Veterinary Research Institute

Abstract AIMS Thrombolytic enzymes represent an accessible and streamlined treatment for acute ischemic stroke, yet current FDA-approved agents, alteplase and tenecteplase, are limited by incomplete recanalization, hemorrhagic risk, and neurotoxicity. We aimed to develop a multidisciplinary workflow for rational design and multi-level testing of thrombolytic proteins to generate a next-generation thrombolytic with improved potency and safety. METHODS AND RESULTS Our pipeline integrated in silico design, biochemical characterization, in vitro thrombolysis, and in vivo validation. Computational screening included rational mutagenesis to reduce side effects, reconstructed ancestral sequences, and database-mined homologs. Selected candidates were biochemically assessed for enzymatic activity, fibrin stimulation, selectivity and affinity, inhibition resistance, and clot penetrability. The most promising variants underwent in vitro clot thrombolysis and in vivo efficacy and safety studies in rats. Brnoteplase emerged as the lead candidate, exhibiting 80-fold enhanced fibrin selectivity, 4-fold higher inhibition resistance, and superior clot penetrability compared to alteplase. In vitro , Brnoteplase demonstrated deeper intrathrombus penetration and a progressive dose–response profile, unlike alteplase, which declined at higher concentrations. In vivo , Brnoteplase achieved higher thrombolysis rates and recanalization frequency across all doses, with extended biological half-life enabling bolus administration. Safety assessments revealed the lowest incidence of severe hemorrhagic transformation and minimal hemispheric asymmetry. CONCLUSIONS We established a robust pipeline for efficient generation and selection of novel thrombolytics and identified Brnoteplase as a promising candidate combining improved fibrin selectivity, inhibition resistance, biological half-life, and clot penetration. These features translate into higher thrombolytic efficacy and a promising safety profile in vivo , warranting further preclinical evaluation to confirm its potential as a next-generation thrombolytic for acute ischemic stroke.

The Usutu virus (USUV; Orthoflavivirus, Flaviviridae) is a mosquito-borne pathogen causing fatal neuroinfections in susceptible wild and captive birds, particularly blackbirds, other passerines, and owls. Zoological gardens provide favourable conditions for the circulation of such viruses due to the proximity of diverse species and limited options for prevention. Following the sudden death of several Chilean tinamous kept in the Brno zoological garden, we tested tissues sampled from 22 bird cadavers (from zoos, private owners, and free-living birds) for the presence of USUV and West Nile virus (WNV) RNA using duplex reverse transcription qPCR. Near-complete whole-genome sequences were acquired from positive samples by next-generation sequencing and subjected to phylogenetic analyses. Furthermore, serum samples from additional zoo animals and privately owned birds were screened for anti-flavivirus antibodies using ELISA and subsequently confirmed by the virus neutralization test. We report fatal USUV infections in multiple bird species from three zoological gardens in the Czech Republic. Duplex RT-qPCR targeting USUV and West Nile virus (WNV) detected USUV RNA in tissues from two Boreal owls (Aegolius funereus), one Eurasian pygmy owl (Glaucidium passerinum), two Brahminy starlings (Sturnia pagodarum), and four Chilean tinamous (Nothoprocta perdicaria). Additionally, three randomly found cadavers of free-living blackbirds (Turdus merula) tested positive. Pathological findings ranged from minimal pathological changes to pronounced hepatosplenomegaly with intestinal bleeding. Phylogenetic analysis of near-complete genome sequences assigned all viruses to the Europe 2 genetic lineage, revealing partial geographic clustering among isolates obtained in this study. Serological testing confirmed exposure in additional birds and demonstrated cross-neutralisation between anti-USUV and anti-WNV-positive sera. In zoological gardens, flavivirus infections can cause substantial losses, even among rarely bred or endangered species. Given the zoonotic potential of both USUV and WNV, documenting their occurrence in avian hosts is important not only for animal health but also for human disease surveillance from the One Health perspective.

