output and average weekly weight throughout the study. At the end of the study, we evaluated macroscopic lesion, histology, cytokine level and leukogram status. The results showed a statistically significant difference among groups. Furthermore, the optimal dose was 400 irradiated oocysts and 1.00 mg of inulin. Moreover, an X-ray could reduce the virulence of E. tenella oocytes. Inulin alone or combined with attenuated oocysts showed an acceptable effect on evaluated parameters.

Abstract Sepsis is an acute condition caused by the systemic inflammatory response syndrome to an infection. There are very few drugs that could improve the severe conditions in patients with sepsis. Hence, it is important to consider different treatment options. In this survey, we studied the effect of adenosine N1-oxide (ANO) and pioglitazone on rats with cecal ligation and perforation (CLP). They were randomly divided to four groups (n = 10) including Group A: as control group receiving normal saline, Group B: the rats with CLP as surgical control group, Group C: the rats receiving ANO, and Group D: the rats receiving pioglitazone. Interleukin (IL) -6, IL-1β, tumor necrosis factor alpha (TNF-α), nitric-oxide (NO) in serum blood and superoxide dismutase (SOD), catalase (CAT) malondialdehyde, (MDA) and myeloperoxidase (MPO) in liver and spleen homogenates were measured. The amount of antioxidant enzymes in the spleen and liver, and finally cell viability and rats’ survival were investigated. The measurement of blood serum nitric-oxide and survival of all groups of rats were also performed. The results indicated that both drugs could cause a decrease in IL-1β, IL-6, TNF-α and NO in rat blood serum and MDA and MPO in the liver and spleen homogenates, however, a significant increase in SOD and CAT in the liver and spleen homogenates in rats that received ANO and pioglitazone was observed compared to rats with CLP group. Cell viability and rats’ survival were significantly improved in rats that received ANO and pioglitazone compared to rats with CLP group. Adenosine N1-oxide showed stronger and more effective effects.

Abstract Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.

Abstract The main objectives of this study were to determine the occurrence and potential causative factors of Immune-mediated hemolytic anemia (IMHA) in native cattle and water buffaloes from southwest of Iran. Fifty-three anemic animals (37 cattle and 16 buffaloes) were studied. A full clinical history and physical examinations were undertaken for all animals. Four clinically healthy cattle and four healthy buffaloes were also used as control animals. Blood samples were subjected to a complete blood count, Coombs’ test, erythrocyte osmotic fragility test and serum biochemical analysis. IMHA was diagnosed in 12 (32.43%) cattle and 6 (37.50%) buffaloes based on the Coombs’ test. Underlying or concurrent diseases, including theileriosis, anaplasmosis, vaccination, and pneumonia were detected in 11 cattle and four buffaloes. Primary or idiopathic IMHA was identified in one cattle and two buffaloes that their Coombs’ test was positive. Hematologic and biochemical findings in the cattle with IMHA included a nonregenerative anemia, leukopenia, thrombocytopenia, increased osmotic fragility, hyperbilirubinemia and elevated serum alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase activities. It can be concluded that IMHA occurs in a significant proportion of anemic cattle and river buffaloes in southwest of Iran. The occurrence of IMHA in both cattle and buffaloes is mostly secondary to infectious diseases especially theileriosis and anaplasmosis. Clarification of the mechanisms of primary or idiopathic and secondary IMHA in cattle and buffaloes require further studies.

Abstract Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens.In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

