Veterinary Research Forum

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 described 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 CSCC. Despite complete initial removal, tumor recurrence was noted seven months postoperatively. The owner declined further chemotherapy and surgical intervention. This report detailed the clinical case of a massive, CSCC located in the ventral abdominal/inguinal mammary region of a male Pitbull dog. Although CSCC is a common neoplasm in canines, this case was distinguished by its extreme size, secondary infection and atypical anatomical presentation in a male dog. The report highlighted the unique clinical challenges associated with managing an unusually severe manifestation of a common tumor and provided a comprehensive overview of the surgical, radiological and histopathological findings.

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 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 This study explored the effects of different levels of hempseed meal(HSM) supplementation in laying hen diets, with and without a multienzyme supplement, on egg production, quality, yolk fatty acid composition. A total of 144 White Leghorn hens, aged 42 weeks, were divided into eight groups with six replicates each, and the experimental groups were fed with four HSM levels (0%,5%,10%,15%) with or without 1000mg multienzyme supplement per kg feed for a period of 84 days trial. Results indicated that with or without multienzyme did not significantly affect egg production, weight, mass, feed conversion ratio. Feed intake was consistent across HSM levels, but cubic interaction between multienzymes and HSM was observed(p<0.05). Eggshell parameters were unaffected by HSM or multienzyme supplementation. Yolk color(L* value) showed a cubic response to HSM levels, peaking in 5% treatment. The addition of multienzymes and HSM significantly decreased the a*value(p<0.05), while the b*value increased quadratically with HSM supplementation(p<0.05). Yolk fatty acid composition exhibited linear and cubic effects from HSM and multienzyme HSM interaction(p<0.05), with increased total polyunsaturated fatty acids(ƩPUFA) and decreased total monounsaturated fatty acids(ƩMUFA)(p<0.05). The omega-6/omega-3 ratio was linearly influenced by HSM supplementation(p<0.05). In summary, multienzyme supplementation alone did not significantly alter production or quality parameters, but interacted with HSM to affect feed intake, yolkcolor(a* value), and fatty acid composition. These findings suggest that HSM supplementation may improve the quality of egg yolk, although it does not affect overall egg production, and may therefore be viable option to improve the fatty acid composition of eggs.

Abstract Phthalates are endocrine-disrupting chemicals widely used in numerous consumer products, making human and animal exposure a significant public health concern.
This study evaluated the protective effects of royal jelly (RJ) against dibutyl phthalate (DBP)-induced ovarian damage. Forty female Wistar albino rats were randomly allocated into eight groups (n=5 per group): control (corn oil), DBP (500 mg kg-1), RJ (200 mg kg-1), Quercetin (QCN) (50 mg kg-1), DBP + RJ (100, 200, and 300 mg kg-1), and DBP + QCN (50 mg kg-1) for 28 consecutive days by gavage. QCN was used as a positive antioxidant control. RJ was dissolved in normal saline, whereas QCN and DBP were dissolved in corn oil. The levels of testosterone, dehydroepiandrosterone (DHEA) and TNF- in the serum were measured. Oxidative and nitrosative stress biomarkers (MDA, NO, TTM) were assessed in the ovarian tissue. The histopathological alterations of ovarian tissue and ovarian index were also evaluated. RJ significantly reduced DBP-induced elevations in serum testosterone and DHEA levels. Compared with the DBP group, RJ treatment significantly restored the ovarian index and reduced serum TNF-α level. RJ treatment significantly attenuated DBP-induced oxidative and nitrosative stress in ovarian tissue.
DBP disrupted normal ovarian architecture, characterized by increased atretic and cystic follicles and reduced corpora lutea formation; these alterations were markedly ameliorated by RJ. In conclusion, RJ attenuated DBP-induced ovarian toxicity, as evidenced by reduced oxidative/nitrosative stress, decreased serum TNF‑α, and lowered serum testosterone and DHEA levels.

