Babesia spp. and Theileria spp. are important emerging causes of disease in dogs. Alongside these domesticated hosts, there is increasing recognition that these piroplasms can also be found in a range of wild animals with isolated reports describing the presence of these pathogen in foxes (Vulpes vulpes) and captive grey wolves (Canis lupus). The prevalence and impact of these infections in free-ranging populations of canids are unknown. To gain a better insight into the epidemiology and pathogenesis of piroplasm infections in free-ranging grey wolves, pathological and molecular investigations into captive and free-ranging grey wolves in Croatia were performed. The carcasses of 107 free-ranging wolves and one captive wolf were the subjects of post-mortem investigations and sampling for molecular studies. A blood sample from one live captured wolf for telemetric tracking was also used for molecular analysis. PCR amplification targeting the 18S RNA gene revealed that 21 of 108 free-ranging wolves and one captive animal were positive for Theileria/Babesia DNA. Subsequent sequencing of a fragment of the 18S RNA gene revealed that 7/22 animals were positive for Babesia canis while the other amplified sequence were found to be identical with corresponding 18S rDNA sequences of Theileria capreoli isolated from wild deer (15/22). Haematological and cytological analysis revealed the presence of signet-ring shaped or pear-shaped piroplasms in several animals with the overall parasite burden in all positive animals assessed to be very low. Pathological investigation of the captive animal revealed fatal septicemia as a likely outcome of hemolytic anaemia. There was little or no evidence of hemolytic disease consistent with babesiosis in other animals. Importantly, the presence of B. canis in free-ranging grey wolves has not been described before but has been reported in a single fox and domestic dogs only. That B. canis infections cause disease in dogs but have little impact on wolf health possibly suggests that the wolf is the natural and the domestic dog is a secondary host. Surprisingly, the frequent finding of Theileria capreoli in wolves suggests that this Theileria species is not restricted to ungulates (cervids) but commonly infects also this carnivore species. Nevertheless, the potential role that these asymptomatically infected animals may play in the dispersal of these pathogens to susceptible sympatric species such as domesticated dogs requires further investigation.

Classification of Babesia parasites has traditionally relied on morphological differentiation based on piroplasm size and shape. Molecular typing has subsequently revealed a more complex taxonomy for these piroplasms than previously thought. To evaluate the factors that influence the morphology of Babesia species upon microscopic examination and hence, their taxonomic classification, we performed detailed characterizations of piroplasms from archival and prospective collections of cytological samples of dogs with piroplasmosis before and after death. Merozoite morphology and time of parasite disappearance following imidocarb dipropionate was also investigated. The study was divided into a (i) review of archived cytological slides from confirmed cases of canine piroplasmosis, and (ii) a prospective study of smears and tissue imprints from 15 recently necropsied dogs. The latter group could be further sub-divided into a non-treated group and an imidocarb dipropionate-treated group. Exact times of treatment before death were reviewed. Additional blood smears prepared from the live dogs and taken before therapy were also evaluated in the latter group. Parasite burden per each slide was determined in both studies. The shape and size of merozoites were described from blood smears taken while the dogs were alive and from different organs during necropsy. The results of all measurements were statistically analyzed. The morphology and size of merozoites from live dogs corresponded to that of previously described ‘large’ Babesia. The morphology and size of merozoites were significantly different (P < 0.001) in postmortem samples, however, and more consistent in shape and size with piroplasm cells previously referred to as ‘small’ Babesia. PCR and sequencing confirmed B. canis as the causative agent of disease in all investigated dogs, including in postmortem negative tissue imprints from dogs treated at least 24 h before death. Changes in the morphology of ‘large’ B. canis to ‘small’-like Babesia observed by light microscopy appear to represent a common postmortem change. Classification of Babesia parasites into ‘large’ and ‘small’ Babesia using only microscopy of postmortem slides should be treated with caution. PCR-based methodologies for detection and molecular typing of Babesia spp. may prove valuable for investigating suspected cases of babesiosis following necropsy.