Isolation and identification of Mycoplasma hyorhinis and virulence evaluation of its field isolates

Abstract

Introduction: As a prevalent swine pathogen worldwide, Mycoplasma hyorhinis (M. hyorhinis, Mhr) is associated with various diseases, including multiple serositis, pneumonia, arthritis, and otitis media. It is also linked to the porcine respiratory disease complex (PRDC).

Methods: M. hyorhinis prevalence in 2022 Chinese lung samples was assessed by species-specific PCR, followed by isolation and purification of field strains, followed by genetic characterization via multilocus sequence typing (MLST). Pathogenicity evaluation of three isolates (ZZ-1, GD-1 and AH-1) was evaluated using controlled piglet infection trials.

Results: Mhr detection in clinical lung samples showed 31.77% prevalence. Three isolates (ZZ-1/ST166, GD-1/ST167, AH-1/ST144) were characterized by MLST. Piglet infection trials confirmed Mhr-induced polyserositis, pneumonia, and arthritis, with strain-dependent virulence variation observed.

Discussion: This study confirms M. hyorhinis as a high-prevalence pathogen (31.77%) in Chinese swine herds. Animal infection models demonstrated virulence variation among different Mhr strains. These findings contribute to identifying and assessing the threats posed by different strains to pig health, guiding the development of clinical prevention and control strategies.

Keywords: Mycoplasma hyorhinis; identification; infection; isolation; positivity rate.

Figure 1

Month-based summary of data collection.

Figure 2

Identification of M. hyorhinis isolates. (A) Colony morphology of the three Mhr strains on solid media (40×), exhibiting similar “fried egg” colonies. (a) ZZ-1 strains; (b) GD-1 strains; (c) AH-1 strains; (d) Transmission electron microscopy (TEM) image of Mhr ZZ-1 strain (Scale bar: 1 μm). The image reveals the typical pleomorphic structure of the Mycoplasma genus, with the cell wall-lacking structures with a granular interior. (B) PCR identification of Mhr targeting the P37 gene (550 bp) and 16S rRNA gene (1450 bp). M, DNA marker; lane 1–3, ZZ-1 strain, GD-1 strains and AH-1 strains’ P37 gene amplification; lane 4, negative control; lane 5, positive control (P37 gene); lane 6–8, ZZ-1 strain, GD-1 strains and AH-1 strains’ 16S rRNA gene amplification; lane 9, negative control; lane 10, positive control (16S rRNA gene).

Figure 3

Clinical symptom observation. (A) Body temperature of piglets after infection. Temperatures were monitored daily, with peak temperatures at 2 dpi for all infection groups. At 7 dpi, body temperatures in all groups returned to below 40°C. The control group exhibited no significant temperature abnormalities throughout the study. (B) Average daily weight gain (ADWG) during the 28-day experimental period. Data are presented as mean ± standard deviation (SD). The ZZ-1 strain and AH-1 strain groups showed lower ADWG than the control group, while the GD-1 strain group displayed no significant difference in ADWG compared to the control group. (C) Total clinical observation scores of each group. Data are expressed as mean ± standard deviation (SD). Total scores in ZZ-1 and AH-1 strain groups were significantly increased compared to that in the control group, for which no signs of pathological changes were detected (*p < 0.05). (D) Abnormal respiration, cough, and lameness scores of each group. (E) Swollen joints observed in the ZZ-1 strain group. (F) Persistent and pronounced lameness in the ZZ-1 strain group.

Figure 4

Postmortem observation scores of each group. (A) Peritonitis scores of each group. (B) Pleuritis scores of each group. (C) Pericarditis scores of each group. (D) Arthritis scores of each group. (E) Pneumonia scores of each group. (F) Total Postmortem observation scores of each Group. 9.33 ± 0.47, 1.00 ± 0.82, 8.67 ± 2.62, 0.00 ± 0.00 in ZZ-1, GD-1, AH-1 strain groups and control group, respectively. Data are presented as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. (G) Number of positive animals.

Figure 5

Pathological changes caused by M. hyorhinis infection. (A) Pericarditis: the arrow indicates the thickening of the pericardium, increased pericardial effusion, and cheese-like exudate between the heart and pericardium. (B) Pericarditis: the arrow highlights a close adhesion between the pericardium and the heart. (C) Pneumonia: the arrow highlights the shrimp-like lung lesions observed on the dorsal side. (D) Pleuritis: the arrow shows fibrinous adhesions between the lung lobes and pleura. (E) Peritonitis: the arrow highlighting the cheese-like exudate in the abdominal cavity. (F) Arthritis: the arrow points to the abnormal synovial membrane with cheese-like exudate in the articular cavity. (G) H&E-stained hepatization-lung tissue of ZZ-1 strain group (Z1), showing obscured or absent alveolar structure, alveolar spaces filled with fibrinous exudates, accompanied by extensive inflammatory cell infiltration and thickened alveolar septa. (H) Lung tissue of control group (C11), showing intact alveolar structure, clear alveolar spaces, thin alveolar septa, and minimal inflammatory cell presence.