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High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Detection of feline upper respiratory tract disease pathogens using a commercially available real-time PCR test
A Litster1, C C Wu2, C M Leutenegger3
1Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, 625 Harrison St., West Lafayette, IN 47907, USA.
Abstract:
Feline herpesvirus (FHV-1), feline calicivirus (FCV), Bordetella bronchiseptica (Bb), Chlamydia felis (Cf) and Mycoplasma felis (Mf) are common infectious agents identified in cats with upper respiratory tract disease (URTD). Each of these agents can either act as primary pathogens or cause subclinical infections, and pathogen identification can be used to prevent disease transmission in shelters, or to manage individual cats with recurrent URTD. The aim of this study was to compare pathogen detection rates using real-time PCR testing and virus isolation (VI) or bacterial culture in conjunctival, nasal and oropharyngeal swabs from 18 shelter-housed cats with clinical URTD. Co-infections were common; FHV-1 was most prevalent and Cf and FCV were least prevalent. Agents detected by PCR were FCV 2/18 (11%), FHV-1 17/18 (94%), Bb 8/18 (44%) and Mf 15/18 (83%). Agents detected by VI and bacterial culture were FCV 1/18 (6%), FHV-1 12/18 (67%), Bb 8/18 (44%) and Mf 12/18 (67%). Agreement between PCR results and the other two methods was: FHV-1, 57.4%; FCV, 98.1%; Bb, 75.0%; Mf, 60.0%. Discordancies included PCR-positive, VI-negative (FCV, n = 1/54, 1.9%; FHV-1, n = 23/54, 42.6%), PCR-positive, culture-negative (Bb, n = 6/36, 16.7%; Mf, n = 13/36, 36.1%) or PCR-negative, culture-positive (Bb, n = 3/36, 8.3%; Mf, n = 2/36, 5.6%) results. A combination of an oropharyngeal swab and either a conjunctival or a nasal swab submitted for PCR testing was able to detect all infectious agents tested for in each cat. PCR testing was a sensitive and convenient method of detection of infectious agents in cats with clinical signs of URTD.
Insights
Real-time PCR testing effectively detects common infectious agents causing feline upper respiratory tract disease (URTD). This method, utilizing oropharyngeal, conjunctival, or nasal swabs, proved more sensitive than traditional virus isolation or bacterial culture for diagnosing URTD in cats.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Feline upper respiratory tract disease (URTD) is commonly caused by infectious agents like feline herpesvirus (FHV-1), feline calicivirus (FCV), Bordetella bronchiseptica (Bb), Chlamydia felis (Cf), and Mycoplasma felis (Mf).
- Accurate identification of these pathogens is crucial for disease prevention in shelters and managing recurrent URTD in individual cats.
- Co-infections with multiple agents are frequent in cats presenting with clinical URTD.
Purpose of the Study:
- To compare the diagnostic performance of real-time PCR with conventional virus isolation (VI) and bacterial culture for detecting common URTD pathogens in cats.
- To evaluate the efficacy of different swab types (conjunctival, nasal, oropharyngeal) for pathogen detection using PCR.
Main Methods:
- Real-time PCR, virus isolation, and bacterial culture were performed on conjunctival, nasal, and oropharyngeal swabs from 18 shelter-housed cats with clinical URTD.
- Detection rates and agreement between PCR and VI/culture were calculated for FHV-1, FCV, Bb, and Mf.
- The study analyzed discordant results to understand discrepancies between diagnostic methods.
Main Results:
- Real-time PCR identified FHV-1 in 94% of cats, Mf in 83%, Bb in 44%, and FCV in 11%.
- VI/culture detected FHV-1 in 67%, Mf in 67%, Bb in 44%, and FCV in 6%.
- PCR demonstrated higher sensitivity for FHV-1 and FCV compared to VI/culture, with good agreement for FCV (98.1%) and Bb (75.0%). A combination of oropharyngeal and conjunctival/nasal swabs for PCR detected all agents in every cat.
Conclusions:
- Real-time PCR is a sensitive and convenient method for detecting infectious agents associated with feline URTD.
- A combined sampling strategy using oropharyngeal swabs alongside conjunctival or nasal swabs for PCR testing ensures comprehensive pathogen detection.
- PCR offers advantages over traditional methods, particularly for detecting FHV-1 and FCV, aiding in URTD diagnosis and management.
