Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78–78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli- Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission

Pathological findings associated with Dipetalonema spp. (Spirurida, Onchocercidae) infection in two species of Neotropical monkeys from Brazil

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil