Evaluation of the Virulence of Low Pathogenic H9N2 Avian Influenza Virus Strains in Broiler Chickens.

Our study aimed to investigate the virulence of three recent H9N2 LPAIV strains belonging to the G1 lineage, isolated from field infections in North Africa and the Middle East. Three-week-old commercial broiler chickens (in total 62) were included and randomly allocated into three infected test groups and one control group. Each test group was inoculated intranasally/intratracheally with one of the three H9N2 isolates at a dose of 108 EID50 virus. The control group received phosphate-buffered saline (PBS) via the same route of application. The pathogenicity was evaluated based on clinical signs and gross pathological and histopathological lesions, the viral antigen load was assessed through immunohistochemistry staining (IHC), and a semi-quantitative detection of the genetic material was conducted via a real-time PCR. Our findings confirmed the obvious respiratory tract tropism of the virus strains with variable renal tropism. In contrast to the highly pathogenic AIVs, the tested H9N2 strains did not show replication in the central nervous system. The virus presence and lesions, mainly in the respiratory tract, were predominant on dpi 5 and significantly reduced or disappeared by dpi 11. A clear difference was demonstrated among the three isolates: the A/chicken/Morocco/2021/2016 strain proved to be significantly more virulent than the Egyptian and Saudi Arabian ones, which showed no remarkable difference.

Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry.

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

In Situ Hybridization of Feline Leukemia Virus in a Case of Osteochondromatosis.

Osteochondromatosis, also known as multiple cartilaginous exostosis, polyostotic osteochondroma, and multiple osteochondromas, comprises one-fifth of all primary bone tumors in cats, with no breed or sex predisposition or hereditary pattern. Unlike in dogs, horses, and humans, it is predominantly seen in young cats (2-4 years old), after the maturation of the skeleton. Although the pathogenesis of osteochondromatosis is not fully understood, it is considered to be related to infection by feline leukemia virus (FeLV) or other retroviruses, such as the feline sarcoma virus. However, the presence of viral particles within tumor lesions has only been demonstrated by electron microscopy. The malignant transformation of osteochondromas, most typically to osteosarcoma or chondrosarcoma, has also been attributed to the viral infection. Here we report the case of osteochondromatosis in a 3.5-year-old male domestic European shorthair cat with concurrent FeLV infection confirmed by polymerase chain reaction. Viral RNA was visualized in representative tissues (spleen, mesenteric lymph node, liver, kidney, lung, brain) and in the osteochondromas with RNAscope in situ hybridization, which supports that FeLV infection may be involved in the pathogenesis of osteochondromatosis.

"The SOFTVETS Competence Model" - a preliminary project report.

Aim: Recent developments measured using statistics and surveys among veterinarians show that integrating key competence training into veterinary education is becoming increasingly important. This article describes the collaborative development process of the first work package within the SOFTVETS project. The SOFTVETS project aims to create a competence model and an ideal version of a soft skills curriculum that can be implemented in veterinary higher education throughout Europe. Method: In the course of a desk research phase, a literature review and an inventory of the current practice of key competence training within veterinary education was carried out. An initial set of recommendations for three competence areas was developed using the Handbook for Internal Quality Management in Competence-Based Higher Education. Finally, an alternating sequence of individual and collaborative expert reviews was carried out. Result: Experts from five European countries participated in the process. The derived competence model consisted of the following three competence areas with the corresponding number of defined competences: ten communication, nine entrepreneurial and eight digital competences. Conclusion: In the next work packages, learning objectives, teaching and assessment methods will be collected. Training concepts for facilitators to provide professional competence training will be established. In addition, an evaluation toolkit will be developed to standardise the implementation, evaluation and assessment of competence training events. The SOFTVETS competence model should help educators to be able to integrate the training of key competence training into the veterinary curriculum. This detailed list of competences can also be used as a tool to identify existing deficiencies and thus enable further curricular changes.

Prevalence of feline leukaemia virus and feline immunodeficiency virus in domestic cats in Ireland.

Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting felid species worldwide. A study was performed over a period of 5 months in Ireland with the aim to get an updated and more realistic prevalence of these retroviruses. A total of 183 EDTA-anticoagulated whole-blood samples were collected from cats distributed between 10 clinics. The samples were tested using both point-of-care enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Basic clinical data and vaccination history were also recorded for the sampled cats. The results of ELISA tests showed a prevalence of 10.4 and 3.3% for FIV and FeLV, respectively, and an apparent prevalence of 9.3% for FIV and 11.6% for FeLV with PCR. Phylogenetic analysis of the partial polymerase (pol) gene sequences obtained from 8 FIV-positive strains showed that all but one of the Irish strains belonged to FIV subtype A, and one to subtype B. The overall mean genetic similarity between the analysed strains was 91.15%.