Abstract Superficial digital flexor (SDF) tendonitis is a leading cause of lameness in race and polo horses. Traditional therapies do not always prove effective in eliminating tendinopathy, thus culminating in tendon re-injury, persistent lameness, and performance shortfalls in affected horses. This study assessed therapeutic outcomes of conventional therapies versus extracorporeal shockwave therapy (ESWT), applied on a total of 18 race and polo horses affected with chronic tendinitis. The horses were divided into three groups (n=6): Group A (control, sham treated), Group B (blister treated), and Group C (shock wave treated with 500–1500 pulses, every two weeks for a total of 4-6 treatments). Follow-up clinical examinations revealed that warmth, swelling, and pain scores were significantly lower for the shock wave Group C (P<0.01). Shock wave therapy proved superior in providing an early analgesic effect (day 28) versus the late analgesia provided by blister group B. Lameness scores were likewise significantly lower for the shock wave group C (P<0.01), with an early return to full performance (day 84) versus delayed performance (day 98) and persistent lameness in the blister group B. Assessment of the injured, treated site, through color Doppler ultrasound at the maximum injury zone (MIZ), revealed significant Doppler signals suggestive of vascularization and early healing in the ESWT Group C (P<0.01) versus the absence of such signals in the blister and control groups (B and A), respectively. Conclusively, shockwave therapy proves to be superior to blistering, owing to its greater efficacy, humane approach, early regeneration, and lack of reported complications.

Abstract The aim of this study was to evaluate if amino acids (aa) or endogenous lipids may be used as unique oxidative substrates for porcine oocyte in vitro maturation (IVM). Immature porcine cumulus-oocyte complexes were in vitro matured in NCSU-37 (without pyruvate and glucose, negative control), NCSU-37+L-carnitine (lipid catabolism stimulator), NCSU-37+etomoxir (lipid catabolism inhibitor), NCSU-37+aa, NCSU-37+aa+salicylate (aa catabolism inhibitor), or NCSU-37+glucose (positive control). Oocyte nuclear and cytoplasmic maturation were determined by metaphase II rate and cleavage and blastocyst production rates, respectively. The use of endogenous lipids was determined by evaluating oocyte lipid content after IVM using Nile Red stain, while the use of aa was evaluated measuring ammonia production using spectrophotometry. The medium supplemented with L-carnitine showed a higher nuclear maturation rate (35.3%) compared to those without oxidative substrates (8%) or added with etomoxir (0%) and an increase in lipid consumption respect to the other treatments. Supplementation with aa during IVM resulted in a faster meiotic progression (52.4% of metaphase II) and a higher level of residual ammonia compared with the other groups. No significant differences, however, were detected for cytoplasmic maturation rates between oocytes matured in the presence of aa, L-carnitine or glucose. Our results indicate that both endogenous lipids or aa may be used as unique substrates for porcine oocyte metabolism during IVM, sustaining nuclear and cytoplasmic maturation.

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 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 Infectious coryza, caused by Avibacterium paragallinarum, poses a significant economic threat to the global poultry industry. Although infectious coryza is present in Iran, there is limited molecular data characterizing the circulating strains, particularly regarding key hemagglutinin genes such as hagA. This study aimed to investigate the genetic diversity and phylogenetic relationships of the Av. paragallinarum isolates recovered from commercial and backyard poultry in Iran by sequencing the hagA gene and comparing the findings with those of global strains. Twenty-three Av. paragallinarum isolates collected from Iranian poultry farms underwent PCR amplification and sequencing of the hagA gene. Nucleotide and deduced amino acid sequences were analyzed for homology, subjected to BLAST searches, and used for phylogenetic reconstruction. The isolates showed high sequence identity, ranging from 94.10 - 99.90% at the nucleotide level and 92.10 - 100% at the protein level. Phylogenetic analysis classified the isolates into five distinct clusters (Iran-1 to Iran-5). The largest cluster (Iran-1) showed a strong affiliation with strains from China and India, whereas the other isolates clustered with strains from South Korea, Germany, and Japan. Notably, the Iran-5 group exhibited high similarity to a non-typable strain from USA. Additionally, eight isolates encoded truncated HagA proteins, each consisting of 344 rather than 345 amino acids. This study demonstrates the circulation of genetically diverse Av. paragallinarum strains in Iran. These findings highlight the need for further molecular investigations to improve control measures and assess the efficacy of the vaccines currently used in the country.