Abstract Monoclonal antibodies (MAbs) are invaluable molecules which have several advantages over polyclonal immunoglobulins (Igs) including consistency and higher specificity and hence can be used in biological researches, diagnosis and treatment of diseases. The present study was conducted to produce monoclonal antibody against chicken IgG.TheIgG molecules were purified from chicken serum and used as antigens to immunize several mice. Thereafter, a well-immunized mouse was chosen and used for fusion process. After production of hybridoma cells, several rounds of cloning were carried out and produced MAbs were examined by various immunological assays including enzyme-linked immunosorbent assay (ELISA) and western and dot blotting. Assessment of grown hybridomas indicated that only one clone (5B8) has produced desired MAb against chicken IgG. Meanwhile, using an indirect ELISA, it was shown that this MAb successfully recognizes chicken IgG molecules attached to influenza virus nucleoprotein. Evaluation of cross reactivity of MAb 5B8 with several avian serum samples revealed that this molecule specifically identifies chicken antibody molecules. However, it also recognized turkey antibodies with less affinity. In addition to research applications like isolation and purification of chicken and turkey IgG molecules, such a MAb can be applied to design and development of various immunoassays (e.g. ELISA) in these avian species.

Abstract Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')₂ can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')₂ fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')₂ and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')₂ was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')₂ fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')₂ and polyclonal anti-IgG F(ab')₂ are useful tools in biomedical and biochemical researches and diagnostic kits.

Abstract Members of gram-negative bacteria family Pasteurellaceae, include a large number of important economically human and veterinary pathogens. Organisms belonging to the family can colonize in mucosal surfaces of the respiratory, alimentary, genital tracts and cause diseases in various mammals, birds, and reptiles. Hemorrhagic septicemia is an acute disease of cattle and buffaloes in tropical countries caused by Pasteurella multocida serotype B:2. In the present study, the possible bactericidal activity of immune calf sera in the presence and absence of complement system was investigated. The results showed that P. multocida B:2 is highly resistant to positive serum, containing high levels of IgG and IgM obtained from calves after vaccination, and complement activity in normal fresh calf serum. This organism also grew rapidly in the normal fresh calf serum and the mixture of positive serum as well as normal fresh calf serum. As a control test an E. coli strain was subjected to the same experiment and found completely sensitive to the bactericidal activity of complement in calf and guinea pig fresh sera. Results were indicative of the presence of inhibitory mechanism(s) in P. multocida B:2 against bactericidal activity of immune calf serum and complement system.

Abstract Bovine ephemeral fever is an acute and arthropod-borne viral disease of cattle and water buffalo which occurs seasonally in most of the world tropical and subtropical regions. The epizootic feature of the disease has been reported in Iran with serious economic consequences. The surface glycoprotein G of bovine ephemeral fever virus (BEFV) is composed of 4 antigenic sites (G1-G4) and plays the main role for eliciting neutralizing antibodies and protective immunity. The G1 – epitope is a linear antigenic site and conserved among BEFV strains. In order to develop an ELISA test based on G1-epitope as coating antigen, this study was carried out to express the recombinant G1-epitope of BEFV in prokaryotic system. Using PCR and specific primers, a length of 88 amino acid of the G glycoprotein of BEFV including G1- epitope was amplified and cloned into the expression vector pGEX-4T-1, with the GST moiety. The recombinant plasmid (pGEX-4T-1-G1) was then transformed into Escherichia coli BL₂₁ and expression of fusion protein was induced by 0.10 mM IPTG. The maximum expression of the fusion protein was obtained at 16 hr post induction as verified by SDS-PAGE electrophoresis, and it was also confirmed that this protein bearing G1- epitope is sufficiently biologically active to bind to anti-BEFV serum in western blot experiment.

Abstract Avian infectious bronchitis (IB), caused by a gammacoronavirus, is an OIE-listed (List B) disease and characterized by respiratory and renal involvements, causing high mortality, and economic loss in both layers and broilers. In comparison with other diagnostic methods, real-time polymerase chain reaction (RT-PCR) and conventional RT-PCR are potent, more sensitive and faster techniques for infectious bronchitis virus (IBV) detection. This research was conducted to detect IBV using specific primers of IB in three governorates (Basra, Thi-Qar and Muthana) in the south of Iraq. Tracheal specimens were collected from 46 IB suspected commercial broiler flocks. XCE2+ and XCE2- Primers, which amplify all IBV serotypes, were used. Primers MCE1+, BCE1+ and DCE1+ were used to amplify the specific nucleotide sequences of Massachusetts, 793/B and D274 genotypes, respectively. The results of real-time RT-PCR of this study showed that 34 (74.00%) out of 46 infected flocks were positive to IBV. The results of nested PCR showed that 50.00% and 5.89% of positive samples were belonged to genotypes 793/B and Massachusetts, respectively, and the remaining positive (44.11%) were unknown. The results indicate presence of  Massachusetts and 793/B IBV strains in commercial broilers in southern Iraq.

