Veterinary Research Forum

Abstract Streptococcus agalactiae is a Gram-positive bacterial pathogen accountable for disease outbreaks in tilapia, resulting in substantial economic losses in aquaculture. In this study, a 10-round whole-cell Systemic Evolution of Ligands by Exponential Enrichment procedure was conducted to isolate and characterize single-stranded DNA aptamers with specific binding affinity to S. agalactiae. The enriched single-stranded DNA libraries were sequenced by high-throughput sequencing, yielding a total of 180,987 reads, of which 126,933 included unique aptamer sequences. The most frequent sequences were selected for further analysis. Among them, SA1 had the highest frequency, with 231 reads, whereas SA2 showed a lower number of 16 reads. These findings underscored the validity of combining whole-cell Systemic Evolution of Ligands by Exponential Enrichment, high-throughput sequencing and aptamer-structure analysis to explore the interaction between aptamers and bacterial cell surfaces. The selected aptamers not only showed promise for the detection of S. agalactiae but also served as molecular probes for identifying surface-exposed proteins, studying host-pathogen interactions and guiding targeted drug delivery in aquaculture-related applications.

Abstract Bone tissue engineering offers a potential alternative to conventional grafting by combining biocompatible scaffolds with biological stimulants. Polycaprolactone (PCL) is a biodegradable polyester with good mechanical strength and biocompatibility. Hydroxyapatite (HA) is a calcium phosphate mineral that is a major component of bone and has excellent bioactivity and biocompatibility. This research investigated the histopathological effects of PCL-HA nanocomposite scaffolds along with their combination with platelet-rich fibrin (PRF), on the regeneration of bone in rabbit calvarial bone defects. Four circular full-thickness bone defects of 5.00 mm in diameter were created on the calvarial bone of 15 male New Zealand white rabbits. Three defects were filled with PRF, PCL-HA, and PCL-HA/PRF, and one defect was served as a control with no filler. Histopathological evaluations were conducted at 4-, 8-, and 12-weeks post-implantation. Data were evaluated using the Kruskal-Wallis and Mann-Whitney U tests. Significant differences were observed between the treatment and control groups regarding bone regeneration throughout all 12^th weeks studied. In the 4^th weeks, no significant differences in bone regeneration were noted among the treatment groups. In the 8^th weeks, most new bone formation was observed in the PCL-HA/PRF group. Both the PCL-HA and PCL-HA/PRF groups significantly improved bone regeneration compared to the control and PRF groups, with the PCL-HA/PRF group demonstrating the greatest bone formation, and vascularization and the lowest inflammation by the 12^th week. Thus, PCL-HA/PRF could be considered as a suitable alternative to bone grafts and could be increasingly utilized in orthopedic surgery and bone tissue engineering.

Abstract The present study evaluated the protective effects of royal jelly (RJ) on methotrexate (MTX)-induced testicular damage in rats, focusing on oxidative stress and autophagy. Methotrexate, a folic acid analogue used in cancer and autoimmune treatments, impairs spermatogenesis via oxidative stress and apoptosis. Twenty-four male Wistar rats were randomized into four groups: Control (normal saline, 35 days), MTX (0.30 mg kg-1, gavage, three times per week, 35 days), MTX + RJ (0.30 mg kg^-1 MTX + 0.10 mg kg^-1 RJ, gavage, three times per week, 35 days), and RJ (0.10 mg kg^-1, gavage, three times per week, 35 days). After 35 days, rats were euthanized and testicular tissue was analyzed via histopathology, immunohistochemistry for LC3-I/II expression in germ cells and qRT-PCR for mRNA expression of autophagy-related genes (Beclin-1, Atg7, LC3-I). Histopathological findings revealed that MTX caused severe interstitial edema, coagulative necrosis and disrupted spermatogenesis with reduced seminiferous tubule diameter, epithelial thickness, tubular differentiation index (TDI) and spermiogenesis index. Co-administration of RJ significantly improved seminiferous tubule morphology, diameter, epithelial thickness, TDI and spermiogenesis index. Immunohistochemistry showed a significant increase in LC3-I/II+ germ cells (spermatogonia, spermatocytes, spermatids) in the MTX group which was markedly reduced in the MTX + RJ group. Similarly, qRT-PCR analysis demonstrated elevated mRNA levels of Beclin-1, Atg7, and LC3-I in the MTX group which were significantly reduced in the MTX + RJ group. These findings suggested that RJ mitigated MTX-induced testicular damage by reducing oxidative stress and autophagy, thereby, preserving spermatogenesis and testicular integrity.

Abstract To mitigate metabolic disorders and skeletal abnormalities associated with rapid growth in broilers, and to reduce reliance on antibiotics, strategies involving feed restriction and non-antibiotic dietary additives are increasingly explored. This study investigated the effects of various feeding methods and additives on weight gain, feed intake, feed conversion ratio, carcass characteristics, intestinal morphology and ileal digestibility in broiler chickens. The research involved 480 one-day-old Ross 308 male broiler chickens. A 2 × 5 factorial experiment, arranged in a completely randomized design, incorporating two feeding methods (without and white quantitative restriction) and four types of additives including thyme essential oil (300 g per ton), oregano essential oil (300 g per ton), equal weight ratio of thyme and oregano essential oil (300 g per ton) and probiotic (250 g per ton) along with control diets (without additives). Dietary supplementation with oregano essential oil significantly increased weight gain compared to the other additive groups. While the feeding method, additives and their interactions significantly influenced feed intake in chickens and the feed conversion ratio was primarily affected by the type of additive. Supplementation with any of the tested additives led to reduced total feed intake and improved final feed conversion ratio compared to the control group. Histological analysis revealed that chickens receiving oregano exhibited the highest villus height to crypt depth ratio. In conclusion, this research suggested that quantitative feed restriction, particularly when combined with specific dietary additives such as oregano essential oil could significantly improve broiler growth performance, feed efficiency and intestinal morphology, offering potential alternatives to conventional practices.

Abstract Crocetin (CRT) is one of the active chemical compounds of saffron and has many biological effects such as antioxidant property. The present study investigated the effects of CRT on crushed sciatic nerve function. Vitamin (Vit) C was used as an antioxidant drug. Thirty rats were divided into six groups including intact, sham, crush, CRT 7.50, CRT 30.00 and Vit C 100. Nine other rats with no surgery were scheduled in three groups to receive 7.50 and 30.00 mg kg^-1 CRT and 100 mg kg^-1 Vit C. In anesthetized rats, right sciatic nerve was crushed using a small hemostatic forceps. Sciatic functional index values on days five, 10, 15 and 20 after crush were accelerated, the severities of sciatic nerve degeneration and gastrocnemius muscle atrophy were ameliorated, and the increased malondialdehyde level and the decreased superoxide dismutase activity in the serum were restored by 20 consecutive days of oral administration of 30.00 mg kg^-1 CRT and 100 mg kg^-1 Vit C. No significant differences were observed between 30.00 mg kg^-1 and 100 mg kg^-1 Vit C. The groups that did not have surgery but received CRT (7.50 and 30.00 mg kg-1) and Vit C (100 mg kg-1) showed no behavioral, histopathological and biochemical alterations when compared to intact group. It was concluded that CRT and Vit C produced similar improving effects on crushed-injured sciatic nerve function. Inhibition of oxidative stress, enhancement of endogenous antioxidant activity might be involved in improving effects of CRT and Vit C.

Abstract Bovine viral diarrhea virus is a pestivirus of the Flaviviridae family including two biotypes, cytopathic (CP) and non-CP (NCP). This study aimed to evaluate DNA damage and apoptosis in Madin-Darby bovine kidney cells following infection with both biotypes. The MDBK monolayers were inoculated with a final dose of virus (1.00 × 10³ Tissue Culture Infectious Dose 50% mL-1) and incubated for 24 hr. DNA strand integrity was assessed using alkaline single-cell gel electrophoresis, and DNA damage was quantified through tail moment and olive tail moment indices (n = 3). Apoptosis was evaluated using annexin V–fluorescein isothiocyanate/propidium iodide flow cytometry to determine early and late apoptotic cell populations. Both biotypes significantly increased DNA fragmentation compared to the control group. The tail moment values were 15.89 ± 2.13 (control), 57.63 ± 16.20 (NCP), and 68.15 ± 9.93 (CP); while, olive tail moment values were 8.71 ± 1.01 (control), 29.35 ± 9.18 (NCP), and 35.14 ± 6.90 (CP). Apoptosis analysis showed a higher percentage of apoptotic cells in infected groups, with CP biotype of bovine viral diarrhea virus inducing the greatest early and late apoptotic responses, being consistent with its CP nature. Overall, both biotypes caused notable genomic injury and apoptosis in Madin-Darby bovine kidney cells, with CP producing the highest level of damage, confirming single-cell gel electrophoresis combined with apoptosis assays as sensitive tools for detecting virus-mediated genomic instability and supporting their potential application in breeding programs aimed at enhancing resistance to infectious diseases.

