Veterinary Research Forum

Abstract Urinary bladder cancer, the tenth most common cancer globally, exhibits significant variability in the reported expression of estrogen receptor alpha (ERα), a key prognostic marker. Accurate and reproducible assessment of ERα is critical for elucidating its role in bladder cancer biology and identifying therapeutic targets. Aim: This study compared manual scoring with automated analysis using ImmunoRatio software for quantifying ERα expression in a chemically induced bladder cancer model in rats, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Methods: Twenty male Wistar rats were randomly assigned to two groups: a control group (n=8) and a group treated with BBN (BBN; n=12), administered in drinking water at 0.05% for 20 weeks. Animals were sacrificed and urinary bladders were collected for macroscopic and microscopic evaluation. Samples underwent standard histological processing and immunohistochemical analysis to assess ERα expression, quantified both manually and automatically. Results: The immunoexpression of ERα, detected by ImmunoRatio was found to be significantly higher than that obtained through manual counting in both groups (p < 0.0001). ImmunoRatio analysis indicated a higher ERα expression in the control group than in the BBN group (p < 0.001), whereas manual counting showed the opposite tendency, without statistical significance (p > 0.05). Conclusion: Our findings demonstrated that ImmunoRatio resulted in significantly higher ERα immunoexpression values than manual counting in both control and BBN-treated groups, with inconsistent group-wise trends between methods. These findings suggest that, although digital pathology tools offer advantages in standardization and reproducibility, their application requires tissue-specific calibration and validation to ensure accurate and reliable biomarker quantification.

Abstract Foot-and-mouth disease virus (FMDV) continues to pose a major threat to the livestock industry worldwide due to its high transmissibility and substantial economic consequences. For diagnostic and vaccinal purposes, necessitate the exploration of novel immunogenic targets is pivotal. Here, antigenic determinant (an epitope) sequence comprising core of 12 amino acids from VP1 protein of foot-and-mouth disease virus (FMDV) was designed by immuno informatics. To improve the physio-chemical and stability characteristics of the final peptide, additional residues were incorporated until it resulted in the synthesis of a 23-amino acid peptide (VARTLPTSFNYGAIKATRVTDEC, 2.5 Kda and PI 8.17). After chemical synthetizing with 95 % purity, the peptide was conjugated to bovine serum albumin (BSA) via EDC/NHS cross linker (Peptide: EDC: Sulfo-NHS: BSA (carrier protein): 10:40:50:1 in MES buffer (50 Mm)) and formulated with an aluminum hydroxide adjuvant. Twelve male New Zealand White rabbits were randomly assigned into three groups and main group received the peptide-BSA conjugate. Sera were direct to Dot blot, double immunodiffusion (DID) and serum neutralization (SN) tests, and also indirect peptide ELISA. The synthetic peptide elicited strong antibody response by primary positive results in dot blot and DID test (positive in 1:4 serum dilution) as well as it showed cross-reactivity with whole antigen. Although only low SN titers (1:8) were observed. Moreover, the peptide ELISA confirmed specific antibody responses against O serotype (OD of 2.4 at day 55 in 1/10 dilutions). Altogether, rationally designed epitope are promising regimes for the future development of FMDV novel diagnostic tools and vaccine cocktails.

Abstract Interstitial cells of Cajal (ICC) play a crucial role in regulating gastrointestinal motility, functioning as pacemakers in the digestive system of equids. This study described and compared the ICC populations in different regions of the stomachs of equids (horses, donkeys and mules). A total of 30 healthy stomachs from horses, donkeys, and mules were evaluated using immunohistochemistry and high-resolution microscopy to assess various regions of the stomach in these species. The results showed the presence of ICC in all three equid species, with a predominance in the submucosa across the squamous mucosa, glandular mucosa and pyloric antrum. The density and morphology of ICC varied slightly between species, although no significant differences were found. Among the equids studied, donkeys showed the lowest ICC density, while horses exhibited the highest in the evaluated tissues. Four cellular morphologies were identified, three of which were included in the analysis, as toluidine staining revealed that one corresponded to mast cells. Further studies are needed to elucidate dynamics and possible alterations in response to gastric pathologies.

