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

Abstract

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult 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 chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ≈ 4.0 to 4.0 × 10(-5) μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10(-3) μg/μl of DNA for A. abstrusus, 4.9 × 10(-3)μg/μl for T. brevior, and as low as 4.0 × 10(-3) μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

Keywords: Aelurostrongylus abstrusus; Duplex-PCR; Feline lungworms; ITS-2; Phylogeny; Troglostrongylus brevior.