The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7-78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0-6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p<0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p=0.219) or age groups (<2 years vs. ≥ 2 years) (p=1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs.

BackgroundHepatozoon canis is a protozoan tick-borne pathogen of dogs and wild canids. Hepatozoon spp. have been reported to infect foxes in different continents and recent studies have mostly used the polymerase chain reaction (PCR) for the detection and characterization of the infecting species. Surveying red foxes (Vulpes vulpes) may contribute to better understanding the epidemiology of canine vector-borne diseases, including hepatozoonosis caused by H. canis in domestic dogs. The present study investigated the prevalence of Hepatozoon spp. by means of histopathology and molecular analysis of different tissues in red foxes from different parts of Portugal.MethodsBlood and tissues including bone marrow, heart, hind leg muscle, jejunum, kidney, liver, lung, popliteal or axillary lymph nodes, spleen and/or tongue were collected from 91 red foxes from eight districts in northern, central and southern Portugal. Tissues were formalin-fixed, paraffin-embedded, cut and stained with hematoxylin and eosin. Polymerase chain reaction (PCR) amplified a ~650 bp fragment of the 18S rRNA gene of Hepatozoon spp. and the DNA products were sequenced.ResultsHepatozoon canis was detected in 68 out of 90 foxes (75.6%) from all the sampled areas by PCR and sequencing. Histopathology revealed H. canis meronts similar in shape to those found in dogs in the bone marrow of 11 (23.4%) and in the spleen of two (4.3%) out of 47 foxes (p = 0.007). All the 11 foxes found positive by histopathology were also positive by PCR of bone marrow and/or blood. Positivity by PCR (83.0%) was significantly higher (p < 0.001) than by histopathological examination (23.4%) in paired bone marrow samples from the same 47 foxes. Sequences of the 18S rRNA gene of H. canis were 98–99% identical to those in GenBank.ConclusionsHepatozoon canis was found to be highly prevalent in red fox populations from northern, central and southern Portugal. Detection of the parasite by histopathology was significantly less sensitive than by PCR. Red foxes are a presumptive reservoir of H. canis infection for domestic dogs.