Supplementary data – Mycoplasma hyorhinis vaccine strain – Nagy, 2026hdl:21.15109/ARP/3IIFGYARP2026-03-251Nagy, Eszter Zsófia; Szeredi, Levente; Földi, Dorottya; Belecz, Nikolett; Kovács, Áron Botond; Sulyok, Kinga Mária; Grózner, Dénes; Wehmann, Enikő; Bányai, Krisztián; Marton, Szilvia; Tenk, Miklós; Kreizinger, Zsuzsa; Gyuranecz, Miklós, 2026, "Supplementary data – Mycoplasma hyorhinis vaccine strain – Nagy, 2026", https://hdl.handle.net/21.15109/ARP/3IIFGY, ARP, V1Supplementary data – Mycoplasma hyorhinis vaccine strain – Nagy, 2026hdl:21.15109/ARP/3IIFGYNagy, Eszter ZsófiaSzeredi, LeventeFöldi, DorottyaBelecz, NikolettKovács, Áron BotondSulyok, Kinga MáriaGrózner, DénesWehmann, EnikőBányai, KrisztiánMarton, SzilviaTenk, MiklósKreizinger, ZsuzsaGyuranecz, MiklósLP2022–6/2022RRF-2.3.1-21-2022-00001ARPKreizinger, ZsuzsaKaján, Győző László2026-03-25Agricultural Sciences1-Methyl-3-nitro-1-nitrosoguanidineLive attenuated vaccineMycoplasma hyorhinisNTGPorcineTemperature-sensitive strain<b>Background</b> <i>Mycoplasma</i> (<i>M</i>.) <i>hyorhinis</i> causes substantial economic losses in swine. Currently, prevention and treatment rely on minimizing risk factors and administering antibiotics, as no vaccines are commercially available in Europe. However, antibiotics often cannot fully eliminate the bacteria. The development of an effective vaccine could lead to a potentially long-term control method. <b>Materials and methods</b> A temperature-sensitive <i>M. hyorhinis</i> strain was developed using 1-methyl-3-nitro-1-nitrosoguanidine (NTG) mutagenesis. The immunogenicity and efficacy of this vaccine candidate clone were evaluated in combination with an adjuvant. Three-week-old piglets were immunized with the candidate vaccine strain, and the vaccination site was monitored daily. At six weeks of age, the pigs were challenged intravenously on two subsequent days. Daily clinical examinations were conducted, with blood and nasal swabs collected weekly throughout the study for <i>M. hyorhinis</i> enzyme-linked immunosorbent assay (ELISA), real-time PCR analysis, and isolation. Three weeks post-challenge, the animals were euthanized for gross and histopathological examinations. Body temperature was recorded daily, and body weight was measured upon arrival, and then at six and nine weeks of age. <b>Results</b> Vaccination significantly reduced clinical signs (p = 0.03), as well as gross pathological (p = 0.01) and histopathological (p = 0.005) lesions compared with the positive control group. The vaccinated group exhibited an earlier and higher increase in <i>M. hyorhinis</i>-specific IgG antibody levels post-challenge than the positive control group. However, the vaccine candidate did not mitigate the impact of <i>M. hyorhinis</i> infection on the weight gain. After the challenge (days 21–42), both the vaccinated (p = 0.001) and the positive control (p = 0.003) groups exhibited reduced weight gain compared with the negative control group. <b>Discussion</b> Overall, the attenuated <i>M. hyorhinis</i> strain, combined with the adjuvant, provided protection against <i>M. hyorhinis</i> infection. These results form a basis for the development of a novel vaccine candidate that offers effective prevention.<a href="http://creativecommons.org/licenses/by-nc/4.0">CC BY-NC 4.0</a>The attenuated M2642 clone was deposited at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstraße 7B, 38124 Braunschweig, Germany) (Accession number: DSM 35229).The sequences of the MycSu206 parent strain and the M2642 clone have been deposited in the GenBank database (BioProject number: PRJNA1203880). The attenuated M2642 clone was patented under number P2500036 at the Hungarian Intellectual Property Office.Nagy EZ, Szeredi L, Földi D, et al. Development and efficacy test of a live, attenuated Mycoplasma hyorhinis vaccine candidate strain. Vaccine. 2026;75:128278. doi:10.1016/j.vaccine.2026.12827810.1016/j.vaccine.2026.128278Nagy EZ, Szeredi L, Földi D, et al. Development and efficacy test of a live, attenuated Mycoplasma hyorhinis vaccine candidate strain. Vaccine. 2026;75:128278. doi:10.1016/j.vaccine.2026.128278supplementary_tables_1-14.xlsxSupplementary tables 1-14application/vnd.openxmlformats-officedocument.spreadsheetml.sheetsupplementary_table_10.csvSupplementary table 10. Gross pathological lesion scores of the pigs in the vaccinated, positive control, and negative control groups. Lesions were not observed in the negative control group.text/csvsupplementary_table_11.csvSupplemetnary table 11. Histopathological lesion scores of the pigs in the vaccinated, positive control, and negative control groups.text/csvsupplementary_table_12.csvSupplementary table 12. Optical density (OD) values of the Mycoplasma hyorhinis in-house ELISA in the vaccinated, positive control, and negative control groups.text/csvsupplementary_table_13.csvSupplementary table 13. Results of the Mycoplasma hyorhinis qPCR from the samples taken during the gross pathological examination from the vaccinated, positive control, and negative control groups. The table also presents the quantification cycle (Cq) values of the positive samples and the organs where pathological lesions were observed (highlighted with a *).text/csvsupplementary_table_14.csvSupplementary table 14. Results of Mycoplasma hyorhinis isolation and multiple-locus variable-number tandem-repeat analysis (MLVA). MLVA was performed following established protocols.text/csvsupplementary_table_1A.csvSupplementary table 1A. Mastermix compositions of the applied qPCR assays.text/csvsupplementary_table_1B.csvSupplementary table 1B. Thermal protocols of the applied qPCR assays.text/csvsupplementary_table_2.csvSupplementary table 2. Scoring system for the clinical signs.text/csvsupplementary_table_3.csvSupplementary table 3. Scoring system for the pathological lesions and evaluation of the vaccination site.text/csvsupplementary_table_4.csvSupplementary table 4. Scoring system for the histopathological lesions.text/csvsupplementary_table_5.csvSupplementary table 5. Selection of the 1-methyl-3-nitro-1-nitrosoguanidine (NTG) treated, temperature-sensitive (ts+) Mycoplasma hyorhinis clone.text/csvsupplementary_table_6.csvSupplementary table 6. Detailed information on the polymorphisms (deletions, insertions, and Single Nucleotide Polymorphisms (SNPs)) in the genome of the temperature-sensitive (ts+) Mycoplasma hyorhinis clone compared to its parent strain.text/csvsupplementary_table_7.csvSupplementary table 7. Body temperature changes of the pigs during the immunization and the challenge.text/csvsupplementary_table_8.csvSupplementary table 8. Clinical sign scores of the vaccinated, positive control, and negative control pigs. Clinical signs were not observed in the negative control group.text/csvsupplementary_table_9.csvSupplementary table 9. Weight gain (kg) during the experiment in the vaccinated, positive control, and negative control groups.text/csv