In situ hybridization of feline leukemia virus in a primary neural B-cell lymphoma.

An 8-y-old castrated male, outdoor European shorthair cat was presented with a history of hindlimb weakness and paralysis. Disease progression was continuous from the onset; deep algesia disappeared at the final stage. Radiography of the vertebral column was unremarkable; along with patient history and physical examination results, magnetic resonance imaging suggested inflammatory lesions in the spinal cord, although neoplasia could not be ruled out. Feline leukemia virus (FeLV) positivity was confirmed by a serum ELISA prior to euthanasia. Upon postmortem examination, hemorrhages were present in the spinal cord at the level of vertebrae T7-8. Histologic and immunohistochemical analysis revealed primary diffuse large B-cell lymphoma of the spinal cord with multifocal myelomalacia and hemorrhages. To determine the presence of a pathogen within the lesion, we developed a novel in situ hybridization protocol for FeLV (RNAscope). The reaction revealed large amounts of FeLV viral RNA in the tumor cells.

Modulation of nose-to-brain delivery of a P-glycoprotein (MDR1) substrate model drug (quinidine) in rats.

During the last decades several new drug formulations were developed to target the central nervous system (CNS) from the nasal cavity. However, in these studies less attention was paid to the possible drug-drug interactions in case of multi-drug therapy. In our pilot study first we compared a nasal solution and a nasal gel to demonstrate their distribution in the nasal cavity (3D printed rat skull model and histology). Due to the aspiration induced high mortality at administration of nasal solution the study was continued only with the gel formulation of quinidine. The aim of our experiments was to identify the possible functional role of P-glycoprotein (P-gp) in the drug absorption in nasal cavity and to test drug-drug interactions at nose-to-brain delivery. Therefore, a P-gp substrate model drug, quinidine was tested by intranasal (IN) administration in presence of PSC-833 (specific P-gp inhibitor) given intravenously (IV) or IN and adrenaline (IN) at low (50 ng) or high (20 µg) dose. In control animals the brain penetration of quinidine was at the level of detection limit, but in combination therapy with IV PSC-833 the brain levels increased dramatically, similarly to high dose IN adrenalin, where due to vasoconstriction peripheral distribution was blocked. These results indicate that P-gp has an important role in drug absorption and efflux at nasal cavity, while adrenaline is also able to modify the penetration profile of the P-gp substrate model drug at nasal application as it decreases nose-to-blood absorption, letting more quinidine to reach the brain along with the nasal nerves.

Prevalence of feline immunodeficiency virus and feline leukaemia virus in domestic cats in Hungary.

OBJECTIVES: Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting cats worldwide. The objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in Hungary and to characterise the phylogenetic relationships of FIV strains. METHODS: A total of 335 anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of FIV and FeLV with two methods: ELISA and PCR. Statistical analysis was carried out to analyse the data obtained. Sequencing and phylogenetic analysis of partial polymerase (pol) gene sequences was performed to describe circulating FIV subtypes. RESULTS: Statistical analysis showed 11.8% and 9.9% true prevalence of FeLV and FIV, respectively, with ELISA. The apparent prevalence calculated from the PCR results were 17.3% for FeLV and 13.1% for FIV. Phylogenetic analysis of partial pol gene sequences obtained from 22 FIV strains showed that all observed Hungarian strains belonged to FIV subtype B. The strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. The overall mean genetic similarity between the analysed strains was 98.2%. CONCLUSIONS AND RELEVANCE: We report the first thorough overview of the prevalence of FeLV and FIV in Hungary, which is relatively high, and give insight into the genetic diversity of Hungarian strains of FIV.

Assessment Tools for Feedback and Entrustment Decisions in the Clinical Workplace: A Systematic Review.

