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.

Summary: Mycoplasma hyorhinis is an emerging swine pathogen bacterium with high prevalence worldwide. The main lesions caused are arthritis and polyserositis and the clinical manifestation of the disease may result in significant economic losses due to the decreased weight gain and enhanced medical costs. Our aim was to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate. Five-week-old, Choice hybrid pigs were inoculated on two consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal route (Group IV-IP). Mock infected animals were used as control (Control Group). After challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. ELISA test was used to detect anti- M. hyorhinis immunoglobulins in the sera of all animals. Pericarditis and polyarthritis were observed in both challenge groups, however the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores and ELISA titres. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the Control Group. All re-isolated strains were the same or a close genetic variant of the original challenge strain. Our results indicate that both challenge routes are suitable for modelling the disease. However, due to the more severe pathological lesions and the more natural-like route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection.