This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.
Researchers José Luis Arnal, Ana Belén Fernández, Sonia Lacotoure, Alfredo Ángel Benito, Sofía Lázaro, and Marcelo Gottschalk share their study evaluating oral fluids combined with qPCR for detecting and serotyping A. pleuropneumoniae in swine herds.
Key Points
- Asymptomatic pigs infected with Actinobacillus pleuropneumoniae (A. pleuropneumoniae) often harbor multiple serotypes in their tonsils, but microbiological isolation is challenging
- Oral fluids is a non-invasive sample type, effective for identifying A. pleuropneumoniae serotypes at the herd level with qPCR
- Combining qPCR analysis of oral fluids with serological methods enhances pleuropneumonia surveillance and management
Introduction
Porcine pleuropneumonia, caused by A. pleuropneumoniae, significantly affects the global swine industry. Nineteen distinct serotypes exist, many lacking cross-protection. Asymptomatic infected pigs appear healthy but spread infection. Detecting serotypes in these carriers is critical yet challenging. Oral fluid sampling, a non-invasive, cost-effective method, shows promise as a complementary diagnostic tool. This study evaluates oral fluids combined with qPCR for detecting and serotyping A. pleuropneumoniae in swine herds.
Material & Methods
The samples were obtained from six swine farms in Quebec, Canada. Farms N1, N2, and N3 were classified as free of A. pleuropneumoniae based on routine serological records and the absence of clinical symptoms, while farms P1, P2, and P3 were confirmed positive through prior serological testing. The tonsil samples were collected from 30 finishing pigs per farm at the slaughterhouse and stored at −20°C until analysis. Then, a variable number (from 5 to 10 per farm) of oral fluids samples were collected using ropes placed in pens 1–2 weeks before slaughter. These samples were frozen at −20°C until testing. Additionally, 30 serum samples from farms P1, P2, and P3 were collected 10 weeks after tonsil and oral fluid sampling, though from different animal batches.
The tonsils were directly sampled with 25 mg of tissue as well as through brushing the surface with a cytology brush. Both materials were plated on chocolate agar plates and incubated at 37°C for 24 h. The oral fluids were thawed overnight at 4°C, clarified and processed. EXOone A. pleuropneumoniae qPCR kit (Exopol, Spain) was used to detect A. pleuropneumoniae in various samples. The kit includes an endogenous control to ensure quality. qPCR targeted the 19 known serotypes in tonsil brushings, cultures, and oral fluids using serotype-specific EXOone kits (Exopol, Spain) with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).
The serological tests were conducted via ELISA tests based on LPS O-chain purified antigen were employed to detect antibodies against A. pleuropneumoniae serotypes/serogroups: 1(9,11); 5a, 5b; 2; 3(6,8,15,17); 7(4); 10; 12; 13; and 14.
Results
Negative Farms: All samples tested negative for A. pleuropneumoniae serotypes by qPCR. Endogenous controls validated sample processing.
Positive Farms: qPCR detected A. pleuropneumoniae in tonsils, cultures, and oral fluids from three positive farms. Cq values were higher in oral fluids, reflecting lower bacterial loads.
Serotype Detection:
- Positive Farm 1: Serotypes 2, 8, 7, 12, and 19 detected, serotype 7 was most prevalent
- Positive Farm 2: Serotypes 2, 5, 7, 12, and 17 detected. Serotype 2 had low prevalence in cultures but identified via qPCR. Tonsils from this farm showed highest serotype diversity.
- Positive Farm 3: Predominantly serotype 7, some serotypes detected in tonsils but not oral fluids
Conclusion
qPCR effectively detects A. pleuropneumoniae serotypes and tonsil brushings offer practical in vivo sampling. Oral fluids provide insights into herd-level serotype diversity and prevalence. Using oral fluid qPCR and serological methods improves diagnostic accuracy and surveillance. This integrated approach is a proactive strategy for managing A. pleuropneumoniae and enhances swine herd health management.
Read the Full Paper: https://doi.org/10.1016/j.vetmic.2024.110268