BACKGROUND: Entrustable Professional Activities (EPAs) combine feedback and evaluation with a permission to act under a specified level of supervision and the possibility to schedule learners for clinical service. This literature review aims to identify workplace-based assessment tools that indicate progression toward unsupervised practice, suitable for entrustment decisions and feedback to learners. METHODS: A systematic search was performed in the PubMed, Embase, ERIC, and PsycINFO databases. Based on title/abstract and full text, articles were selected using predetermined inclusion and exclusion criteria. Information on workplace-based assessment tools was extracted using data coding sheets. The methodological quality of studies was assessed using the medical education research study quality instrument (MERSQI). RESULTS: The search yielded 6,371 articles (180 were evaluated in full text). In total, 80 articles were included, identifying 67 assessment tools. Only a few studies explicitly mentioned assessment tools used as a resource for entrustment decisions. Validity evidence was frequently reported, and the MERSQI score was 10.0 on average. CONCLUSIONS: Many workplace-based assessment tools were identified that potentially support learners with feedback on their development and support supervisors with providing feedback. As expected, only few articles referred to entrustment decisions. Nevertheless, the existing tools or the principals could be used for entrustment decisions, supervision level, or autonomy.

Impact of Virtual Patients as Optional Learning Material in Veterinary Biochemistry Education.

Biochemistry and physiology teachers from veterinary faculties in Hannover, Budapest, and Lublin prepared innovative, computer-based, integrative clinical case scenarios as optional learning materials for teaching and learning in basic sciences. These learning materials were designed to enhance attention and increase interest and intrinsic motivation for learning, thus strengthening autonomous, active, and self-directed learning. We investigated learning progress and success by administering a pre-test before exposure to the virtual patients (vetVIP) cases, offered vetVIP cases alongside regular biochemistry courses, and then administered a complementary post-test. We analyzed improvement in cohort performance and level of confidence in rating questions. Results of the performance in biochemistry examinations in 2014, 2015, and 2016 were correlated with the use of and performance in vetVIP cases throughout biochemistry courses in Hannover. Surveys of students reflected that interactive cases helped them understand the relevance of basic sciences in veterinary education. Differences between identical pre- and post-tests revealed knowledge improvement (correct answers: +28% in Hannover, +9% in Lublin) and enhanced confidence in decision making ("I don't know" answers: -20% in Hannover, -7.5% in Lublin). High case usage and voluntary participation (use of vetVIP cases in Hannover and Lublin >70%, Budapest <1%; response rates in pre-test 72% and post-test 48%) indicated a good increase in motivation for the subject of biochemistry. Despite increased motivation, there was only a weak correlation between performance in final exams and performance in the vetVIP cases. Case-based e-learning could be extended and generated cases should be shared across veterinary faculties.

Am I ready for it? Students' perceptions of meaningful feedback on entrustable professional activities.

BACKGROUND: Receiving feedback while in the clinical workplace is probably the most frequently voiced desire of students. In clinical learning environments, providing and seeking performance-relevant information is often difficult for both supervisors and students. The use of entrustable professional activities (EPAs) can help to improve student assessment within competency-based education. This study aimed to illustrate what students' perceptions are of meaningful feedback viewed as conducive in preparing for performing EPA unsupervised. METHODS: In a qualitative multicentre study we explored students' perceptions on meaningful feedback related to EPAs in the clinical workplace. Focus groups were conducted in three different healthcare institutes. Based on concepts from the literature, the transcripts were coded, iteratively reduced and displayed. RESULTS: Participants' preferences regarding meaningful feedback on EPAs were quite similar, irrespective of their institution or type of clerkship. Participants explicitly mentioned that feedback on EPAs could come from a variety of sources. Feedback must come from a credible, trustworthy supervisor who knows the student well, be delivered in a safe environment and stress both strengths and points for improvement. The feedback should be provided immediately after the observed activity and include instructions for follow-up. Students would appreciate feedback that refers to their ability to act unsupervised. CONCLUSION: There is abundant literature on how feedback should be provided, and what factors influence how feedback is sought by students. This study showed that students who are training to perform an EPA unsupervised have clear ideas about how, when and from whom feedback should be delivered.

Utilization and acceptance of virtual patients in veterinary basic sciences - the vetVIP-project.