Inter-laboratory trials were carried out to evaluate the reproducibility and estimate test performance characteristics of the Phagomagnetic separation (PhMS)-qPCR assay, a novel phage-based assay recently developed as a rapid alternative to culture for detecting viable Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk and feces. Unique reagents and a detailed instruction manual required for the PhMS-qPCR assay were provided to five European veterinary diagnostic laboratories by Rapid-Myco Technologies Limited. Milk and feces test panels were prepared at NEIKER and distributed to participant laboratories between April–June 2023 and March–May 2024, respectively. Each test panel comprised of MAP-spiked and/or naturally infected bovine milk or feces samples (18 samples per panel on two separate occasions for each sample matrix). The six participant laboratories (including organizer) performed automated or manual PhMS and used whatever qPCR instrument they had available. All laboratories used the IDEXX RealPCR MAP DNA test for the qPCR part of the assay. Generally, substantial agreement was observed overall between PhMS-qPCR results and reference culture results for spiked milk (Kappa value 0.5982) and naturally MAP-infected feces (Kappa value 0.7780 using an amended protocol). Preliminary estimates of the detection (analytical) sensitivity (Se), detection specificity (Sp) and trueness (T) of the PhMS-qPCR assay applied to bovine milk and feces were obtained. The mean Se, Sp, and T values across six laboratories were 93.1%, 67.9%, and 88.7% when milk was tested and 84.1%, 93.7%, and 88.9% when feces was tested. Overall, the PhMS-qPCR assay performed well in multiple laboratories and test reproducibility was demonstrated (Cohen's Kappa ≥0.6–1.000). The estimates of performance characteristics of the PhMS-qPCR assay are generally acceptable for a potential diagnostic test. Hence the PhMS-qPCR assay shows considerable promise as a rapid test to detect viable MAP in veterinary specimens such as milk and feces. Further and fuller validation of the assay will continue to assess its diagnostic potential.

Theileria, Babesia, and Anaplasma species caused haemoparasitic infections which represent major constraints to cattle and buffalo productivity in subtropical and tropical areas. This study has evaluated the prevalence and risk factors of these hemoparasites in Lahore District, Punjab, Pakistan, through microscopic examination of blood smears of 2,176 cattle and buffaloes, with reference to associated risk factors, including age, sex, and management system. The total prevalence was 10.3% (224/2,176 positive samples). Among positive Theileria spp. were the most predominant positives (94.6% 212/224), followed by Babesia spp. (3.6% 8/224), and Anaplasma spp. (1.8% 4/224) meanwhile, there were no positive results of Trypanosoma spp. or mixed infections. The rates of infection were notably higher in animals below 2 years of age (15.6%, 109/700) than in those at age 2 years and older (7.8% 115/1476; p=0.001) and in females (11.6%, 160/1376) than in males (8.0, 64/800; p=0.01-0.04), probably because of immunological immaturity and reproductive stress The prevalence of animals in extensive/ grazing systems was almost twice (13.9%, 136/976) when compared to semi-intensive stall-fed systems (7.3%, 88/1200; p=0.003), and this indicates higher exposure to ticks in open grazing. These results affirm that tropical theileriosis is endemic in peri-urban Punjab with less burdens of babesiosis and anaplasmosis potentially attributed to the ecological conditions of vectors and their management. The microscopy is probably underestimating actual prevalence because there is low parasitemia in the carriers.

Fluconazole (FLU) is a widely used antifungal agent frequently detected in surface waters because of its extensive use in medicine, agriculture, and personal care products.Despite concerns about its persistence and developmental toxicity in aquatic species, its effects on amphibians remain poorly understood. This study aimed to assess the developmental and molecular effects of FLU and its structural core, 1,2,4-triazole (TRI), in amphibian embryos. Xenopus laevis embryos were exposed to FLU or TRI and evaluated for mortality, hatching rate, heart rate, body length, malformation incidence, and changes in gene expression. Even at low micromolar concentrations, both azoles altered the expression of Wnt- and BMP-associated genes, indicating disruption of these signaling pathways. At higher micromolar concentrations, these molecular changes were accompanied by early signs of developmental abnormalities, which intensified at the highest doses. Observed phenotypes included reduced head size, altered skin pigmentation, prolonged body length, changes in heart rate, and mild digestive tract malformations. These findings demonstrate that even the core structural motif TRI can disrupt key developmental signaling pathways in vertebrate embryos, underscoring the need for closer monitoring of azole compounds in aquatic environments. Given the fundamental role of these pathways in vertebrate development, the results raise concerns about potential risks from long-term or prenatal exposure to azoles, in both environmental and clinical contexts.

