First report of Anaplasma platys infection in red foxes (Vulpes vulpes) and molecular detection of Ehrlichia canis and Leishmania infantum in foxes from Portugal

Abstract

Background: The bacteria Anaplasma platys and Ehrlichia canis and the protozoan Leishmania infantum are vector-borne agents that cause canine vector-borne diseases, some of which are zoonotic. The present survey investigated the prevalence of Anaplasma, Ehrlichia and Leishmania in red foxes (Vulpes vulpes) from Portugal by molecular analysis, in order to evaluate the epidemiological role of these canids as reservoirs of infection.

Methods: Blood and/or bone marrow samples were collected from 78 red foxes obtained in eight districts of northern, central and southern Portugal. Real-time polymerase chain reactions (PCR) amplified a 123 bp fragment of the 16S rRNA gene of Anaplasma spp. and Ehrlichia spp. and a 265 bp fragment of the L. infantum internal transcribed spacer one (ITS1) region of the rRNA operon evaluated by PCR-high resolution melt analysis (PCR-HRM), with sequencing of the DNA products. A phylogenetic analysis was carried out to compare these to other sequences from Anaplasma spp. and Ehrlichia spp. deposited in GenBank.

Results: A. platys was detected in 10 (14.5%) and E. canis in two (2.9%) out of 69 foxes; and L. infantum was detected in one (1.3%) of the 78 foxes. The prevalence of A. platys was significantly different from the prevalence of E. canis (p=0.016) and from that of L. infantum (p=0.002). No co-infections were found in any one of the 78 foxes. No statistically significant differences were found between the type of sample (blood and bone marrow), geographic regions (north/centre and south), age (<2 years and ≥2 years) and gender for any one of the agents.

Conclusions: This is the first known report of A. platys in red foxes worldwide, as well as the first molecular evidence of E. canis in foxes from Portugal. The moderate prevalence of A. platys suggests that red foxes may play a role in the epidemiology of infection with this bacterium and serve as a reservoir for domestic dogs.

Figure 1

Maximum likelihood tree phylogram comparing 385 bp sequences of the 16S DNA sequences from Anaplasma spp. and Ehrlichia spp. deposited in GenBank®. The GenBank® accession numbers, animal source and country of origin from which the sequences were derived are included for each sequence. Bootstrap values higher than 60% are indicated. New sequences derived from the present study are marked in bold letters. The 385 bp sequences included in the phylogenetic analysis were formed by a combination of two non-overlapping 16S rRNA fragments amplified by the E.c-16S fwd/E.c-16S rev primers and EHR16SD/EHR16SR primers. 305 bp DNA sequences included in the phylogram from one fragment of the combined fragment sequences were deposited in GenBank® as accession numbers KP717550, KP717551 (R02B, R16B) for A. platys and KP717552 (07B_UE08) for E. canis.