Context: In medical and veterinary medical education the use of problem-based and cased-based learning has steadily increased over time. At veterinary faculties, this development has mainly been evident in the clinical phase of the veterinary education. Therefore, a consortium of teachers of biochemistry and physiology together with technical and didactical experts launched the EU-funded project "vetVIP", to create and implement veterinary virtual patients and problems for basic science instruction. In this study the implementation and utilization of virtual patients occurred at the veterinary faculties in Budapest, Hannover and Lublin. Methods: This report describes the investigation of the utilization and acceptance of students studying veterinary basic sciences using optional online learning material concurrently to regular biochemistry and physiology didactic instruction. The reaction of students towards this offer of clinical case-based learning in basic sciences was analysed using quantitative and qualitative data. Quantitative data were collected automatically within the chosen software-system CASUS as user-log-files. Responses regarding the quality of the virtual patients were obtained using an online questionnaire. Furthermore, subjective evaluation by authors was performed using a focus group discussion and an online questionnaire. Results: Implementation as well as usage and acceptance varied between the three participating locations. High approval was documented in Hannover and Lublin based upon the high proportion of voluntary students (>70%) using optional virtual patients. However, in Budapest the participation rate was below 1%. Due to utilization, students seem to prefer virtual patients and problems created in their native language and developed at their own university. In addition, the statement that assessment drives learning was supported by the observation that peak utilization was just prior to summative examinations. Conclusion: Veterinary virtual patients in basic sciences can be introduced and used for the presentation of integrative clinical case scenarios. Student post-course comments also supported the conclusion that overall the virtual cases increased their motivation for learning veterinary basic sciences.

Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.

Seroprevalence of bovine viral diarrhoea virus in Hungary - situation before launching an eradication campaign.

Bovine viral diarrhoea (BVD) is a viral disease appearing in various forms and causing high economic losses in the cattle stocks of Hungary. The aim of the present study was to determine the prevalence of bovine viral diarrhoea virus (BVDV) in Hungary through a monitoring survey carried out on samples collected in cattle-keeping units throughout the country. Since no such survey had been carried out in Hungary during the last thirty years, our study may serve as a basis for later monitoring investigations aimed at following the progress of an expected eradication campaign of BVD. The tests were carried out using an ELISA method, on a total of 1200 blood samples submitted from 54 cattle herds. The herds had not been vaccinated against BVDV before the sampling. Out of the 1200 samples, 521 proved to be positive (43.4%), 40 gave doubtful result (3.3%) and 639 were negative (53.3%). In some stocks the samples were collected from cows having completed several lactation periods, and therefore the seronegativity indicates the BVDV-free status of the given stock. Moreover, among the positive herds we found a few where the seropositivity rate was rather low (< 5%). According to the results of the survey, a rather high portion (about one third) of the cattle-keeping units of Hungary can be regarded as BVDV free, which ratio is much higher than had been expected on the basis of surveys carried out on a lower number of samples and in smaller regions of the country. Hence, the chances of an eradication campaign launched in the near future, or carried out parallel to the IBR eradication programme, are better than previously expected.

Novel mastadenovirus infection and clinical disease in a pygmy marmoset (Callithrix [Cebuella] pygmaea).

We describe the detection and successful isolation of a novel mastadenovirus from a pygmy marmoset (Callithrix [Cebuella] pygmaea) that died following an episode of severe respiratory signs. Pathologic/histopathologic examination revealed hydrothorax and catarrhal bronchopneumonia with pronounced desquamation of the bronchiolar epithelial cells, while in other airways a marked hyperplasia of the epithelial lining and numerous giant cells could be observed. We obtained partial sequence data from the adenoviral DNA-dependent DNA-polymerase gene of the isolated strain and analyses of this region showed the highest level of identity to the recently described bat adenoviruses (strains PPV1 and TJM) and the type 2 canine adenovirus. Similar results were gained by phylogenetic calculations indicating that this novel marmoset adenovirus is only distantly related to reference Old and New World primate adenoviruses and formed a monophyletic group with bat and canine adenoviruses and the equine adenovirus 1. Even though the source of the infection remained unknown, our results could imply the possibility of a cross-species transmission of the virus from an anonymous host to the pygmy marmoset.

Evaluation of mercury and lead content in the liver of the cormorant (Phalacrocorax carbo sinensis) population of Kis-Balaton, Hungary.

Mercury and lead concentrations were measured in the livers of cormorant (Phalacrocorax carbo sinensis), an aquatic bird species living and nesting in the special, highly protected nature conservation area of Kis-Balaton, Hungary. The measurements of metal concentrations were performed by atomic absorption spectrometry using the cold vapour method for mercury and the electrothermal method for lead. Mercury concentrations in the livers were significantly higher in the adult population (4.479 ± 3.336 mg/kg dry matter, DM) than in the juvenile birds (2.682 ± 2.087 mg/kg DM), indicating an increase of bioaccumulation with age. A similar pattern was not observed for lead. There were no statistical differences between males and females either in mercury or in lead concentrations. The average levels of mercury (3.580 ± 2.906 mg/kg DM) and lead (0.746 ± 0.499 mg/kg DM) were statistically different in the liver. No correlation was found between the concentrations of the two heavy metals. Recently, the wild birds have been chronically exposed to subtoxic amounts of metals which have a tendency to accumulate especially in the soft tissues.

