Abstract:

【Objective】This study aims to isolate and identify prominent bacterial respiratory pathogens from samples collected from fattening pigs that have died from respiratory diseases in major domestic pig farming area of middle and eastern China. And identifying these bacterial pathogens will offer valuable evidence for preventing and controlling the significantly prevalent respiratory diseases in recent years. Moreover, the characteristics of Pm were identified, providing reference for the development of Pm vaccines.【Method】The lungs of pigs died from respiratory disease were collected from large-scale farms in major pig farming area of middle and eastern China from 2021 to 2023. Blood agar and TSA were employed for the isolation of pathogens, which were then identified through microbiological and molecular biology methods. Additionally, MLST typing and virulence testing in mice were conducted on Pm isolates. The primers for PCR against seven housekeeping genes of adk, est, gdh, mdh, pgi, pmi and zwf of Pm were designed. Then the productions of amplicons were sequenced and submitted to perform MLST typing. The capsule and lipopolysaccharide typing were detected by PCR. The virulence factor genes were detected by PCR. Single isolates of A type and selected D and F types of Pm were evaluated for virulence in ICR mice. LD₅₀ of JS-65, JS-51 and JS-34 were detected in ICR mice.【Result】A total of 73 Pm isolates were obtained, with an isolation rate of 15.53%. Additionally, 71 SS isolates, 29 APP isolates, and 10 GPS isolates were obtained, with isolation rates of 15.11%, 6.17%, and 2.13% respectively. The typing results indicated that the prevailing subtype among Pm isolates was A:L3, accounting for 55%. Among SS isolates, subtype 9 was the prevailing type, accounting for 38.03%. Among APP isolates, subtype 15 was the prevailing type, accounting for 51.72%. Among GPS isolates, subtype 5/12 was the prevailing type, accounting for 60.00%. Co-infection included Pm+SS, Pm+APP, SS+APP and Pm+APP+GPS, accounting for 16.67% of the total pig population. Three capsule types were isolated: A (67%), D (30%), and F (3%). Two lipopolysaccharide types were found: L3 (56%) and L6 (44%). Nine ST genotypes were identified: ST79, ST50, ST7, ST74, ST13, ST27, ST9, ST287, and ST370, with proportions of 33%, 26%, 16%, 10%, 4%, 4%, 3%, 3%, and 1%, respectively. The results of virulence gene detection showed that the positivity rates of ptfA, fimA, hsf-2, exbB, exbD, tonB, fur, nanH, sodA, and sodC genes were greater than 95%. The positivity rates of hsf-1, pfhA, tadD, hgbA, hgbB, pmHAS, ompA, ompH, oma87, and plpB genes ranged from 40% to 90%. The positivity rates of tbpA and nanB genes were between 10% and 30%; the toxA gene was not detected. The virulence test results indicated that all mice died when exposed to less than 10² CFU of strain A, the mortality rate of mice was between 60% and 100% when exposed to 10³ CFU of strain D, and the mortality rate of mice was 60% when exposed to 5×10³ CFU of strain F. LD₅₀ of JS-65, JS-51, and JS-34 were detected in ICR mice, and the results showed that JS-65 LD₅₀<10 CFU, and JS-51 LD₅₀=6.3 × 10² CFU, JS-34 LD₅₀=3.98 × 10³ CFU.【Conclusion】Based on the bacterial pathogen isolates from 2021 to 2023, the primary pathogen bacterium of the respiratory tract in dead fattening pigs were Pm, SS, APP, and GPS. Pm had the highest number of isolates and isolation rate from lung tissue. The RIRDC identified a Pm strain of ST370 and extended MLST typing data in pigs. PCR typing results showed that the dominant serotype of Pm was A:L3:ST79, which exhibited the highest virulence in ICR mice with a minimum lethal dose of less than 10 CFU. These results serve as the foundation for the development of an inactivated Pm vaccine.

Key words: epidemiology, Pasteurella multocida, identification, molecular typing