Abstract Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease and a potential contributor to Crohn’s disease, presents a significant challenge due to its resistance to conventional antibiotics. This necessitates the development of innovative strategies for prevention and treatment. This study aimed to evaluate the anti-bacterial activity of pathogen-specific antibodies derived from chicken egg yolks (immunoglobulin Y [IgY]) and the postbiotics from lactic acid bacteria against MAP. Immunoglobulin Y antibodies were produced by immunizing hens with formalin-killed MAP strain antigens. The IgY was extracted and purified, and the anti-MAP titers were quantified by indirect enzyme-linked immunosorbent assay. The minimum inhibitory concentration of different concentrations of specific anti-MAP IgY and the mixture of postbiotics (from four different probiotic strains, including Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Pediococcus acidilactici) individually and in combination against MAP was determined at various time intervals. Anti-MAP IgY titers in egg yolks increased within 2 weeks of immunization, reaching peak levels at 6 weeks. Growth inhibition assays revealed that postbiotics concentration as low as 6.25 mg mL^-1 effectively inhibited MAP growth. Anti-MAP IgY demonstrated anti-bacterial activity with a minimum inhibitory concentration of 50.00 mg mL^-1, while the combined IgY-posbiotics treatment achieved MAP growth inhibition at a minimum inhibitory concentration of 3.125 mg mL^-1. The findings of the study suggest that combination therapy with specific IgY and postbiotics may be a promising preventive strategy for controlling MAP infections. Further in vivo studies are needed to elucidate the underlying mechanisms and optimize the application of this approach for broader use in veterinary and human medicine.

Abstract Selenium (Se) and gold (Au) nanoparticles (NPs) possess unique properties such as high absorption ability, low toxicity and minimal environmental persistence. These properties, combined with demonstrated anti-parasitic effects against certain parasites, suggest their potential as candidate for house fly control. This study aimed to evaluate the effectiveness of Se and AuNPs in controlling house flies. We investigated the insecticidal effect of Se and AuNPson Musca domestica using a dipping method. Several concentrations (20.00, 60.00, 100, 400, and 1,000 ppm) were prepared using deionized water. Fifteen larvae per concentration were tested, with each test repeated three times. The larvae were dipped in the NP solutions for 30, 60 and 90 sec in 10.00 mL of the tested concentrations. Distilled water was used for the control group. Larval mortality was recorded 24 hr post-treatment. No mortality was observed in house fly larvae 24 hr after exposure to different concentrations of Se and AuNPs, regardless of the incubation time. Thus, Se and AuNPs showed no insecticidal effect on house fly larvae. In conclusion, Se and AuNPs cannot be used to control house flies. Due to the issues associated with chemical control, it is crucial to search for alternative pesticides.

Abstract Physalopteridae nematodes pose a threat to a broad spectrum of animals including amphibians, reptiles, birds and mammals. The current study was the first molecular and phylogenetic characterization of Physaloptera clausa parasitizing long-eared hedgehogs (Hemiechinus auritus) in Iran. A male road-killed H. auritus was collected from Shahriar City, Tehran province in May 2022 and it was subjected to necropsy. After isolating parasites, they underwent morphological analysis using a light microscope and an identification key. For molecular analysis, the genomic DNA was isolated using the FavorPrepTM Tissue Genomic DNA Extraction Mini Kit. The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. For phylogenetic analysis, these nucleotide sequences were aligned in MEGA 11 based on the lowest bayesian information criterion score. A cluster of parasites was found on the mucosa of the hedgehog stomach. All the nematodes were identified as P. clausa. The sequence obtained in this study has been submitted to GenBank^® with the accession number OR088573. The phylogeny analysis revealed that the genus Physaloptera formed a major clade where P. clausa was clustered with various Physaloptera species, closely related to Turgida, Physalopteroides and Skrjabinoptera genera. Our study specified the necessity for additional taxonomic and phylogenetic research on Physaloptera species and related genera to fully understand the evolutionary dynamics and ecological significance of these parasites.

Abstract Generalized subcutaneous emphysema (GSE) is an uncommon clinical presentation in equids, typically associated with penetrating trauma and rarely compromising systemic health. This case report documents a 4-year-old working mule presenting with diffuse subcutaneous emphysema secondary to an axillary penetrating wound in the right forelimb. Clinical examination revealed pronounced crepitus without concurrent ataxia, dyspnea, dehydration, or gastrointestinal dysfunction. Diagnostic investigations, including ultrasonography, radiography, complete blood count, paracentesis, and bacterial culture, yielded unremarkable hematological findings and no microbial growth. The wound was managed via local debridement, with adjunct systemic therapy, including penicillin G and flunixin meglumine. Complete resolution occurred within five days under stall rest. This case underscores that generalized subcutaneous emphysema in mules may represent a benign, self-limiting condition when infectious etiologies are excluded. Conservative management, including compulsory rest and anti-inflammatory therapy, can facilitate spontaneous resolution while mitigating risks of pneumothorax or gas gangrene. The rarity of non-infectious generalized subcutaneous emphysema in equids warrants further etiopathological investigation to establish species-specific prognostic criteria.