Abstract Pasteurella multocida and Mannheimia haemolytica are significant bacteria of respiratory tract infections in cattle. Identifying whether these bacteria are commensal or pathogenic is crucial for understanding their role in predisposing calves to respiratory tract infections. This study aimed to determine the prevalence and pathogenic characteristics of P. multocida and M. haemolytica in nasal swab samples of clinically healthy calves in Konya, Turkey, and to assess antibiotic resistance profiles. Using conventional culture methods, 5 samples (1.67%) from 4 different farms were positive for P. multocida and 3 samples (1%) were positive for M. haemolytica. PCR of nasal swab samples yielded P. multocida positivity in 17 (5.67%) samples and M. haemolytica positivity in 13 (4.33%), including all culture-positive samples. While all P. multocida isolates were atypical for the capsule, 18% of the PCR-positive samples were L2, 70% L3A, and 12% L8 serotypes. The ompA, omp87, hghA, and tbpA genes were the most prevalent, while the presence of other virulence genes varied. High rates of ampicillin/cloxacillin (100%) and streptomycin (80%) resistance were observed among the isolates. The rates of serotypes A1 and A6 in M. haemolytica PCR-positive samples were 53.85% and 46.15%, respectively. The ssa-1 gene was detected in 33.33% of the isolates. Moderate resistance to trimethoprim/sulfamethoxazole, streptomycin, and neomycin (66.67%) was recorded. The findings revealed the presence of P. multocida and M. haemolytica with various virulence characteristics and antimicrobial resistance profiles in the nasal microbiota of healthy calves, providing novel insights that could inform improved herd management, disease control, and treatment strategies.

Abstract Cutaneous squamous cell carcinoma (CSCC) is a clinically significant skin neoplasm in dogs, typically arising in areas exposed to chronic ultraviolet light. This report describes a 10-year-old intact male Pitbull dog weighing 33.00 kg that presented with a large, ulcerated, necrotic, and hemorrhagic mass in the inguinal mammary region. Initial hematological evaluation revealed marked leukocytosis with segmented neutrophilia, severe toxic changes, along with degenerated neutrophils observed in fine needle aspiration of the mass. Following a course of antibiotic therapy to control the severe infection, the mass was surgically excised using electrosurgery. The preoperative thoracic radiographs ruled out pulmonary metastasis. Histopathological examination confirmed the diagnosis of cutaneous SCC. Despite complete initial removal, tumor recurrence was noted seven months postoperatively. The owner declined further chemotherapy and surgical intervention. This report provides a comprehensive overview of the surgical, radiological, and histopathological findings of a massive cutaneous squamous cell carcinoma located in the ventral abdominal mammary region in a male Pitbull dog.

Abstract Continuous exposure to pesticides may affect ovarian function and folliculogenesis by altered oxidative statute, steroidogenesis and oocyte quality . Sweet apricot kernels (SAK) have been traditionally used as phytotherapeutic agents known for their broad pharmacological potential, including antioxidant, anti-inflammatory, and anticancer properties. This study aimed to assess the protective effect of SAK against ovarian toxicity induced by subchronic dietary exposure to a pesticide mixture (Mix) consisting of trifloxystrobin (TFS) and thiacloprid (THI) in female rats. Thirty-two female Wistar rats were randomly assigned into four groups of 8. Samples in 0.5 ml were given by oral gavage for 100 consecutive days. The control group received distilled water, SAK group including untreated rats received SAK extract (50 mg/kg bw), Mix group received TFS (0.30 mg/kg) + THI (0.20 mg/kg) and SAK + Mix group including mix treated rats supplemented with SAK extract (50 mg/kg bw) . Ovarian tissue samples were collected at the end of experiment for biochemical, histopathological, and immunohistochemical analysis. Pesticide mixture induced ovarian toxicity, characterized by a significant decrease in serum estradiol levels, ovarian total antioxidant capacity (TAC), ovarian follicle counts, Bcl-2 immunostaining, ovarian superoxide dismutase (SOD) and catalase (CAT) activities. In addition, ovarian malondialdehyde (MDA) levels, serum interleukin-6 (IL-6) concentrations, and p53 immunostaining were increased significantly. The co-administration of SAK extract significantly attenuated MDA, restored TAC levels, and modulated inflammation, Estrogen levels, apoptosis-related protein expression, CAT and SOD enzyme activities.

Abstract Babesia caballi is a tick-borne protozoan parasite responsible for causing equine babesiosis, a disease with considerable variation in prevalence across different regions of the world. This report presented a confirmed case of B. caballi infection in a three-year-old mare originating from Gonbad-e Kavus, Iran, highlighting the clinical, laboratory and diagnostic aspects of the disease. The affected mare showed classical clinical signs including persistent fever, generalized weakness, tachycardia and pale mucous membranes, which are indicative of systemic infection and anemia. Laboratory evaluations revealed evidence of hepatic and renal compromise as well as hematological parameters consistent with active inflammatory processes. Microscopic examination of blood smears and molecular techniques such as PCR were employed to confirm the presence of B. caballi, while simultaneously excluding co-infections with other common equine pathogens including Theileria equi and Anaplasma phagocytophilum. This case underscored the critical need to combine conventional diagnostic approaches with molecular methods to achieve accurate detection, effective treatment and appropriate management strategies for equine babesiosis in endemic regions.