Abstract It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the expression of the pluripotent stem cell markers in the mice uterine tissue during different stages of estrous cycles. Twelve virgin female NMRI mice (6 to 8 weeks old) were considered at proestrus, estrus, metestrus and diestrus according to the cell types observed in the vaginal smear and underwent hysterectomy operation. Quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining for pluripotent stem cell markers (SOX2, OCT4, KLF4, and NANOG) were performed. Immunofluorescence staining revealed that expression and localization of the pluripotency markers SOX2, OCT4, KLF4, and NANOG at the protein level were not different throughout estrous cycle. Also, mRNA of pluripotency markers was detected in all tested samples. However, there were no significant differences in their genes expression at each stage and during the estrous cycle. Different hormonal profile during the estrous cycle could not affect expression of pluripotent stem cell markers in uterine tissue.

Abstract Mycoplasma synoviae (MS) is a pathogen responsible for respiratory and locomotor disorders and causes major economic losses in poultry industry. Early and accurate diagnosis of MS infection plays a major role in control of the infection. This study was conducted to characterize Iranian field isolates of MS isolated from broiler chickens of West Azarbaijan province (Northwest of Iran), and differentiate them from vaccine strain MS-H. Two encoding genes, 16S rRNA and vlhA were employed. PCR results using primers related to 16s rRNA and vlhA genes were analyzed and compared. Out of 21 field samples, eight samples (38.0%) were positive using both sets of primers. Amplified products of vlhA gene were sequenced for MS strain identification. The results showed that Iranian field isolates of MS had high nucleotide and amino acid similarity. Iranian field isolates were distinct from vaccine strain MS-H. Results presented in this study showed that characterization of field isolates of MS by sequencing of vlhA gene and is beneficial for strain typing and differentiating them from vaccine strain. To our knowledge, this is the first study characterizing vlhA gene of MS isolates from broiler chickens in the West Azarbaijan province.

Abstract Bovine Viral Diarrhea virus (BVDV) is an important viral pathogen of cattle causing several clinical syndromes. There are usually no pathognomonic clinical signs of BVDV infection. Diagnostic investigations therefore rely on serological detection and virus isolation. Nonstructural protein 3 (NS3) as immunogenic protein of BVDV is genetically and antigenically conserved among different isolates. This protein is therefore a candidate antigen for developing ELISA for serological studies. The aim of this study was to produce monoclonal antibody (MAb) against recombinant NS3 protein. For this purpose, the recombinant MBP-NS3 protein was expressed into expression vector pMalc2x in E. coli and purified using amylose resin chromatography column and the purified protein used as antigen in MAb production. After immunizing Balb/c mice with the recombinant antigen, the mouse showing highest titer of anti-NS3 antibody by indirect ELISA was selected for fusion. Spleen cells of the immunized mouse were fused with SP2/0 myeloma cells using polyethylene glycol. The cells in fusion mix were re-suspended in HAT medium and distributed in 96-well plates. Then culture supernatants of primary clones were screened by indirect ELISA. The positive clones after three times cloning, were selected and the reactivity of the monoclonal antibodies with recombinant and natural antigens was established by Western blotting. Based on these results, it appears that the specific monoclonal antibodies produced against NS3 recombinant antigen may be suitable for developing BVDV laboratory diagnostic assays.

