ORIGINAL PAPER Molecular characterization of Hepatozoon canis from farm dogs in Pakistan Abdullah S. Ahmad 1,2 & Muhammad A. Saeed 1 & Imran Rashid 2 & Kamran Ashraf 2 & Wasim Shehzad 3 & Rebecca J. Traub 1 & Gad Baneth 4 & Abdul Jabbar 1 Received: 18 December 2017 / Accepted: 25 January 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Hepatozoon canis is a tick-borne pathogen of canids, which is distributed worldwide. However, very little is known about this protozoan parasite in Pakistan. This study provides the first molecular evidence of H. canis from farm dogs from three agro- ecological zones of Punjab, Pakistan. A conventional PCR targeting the 18S rRNA gene was used to characterize H. canis from farm dogs from three districts, namely Kasur, Rawalpindi, and Muzaffargarh, in Punjab. Of 341 blood samples tested, 155 (45.5%) were positive for H. canis, 73 (61.3%) from Kasur, 46 (42.5%) from Rawalpindi, and 36 (31.5%) from Muzaffargarh. Phylogenetic analyses revealed that 18S rRNA sequences of H. canis from this study clustered in three clades with those of H. canis from previously published studies to the exclusion of all other Hepatozoon spp. included in the analysis. This study provides the first insight into H. canis from farm dogs in Pakistan. Furthermore, it lays a foundation for future studies of the parasite to assess the impact of canine hepatozoonosis in dogs from various agro-ecological zones in Pakistan where pet ownership of dogs is increasing. Keywords Hepatozoon canis . 18S rRNA gene molecular characterization . Prevalence . Dogs . Pakistan Introduction Hepatozoon represents an important genus of apicomplexan protozoa and consists of more than 300 species, with at least 46 of them infecting mammals (Smith 1996). Hepatozoon canis and Hepatozoon americanum are important tick-borne pathogens which cause canine hepatozoonosis in domestic and wild canids (Gomes et al. 2016). Ixodid ticks are known to transmit H. canis in canids, including Ambylomma ovale in South America (Rubini et al. 2009), and Rhipicephalus sanguineus and Rhipicephalus turanicus in different parts of the world (Baneth et al. 2007; Giannelli et al. 2017), whereas Ambylomma maculatum is the only vector known for H. americanum in the USA (Mathew et al., 1998). The trans- fer of infection has also been reported by vertical transmission for H. canis (Murata et al. 1993) and by predation for H. americanum (Johnson et al. 2009). H. canis and H. americanum infections may result in marked elevation of leukocytes, leading to clinical signs such as pyrexia, inappetence, hyperesthesia, muscle wasting, and weight loss (Baneth 2011). Most H. canis- infected dogs have subclinical infection or mild clinical signs, although a certain subset experience severe and po- tentially fatal disease (Baneth 2011). As the name indicates, H. americanum has only been re- ported to cause clinical disease in dogs in the USA so far (Potter and Macintire 2010), whereas the occurrence of Section Editor: Xing-Quan Zhu Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00436-018-5790-1) contains supplementary material, which is available to authorized users. * Abdul Jabbar jabbara@unimelb.edu.au 1 Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Melbourne, Victoria, Australia 2 Department of Parasitology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan 3 Institute of Biochemistry and Biotechnology, Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan 4 The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Parasitology Research https://doi.org/10.1007/s00436-018-5790-1