Abstract Wild birds play a significant role in the dissemination of micro-organisms, including fungal agents, through feathers, skin debris, and feces, both locally and across regions via migration. Identifying fungal carriage in wild birds is crucial for understanding the prevalence of pathogenic fungi and developing effective strategies to prevent and control mycotic infections. This study investigated fungal carriage in wild pigeons, doves, water birds, birds of prey, and small passerines in Balıkesir province of Türkiye, an ecologically important region characterized by key wetlands, two national parks, and major migratory bird routes. A total of 46 wild bird samples were analyzed by culture method, as the “gold standard”, and several clinically important fungal isolates were obtained and subsequently further analyzed by PCR. Fungi were detected across all bird categories, with 27 birds (58.70%) testing positive for at least one fungal species. In total, 10 distinct fungal taxa were isolated from the wild birds. The most frequently identified organisms were Mucor spp. (28.26%) and Aspergillus niger (6.52%), followed by Sporothrix schenckii (4.35%), Candida glabrata (4.30%), Arthroderma terrestre (2.17%), Trichophyton mentagrophytes (2.17%), Nannizzia gypsea (2.17%), Aspergillus flavus (2.17%), Aspergillus fumigatus (2.17%), Lichtheimia spp. (2.17%), and Beauveria spp. (2.17%). Prevalence was the highest in birds of prey and small passerines (77.78%), moderate in pigeons and doves (58.82%), and the lowest in water birds (27.27%). These results emphasize the role of wild birds as potential reservoirs and vectors of fungal agents, highlighting the importance of continued surveillance and biosecurity measures to mitigate the risk of environmental and zoonotic transmissions.

Abstract Equine glandular gastric disease (EGGD) has limited prevalence studies and scarce understanding of the predisposing factors and pathophysiological mechanisms involved. The objectives of this study were to determine and compare the post-mortem prevalence of EGGD and other gastric lesions in Colombian equids, specifically horses, donkeys, and mules. The study utilized a sample of 300 equids stomachs, with 100 specimens from each group, collected immediately after slaughter. The post-mortem examination included a thorough inspection of the ventral fundus, adjacent area of the margo plicatus, and pyloric antrum of the glandular mucosa, carefully identifying any lesions or abnormalities. The results showed that the overall macroscopic presence of EGGD in the equid population was 68.30% on average, with specific rates of 45.00% in horses, 77.00% in donkeys, and 83.00% in mules, with lesions predominantly affecting the fundus and pyloric antrum regions. Inflammatory processes were observed to be similar between donkeys and horses, while mules exhibited distinct inflammatory patterns in the glandular mucosa. Additional gastric findings, such as gastritis (85.30%), gastric impaction (10.00%), and parasitic infections (13.30%), were also documented during the study. The findings suggested that EGGD was prevalent across all three groups of equids. Although the inflammatory conditions and their distribution were similar in horses and donkeys, differences in the location, frequency, and severity of mucosal lesions were observed among the three species.

Abstract This study investigated the effects of zinc oxide nanoparticles (ZnO-NPs) on the growth, blood parameters, skin mucus, and serum biochemistry of juvenile common carp (Cyprinus carpio). A total of 300 fish with an average weight of 12.00 ± 0.33 g were randomly distributed in 12 fiberglass tanks with a capacity of 300 L and fed with diets supplemented with ZnO-NPs at concentrations of 0 (control), 10.00, 50.00, and 100 mg kg^-1 for 60 days. The results indicated that 50.00 mg kg^-1 ZnO-NPs supplementation significantly improved fish weight, length, specific growth rate, and feed conversion ratio. There was no significant difference in survival rates between the treatments and control group; however, the highest survival rate was observed in the treatment with 50.00 mg kg^-1 ZnO-NPs. This treatment also led to the highest red blood cell counts, hemoglobin levels, mean corpuscular hemoglobin concentration, and lymphocyte percentages. In contrast, the highest neutrophil counts were observed at 100 mg kg^-1 ZnO-NPs. The highest serum biochemical parameters, including total immunoglobulin, lysozyme, and Zn levels were observed in the treatment with 50.00 mg kg^-1 ZnO-NPs. While, in the skin mucus, total immunoglobulin, lysozyme, and alkaline phosphatase significantly increased at groups fed with 10.00 mg kg^-1 ZnO-NPs. Overall, the findings suggest that dietary inclusion of 50.00 mg kg^-1 ZnO-NPs can effectively promote growth and enhance immune responses of C. carpio juveniles, indicating their potential application in aquaculture practices. However, caution is advised at higher concentrations due to the potential adverse effects on fish health.

Abstract This study aimed to identify and characterize nitrifying bacteria and their associated bacteriophages from swiftlet house environments as a preliminary step toward developing biological control strategies for nitrate contamination in edible bird nests,a food safety concern due to the excessive nitrite and nitrate accumulation. A total of 80 environmental samples were collected between October and November 2024 from four swiftlet houses in Central Kalimantan, Indonesia, including 20 samples each from feces, pond water, soil, and edible bird nests (Aerodramus fuciphagus). Phenotypic characterization (culture, Gram staining, and biochemical tests) and genotypic identification (PCR and 16S rRNA gene sequencing) were performed. Bacteriophages were screened using spot tests and plaque assays. The study resulted in the isolation and identification of 15 Nitrobacter isolates. Molecular analysis confirmed all isolates belonged to the Nitrobacter genus. One isolate, Nitrobacter AP6, was selected as a representative host for phage screening due to its consistent growth and plaque clarity. Spot tests revealed four bacteriophages capable of lysing Nitrobacter AP6. These findings demonstrate the feasibility of isolating nitrifying bacteria and phages from swiftlet environments and provide a foundation for future biocontrol development, though further in vitro and in situ validation is required to assess effectiveness and safety.

Abstract Bovine ephemeral fever (BEF) is a re-emerging arbovirus infection, causing substantial economic losses primarily attributed to decreased milk production. BEF is a frequently reported disease in Pakistan, however, there is a lack of scientific data, particularly regarding gene sequencing and phylogenetic analysis of this viral infection in bovines. This research was planned for the molecular detection of the BEF virus in dairy animals of Sindh province, Pakistan. The association of various risk factors, including seasonal variation, species, breed, sex, age, lactation and feeding pattern was also analyzed. A total of 470 blood samples from cattle and buffaloes were collected across different topographical regions of Sindh from 2021-2022; and subjected to molecular detection through RT-PCR. Sequencing of positive RT-PCR amplicons and phylogenetic analysis were then performed. A questionnaire was designed to get comprehensive information about each animal before sample collection to develop an association between various risk factors. RT-PCR amplification of the glycoprotein gene yielded a 420 bp product and the phylogenetic tree revealed that the Pakistani BEF virus isolates clustered closely with strains from Iran, India and Australia indicating a strong genetic resemblance. The overall prevalence was 13.83% (65/470) which significantly higher in cattle (15.49%) than in buffaloes (3.70%), and cattle had significantly higher chance of infection than buffaloes (odds ratio = 4.31, confidence interval = 1.33 - 13.95). This study provided the first comprehensive gene sequencing data on BEF in Pakistan, significantly enhancing the global understanding of the virus through detailed molecular and phylogenetic analysis.

Abstract Corneal ulcers, particularly from alkaline chemical injuries, are a leading ocular complication in animals, accounting for about 20.00% of emergency referrals. These burns damage the conjunctiva and cornea, causing deep ulcers, potential perforation, delayed healing, infections and blindness. Prompt treatment is crucial to accelerate healing and minimize complications like permanent opacity. This study compared the efficacy of blood serums (autologous, homologous, and heterologous) against standard treatment (N-acetylcysteine and ciprofloxacin eye drops) and a control group (Normal saline) in treating induced corneal ulcers in five groups consisting of 10 healthy mixed-breed female dogs. Ulcers were created using 3.00 M sodium hydroxide-soaked filter paper on both eyes. Evaluations on 1, 7, 14, and 21 days assessed quantitative parameters (Total corneal and stromal thickness, ulcer diameter) and qualitative symptoms (photophobia, blepharospasm, discharge, congestion, edema, neovascularization, opacity) via ultrasonography and clinical exams, respectively. Results showed significant time- and treatment-dependent improvements. Heterologous serum notably reduced thickness and ulcer diameter from day seven compared to others. By days 14 - 21, all treatments (serums and drugs) lowered stromal thickness versus control. Qualitative parameters were improved significantly with all treatments from day seven. These findings supported the use of blood-derived serums, especially heterologous serum, to accelerate healing and improving clinical outcomes in canine corneal ulcers.