Abstract The receptors 1 and 2 of fibroblast growth factor (FGFR1 and FGFR2, respectively) have been observed in all types of testicular cells. Culture on extracellular matrix (ECM) has been observed to lead to initiation of differentiation in spermatogonial stem cells (SSCs). The present study was carried out to investigate whether FGFR1 and FGFR2 play a role in SSCs differentiation. Following isolation, bovine testicular cells were cultured on ECM-coated or uncoated (control) plates for 12 days. The gene expression of THY1, cKIT, FGFR1 and FGFR2 was evaluated using quantitative real-time polymerase chain reaction (PCR). Results related to the gene expression of markers of with undifferentiated (THY1) and differentiated (cKIT) spermatogonia implicated stimulation of self-renewal and differentiation in cells cultured on ECM-coated and uncoated plates, respectively (p < 0.05). Concomitantly, the expression of FGFR2 increased during culture in the ECM group (p < 0.05), whereas it did not change in the control group (p > 0.05). As a result, the gene expression of FGFR2 was greater in the ECM than control group (p < 0.05). Nevertheless, FGFR1 expression did not change during culture in the control and ECM groups (p > 0.05). In conclusion, the present study revealed the potential role of FGFR2 in differentiation of SSCs during culture on ECM.

Abstract One of the most important species of the Bartonella genus is B. henselae that causes a zoonotic infection, cat scratch disease (CSD). The main source of the bacteria is cat and the carrier is Ctenocephalides felis flea. One hundred and forty nail and saliva samples were collected from 70 domestic cats. Positive samples for B. henselae were characterized by polymerase chain reaction (PCR) and sequencing. Sequences of gltA gene were trimmed using BioEdit software and then compared with the sequences of the same gene from B. henselae isolated from cats and humans in GenBank database. Phylogenic tree was constructed using CLC Sequence Viewer software and unweighted pair group method with arithmetic mean (UPGMA) method. Molecular assessments showed that five samples out of 70 nail samples (7.14%) and one sample out of 70 saliva samples (1.42%) were genetically positive for B. henselae. At least an 87.00% similarity was seen between the gene sequences from the current study and the reference sequences from the GenBank database. Phylogenic analysis has shown that strains isolated in this study were grouped in a different haplo group, compared to other strains.Among the Asian countries, the prevalence of the bacteria in Iran was close to that in Japan and Turkey. In conclusion, findings of this study showed the prevalence of B. henselae in Iranian cats which is important due to its public health issues, especially for the immunocompromised pet owners.

Abstract In the present study, we described cyto-histopathological features and immunophenotyping of the large B-cell lymphoma in an 8-year-old mixed breed dog with applying the World Health Organization (WHO) system of classification of canine lymphomas. In fine-needle aspiration (FNA), lymph nodes were involved by neoplastic cells of intermediate to large size with deep blue cytoplasm; consist of centroblasts, immunoblast and medium-sized cells. Histopathologically, the follicles and sinuses of lymph nodes were replaced by sheets of numerous immunoblasts (less than 90.0% of total cells) and centroblasts. Numerous mitotic figures were also observed. Immunohistochemical analysis presented that the neoplastic cells express B-cell phenotype CD20 and CD79a, but do not stain for T phenotype CD3. On the basis of cytology, histopathology and immunohistochemical findings, the present tumor was diagnosed as diffuse large B-cell lymphoma, high-grade centroblastic type (DLBCL-CB) according to WHO histological classification. Applying this classification system for diagnosis of canine lymphomas is very useful and has a high accuracy and consistency. However, further co-operative studies between clinicians and pathologists should be performed, in order to improve the effectiveness of this classification.

Abstract This in vitro study was conducted to evaluate lymphocyte blastogenic and cytokine production by bovine peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) mitogens, by using tetrazolium salt and ELISA tests, respectively. The results presented that  Interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-17 and IFN-γ production in response to PWM mitogens was the highest and Con A the lowest amount and the median values of three mitogens were in the following order: PWM > PHA > Con A > cell control. In the case of IL-6, the production of this cytokine was the same amount for PWM and Con A and a lower amount for PHA stimulation. The results of this study not only showed a normal range for the production of these cytokines from PBMCs that were affected by mitogens, but it demonstrated that the bovine immune system at 2.5 to 3 months was post-natally matured enough to mount an effective immune response to mitogens as well as specific antigens.