Abstract Aquaculture has a fundamental role in supporting global food security. In response to the escalating human requirement for protein, the production of aquatic species is expanding rapidly. Conversely, aquatic animals are frequently subjected to a variety of husbandry stress, including handling and high stocking densities. They trigger physiological stress responses negatively affecting biological processes, such as somatic growth, reproduction, and immune function, ultimately resulting in inferior performance and high mortality rates. Although antibiotics are still extensively employed, their persistent administration is associated with the emergence of bacterial resistance, destabilizes endogenous microbiota, and causes chemical residues accumulation within aquatic environments. Therefore, the industry has shifted towards eco-friendly therapeutic strategies. In this context, plant essential oils have emerged as promising feed additives. The EOs contain a rich array of bioactive compounds, possessing anti-bacterial, anti-oxidant, and immuno-stimulatory activities. The administration of EOs has been evaluated in a wide range of aquatic species to characterize their therapeutic potential and establish optimal inclusion levels. Research findings indicate that plant EOs represent effective substitutes for antibiotics, playing prominent roles in growth promotion, oxidative stress mitigation, and immune modulation. The present context reviews the beneficial roles of essential oils in modern aquaculture.

Abstract Micromeria congesta (MC) is a medicinal plant traditionally used in the Şanlıurfa, Adıyaman, and Gaziantep provinces of Türkiye for treating respiratory disorders. Despite its ethnobotanical relevance, its pharmacological effects on lung inflammation remain unclear. This study evaluated the protective effects of MC essential oil (MCEO) against lipopolysaccharide (LPS)-induced acute lung injury in rats, with a focus on inflammation and tissue damage. The MC was collected from its natural habitat, and its EO was extracted by hydro-distillation using a Clevenger-type apparatus. The Gas chromatography-mass spectrometry analysis revealed 28 compounds, with piperitenone oxide (33.97%) and pulegone (22.30%) as the main constituents. Thirty-six Wistar albino rats were randomly assigned into six groups, including three MCEO-treated (12.50, 25.00, and 50.00 mg kg^-1), dexamethasone, LPS-only, and control groups. The MCEO was administered by gavage for seven days, followed by intra-venous LPS on day eight (except for the control group). Blood and lung tissues were collected 6 hr later for biochemical, histopathological, and molecular analyses. The MCEO treatment significantly lowered blood glucose and urea levels compared to the LPS group. It also modulated NLRP3, caspase-3, and tumor necrosis factor alpha genes expressions, as determined by RT-PCR analysis, indicating anti-inflammatory effects. Histopathological findings confirmed a dose-dependent reduction in lung inflammation and tissue damage. The MCEO administration alleviated LPS-induced acute lung injury in rats through anti-inflammatory and protective effects, as confirmed by molecular (NLRP3, caspase-3, and tumor necrosis factor alpha) and histopathological analyses. Additionally, MCEO reduced blood glucose and urea levels, supporting its systemic protective role against endotoxin-induced inflammation.

Abstract Swine colibacillosis, caused by colonization of the piglet small intestine with enterotoxigenic strains of Escherichia coli, imposes a significant economic burden on the swine industry. Due to high mortality rates and the emergence of antimicrobial resistance, it is considered one of the major welfare concerns in the livestock industry. This study elucidated the genetic basis and expression patterns of key antibiotic resistance genes in porcine E. coli isolates with colibacillosis. Intestinal contents from five affected piglets were subjected to high-throughput sequencing, revealing 26 resistance genes across seven antibiotic classes. Four strains (428, 430, 432, 433) were further analyzed for mRNA expression levels of six resistance genes (blaTEM, tetA, tetB, tetM, qnrB, and qnrS) under exposure to ampicillin, tetracycline and ciprofloxacin at three concentrations (0.01, 0.001, 0.0001% w/v). Our findings revealed five distinct expression patterns: (1) dose dependent upregulation/downregulation, (2) inverse response to concentration changes, (3) maximal downregulation at intermediate dose, (4) minimal down-regulation at intermediate dose, and (5) sustained upregulation. Strain 428 uniformly followed pattern one for all resistance genes, in strain 430, tetB followed pattern three while blaTEM matched pattern one, strain 432 exhibited patterns two for all tetracycline- and β-lactam-resistance genes, and pattern four for qnrB; strain 433 showed patterns three for both fluoroquinolone-resistance genes, pattern four for tetB, and pattern five for blaTEM. These findings highlighted strain-specific and dose-dependent gene expression responses in swine colibacillosis. This work underscored the need for tailored antibiotic strategies, targeted interventions and well-validated monitoring frameworks for antibiotic resistance in veterinary.