Effect of chronic T-2 toxin exposure in rabbit bucks, determination of the No Observed Adverse Effect Level (NOAEL).

T-2 toxin (T-2) was administered to adult Pannon White (n = 10/group) male rabbits for 65 days, first in a suspension by gavage (0.05, 0.1 or 0.2 mg/animal/day), and secondly mixed into the feed (0.33 and 0.66 mg/kg feed). In the first experiment 0.1 mg T-2 exposure resulted in temporary decrease in feed intake, slower increase in the gonadotropin-releasing hormone (GnRH) induced testosterone synthesis, slight centrolobular infiltration in the liver and a slight hyperplasia of the Leydig cells. In addition to the temporary feed refusal effect, 0.2 mg T-2 caused a temporary decrease in plasma albumin and urea concentrations, lesser glutathione peroxidase (GPx) activity in the seminal plasma, a greater (by 320%) ratio of spermatozoa with cytoplasmic droplets, slower increase in the GnRH-induced testosterone synthesis, centrolobular infiltration in the liver, slightly hyperaemic testes and increased proliferative activity of the Leydig cells. The two smaller doses applied in feed (0.33 and 0.66 mg/kg) did not cause any significant adverse effect, and no feed refusal was observed. According to these results the No Observed Adverse Effect Level (NOAEL) of T-2 for adult rabbit males was found to be <0.1 mg/animal/day (<0.02 mg/kg b.w./day).

Adenoma of the cloacal scent gland in a California Kingsnake (Lampropeltis getulus californiae).

A multiple simple adenoma causing severe distortion of the tail base was identified in the cloacal scent gland of a female California Kingsnake (Lampropeltis getulus californiae). In addition to the normal epithelial layer of the gland and the skin, the tumour cells in the glandular epithelium also showed cross immunereactivity with humanised anti-cytokeratin antibody. This is the first description of an adenoma in the scent gland of a reptile species. Neither epithelial nor mesenchymal tumours arising from the scent gland of reptiles have been reported previously. This report also highlights the possible use of humanised antibodies on reptile species for the fast, reliable and specific differential diagnosis of tumours.

Developing an online professional network for veterinary education: the NOVICE project.

An online professional network for veterinarians, veterinary students, veterinary educationalists, and ICT (Information and Communication Technology) educationalists is being developed under the EU (European Union) Lifelong Learning Programme. The network uses Web 2.0, a term used to describe the new, more interactive version of the Internet, and includes tools such as wikis, blogs, and discussion boards. Focus groups conducted with qualified and student veterinarians within the project's five founding countries (The Netherlands, Germany, United Kingdom, Hungary, Romania) demonstrated that online professional communities can be valuable for accessing information and establishing contacts. Online networks have the potential to overcome common challenges to face-to-face communities-such as distance, cost, and timing-but they have their own drawbacks, such as security and professionalism issues. The Network Of Veterinary ICt in Education (NOVICE) was developed using Elgg, an open-source, free social networking platform, after several software options had been considered. NOVICE aims to promote the understanding of Web 2.0, confidence to use social software tools, and participation in an online community. Therefore, the Web site contains help sections, Frequently Asked Questions, and access to support from ICT experts. Five months after the network's launch (and just over one year into the project) 515 members from 28 countries had registered. Further research will include analysis of a core group's activities, which will inform ongoing support for and development of informal, lifelong learning in a veterinary context.

Solitary adenoma in the proventriculus of a budgerigar (Melopsittacus undulatus) diagnosed by immunochemistry - short communication.

The authors describe a solitary adenoma in a 5.5-year-old female Budgerigar (Melopsittacus undulatus). The tumour was partially blocking the lumen of the proventriculus and filled it almost completely. Decreased passage of food towards the gizzard and the intestines developed in the bird as result of the obstruction, which periodically hindered the passage, leading to slow emaciation. An epithelial tumour composed of irregular glandular acini was diagnosed by histological examination. Immunohistochemical reaction with pancytokeratin showed a positive cytoplasmic reaction both in the neoplastic and the normal glandular structures.