Loss of skin elasticity and youthful appearance is closely linked to reduced hyaluronic acid (HA) levels in the dermis. While topical HA products offer temporary effects, they do not address the root cause of HA depletion. The present study aims to describe how to induce HA synthesis through fibroblast stimulation. Twelve sows were divided into two groups. One group (n = 9) received four consecutive Radiofrequency (RF) and Targeted Ultrasound (TUS) treatments, the other group (n = 3) received four RF treatments only. Samples were collected via punch biopsy from the treated area of each animal at the baseline, 1-month, and 2-month follow-up. Samples were then processed and prepared for enzyme-linked immunosorbent assay as well as hyaluronic acid binding protein with diaminobenzidine (HABP-DAP) staining, which allows for visualization of HA within tissue. The group receiving RF + TUS treatment has demonstrated an increase from an average of 83.0µg/g at the baseline to 163.0µg/g at the 2-month follow-up of HA concentration in the skin. Histology samples clearly demonstrate higher tissue density with an increase of brown pigment due to the HABP-DAP stain, representing HA in the skin. In comparison, the RF standalone treatment group has shown no significant increase in HA concentrations. Study results show no significant increase in HA production following the RF stand alone treatment. Whereas the RF + TUS treatment group induced a significant HA production response as well as histophysiological changes in the dermis.

Abstract Aquagenic urticaria is an extremely rare dermatological disorder characterised by the rapid onset of pruritic erythematous wheals following cutaneous exposure to water, irrespective of its temperature. A 4‐year‐old female Nili‐Ravi buffalo was presented with a 1‐month history of sudden allergic reactions triggered by water contact. The animal had normal feed and water intake and regular defecation and urination. Upon water exposure, severe urticarial lesions developed on the tongue, eyes, hindlimbs and teats, accompanied by ocular discharge, conjunctivitis and nasal discharge. Clinical examinations revealed normal vital signs, haematobiochemical parameters and thoracic radiography, with the absence of any discernible lesion. Random skin scrapings and dermographic testing were unremarkable. Following the water provocation test, the animal was suspected of aquagenic urticaria. The animal was treated with antihistamines and prednisolone, showing full recovery within 5 days. To our knowledge, this represents the first reported case of aquagenic urticaria in buffaloes.

The paper presents the findings on occurrence of cestode infection in wild carnivores during postmortem examination across different forest ranges in the Nilgiris district of Tamil Nadu, India over a period of sixteen months. A total of 37 wild carnivores comprising of seven dholes, four leopard cats, ten tigers and 16 leopards were examined from December 2023 to March 2025. Postmortem examination was conducted by the forest veterinary officer following standard procedures. The tapeworms were collected, labelled, processed as per standard procedures, identified and photographed. Out of the 37wild carnivores, 11 were found infected with cestodes (29.72%) which were identified as Taenia hydatigena morphologically. This comprised of one leopard cat (25%), two dholes (28.57%), five tigers(50%) and three leopards (18.75%). Out of the 11 infected animals, six were males (54.54%) and five were females (45.45%). Also, among the infected animals, one was below one year of age (9.09%) and 10 were above one year of age (90.90%).

Due to the low populations of Bacteroides sp. in the gut microbiota of sows compared to nursed piglets, sows may not represent an ideal source of Bacteroides sp. for newborn piglets. In this study, we therefore tested the effect of oral administration of a mixture of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis and Bacteroides xylanisolvens on the microbiota development of newborn piglets. Oral administration of such a mixture to piglets within 12 h after parturition did not result in any adverse effects. Sequencing of 16S rRNA showed that 4 days after administration, these species formed approx. 20% of total faecal microbiota and affected the development of gut microbiota in treated piglets. The treatment resulted in an increased abundance of Veillonella caviae, Fusobacterium gastrosuis, Dialister sp., Clostridium jeddahitimonense, C. cadaveris, Butyricicoccus pullicaecorum, Actinobacillus indolicus, A. minor, Streptococcus pasteurianus, S. parasuis, S. equinus, S. pluranimalium, S. thoraltensis and S. suis. On the other hand, administration of the Bacteroides mixture suppressed piglet colonisation by C. disporicum and multiple species from family Prevotellaceae. Bacteroides-treated piglets exhibited significantly higher body weight than untreated controls at 3 months of age. Administration of a mixture of Bacteroides shaped the development of gut microbiota in nursed piglets, which resulted in improved parameters at the end of the pre-fattening period.