Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to asthe feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recentlybeen identified as an agent of bronco-pulmonary infestations in cats. These two parasiteshave a similar biology, share ecological niches, potentially co-infesting cats, but are dif-ficult to be differentiated due to the morphological similarities of their first-stage larvae(L1). This paper describes a molecular tool, based on single-step duplex polymerase chainreaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for thesimultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both specieswere collected from faecal samples, morphologically identified, and single larval specimensisolated. An aliquot of faeces was used as a test sample for a case of mixed natural infes-tation. The duplex-PCR was performed using species-specific forward primer sets for theITS-2 region (i.e., A. abstrusus: 220 bp; T. brevior: 370 bp). The detection limit of the molec-ular assay was also assessed by serial dilutions of DNA from single larvae of both species(from ∼4.0 to 4.0 × 10−5g/l). The duplex-PCR carried out on individual DNA sampleswas able to detect as low as 5.2 × 10−3g/l of DNA for A. abstrusus, 4.9 × 10−3g/l for T.brevior, and as low as 4.0 × 10−3g/l for samples containing both species. Species-specificbands of the expected sizes and two bands were simultaneously amplified from the fae-cal sample containing both species. The phylogenetic analyses of the ITS-2 sequences hereexamined and those available for other metastrongyloids were concordant in clusteringthem with those of other Troglostrongylus brevior and A. abstrusus sequences available inGenBank database. This molecular approach proved to be effective and highly sensitivefor the simultaneous detection of the two lungworms species and it might be used formolecular epidemiological studies and for monitoring therapeutic protocols.