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Development of a PCR test to diagnose Haemophilus parasuis infections
S Oliveira1, L Galina, C Pijoan
1Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul 55108, USA.
Abstract:
A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.
Insights
A new polymerase chain reaction (PCR) test offers faster and more accurate diagnosis of Haemophilus parasuis in swine. This molecular method significantly improves upon traditional techniques for identifying this important swine pathogen.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Swine Health
Background:
- Haemophilus parasuis is a significant respiratory pathogen in swine, causing substantial economic losses.
- Accurate and rapid diagnostic methods are crucial for controlling H. parasuis infections.
Purpose of the Study:
- To develop and validate a species-specific polymerase chain reaction (PCR) assay for the accurate and rapid detection of Haemophilus parasuis.
- To compare the diagnostic performance of the developed PCR test with traditional microbiological techniques.
Main Methods:
- Design of species-specific primers targeting the 16S small subunit ribosomal RNA gene of H. parasuis.
- Optimization and validation of the PCR assay using bacterial isolates and clinical samples from swine.
- Comparison of PCR results with bacterial isolation and identification.
Main Results:
- The developed PCR assay demonstrated high specificity, correctly identifying 31 H. parasuis isolates across various serovars and field strains.
- The test exhibited high sensitivity, detecting as little as 102 bacteria or 0.69 pg of DNA, with no cross-reactivity with 15 other common swine bacterial species (except for a weak band with Actinobacillus indolicus).
- In clinical samples, the PCR test showed a positivity rate of 86.7% (26/30), outperforming bacterial isolation (60%, 18/30).
Conclusions:
- The developed PCR test is a sensitive, specific, and rapid tool for the diagnosis of Haemophilus parasuis infections in swine.
- This molecular diagnostic method offers a significant advantage over conventional microbiological techniques for improved disease management and control in swine herds.