Molecular analysis of individually laser-dissected microfilariae reveals Acanthocheilonema reconditum disguised as Dirofilaria immitis.

Introduction

Canine filarial parasites, including Dirofilaria immitis and Acanthocheilonema reconditum, can cause zoonotic infections in humans. Dirofilaria immitis and Acanthocheilonema reconditum, however, respond differently to drug treatments and, therefore, it is necessary to discriminate between these two parasites during diagnosis.

Methods

Light microscope blood surveys of canines were used to identify the presence of D. immitis and A. reconditum infections and co-infections in dogs that were life-long residents of Manaus (Amazonas, Brazil). The ITS-1 and 5S nuclear ribosomal DNA sequences and the mitochondrial CO1 and 12S gene sequences of filarial parasites were amplified by PCR and then Sanger sequenced for the molecular taxonomic classification of 20 individual microfilariae. These 20 individual microfilariae were isolated by laser dissection from a single canine blood sample, which morphological analysis of microfilariae suggested contained both D. immitis and A. reconditum parasites.

Results

A total of 20 individual microfilariae, which all morphologically appeared as Dirofilaria immitis, had two or more taxonomically informative PCR fragments amplified and Sanger sequenced. More than five ITS-1, 5S, 12S and CO1 DNA sequences were recovered from the twenty individual microfilariae. All recovered sequences showed 100% identity to database reference sequences matching A. reconditum.

Conclusions

Acanthocheilonema reconditum is present in the state of Amazonas and indeed the city of Manaus. Acanthocheilonema reconditum microfilariae are more difficult to distinguish from D. immitis than has previously been reported and it may, in fact, not be possible to reliably discriminate between these two species by the morphological features of their microfilariae alone. Although the possibility that A. reconditum and D. immitis form hybrids cannot be ruled out by this work, no evidence of hybrids or geneflow between these parasites was detected in our study.