Detection and Molecular Characterization of Canine Babesiosis Causative Agent Babesia canis in Naturally Infected Dogs in the Dobrogea Area (Southeastern Romania).

Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.

Clinical Occurrence of Canine Babesiosis in the Coastal Area of the Black Sea (Dobrogea) in Southeastern Romania and Associated Epidemiological Implications.

Canine babesiosis (CB) is a significant tick-borne disease caused by intra-erytrocytic protozoa of the genus Babesia (Apicomplexa: Piroplasmida) and is currently emerging or re-emerging in many European countries. Despite the increasing incidence of clinical CB, little is known of the epidemiology of this disease in Romania. Therefore, the present study was conducted to provide epidemiological data on CB in the coastal area of the Black Sea (Dobrogea), southeastern Romania. For this, 306 owned dogs presented during 2016-2018 at a veterinary clinic in the area and clinically suspected for CB were tested for the presence of intra-erytrocytic piroplasms using Giemsa-stained thin blood smears. Overall, 27.8% (95% CI: 22.83-33.16) of dogs were positive for large piroplasms. The Babesia spp.-positive dogs were 51 males and 34 females aged from 3 mo to 13 yr (mean 4.2 yr; SD = 3.6). A higher rate of infection was registered in Bichon Maltese (n = 13) and mixed-breed (n = 28) dogs. Most cases (76/85; 89.4%) were diagnosed from March to June, with only a few cases registered in November and December (1 case each). Infected dogs displayed different clinical presentations, varying from mild (38.8%) to moderate (29.4%) and severe disease (29.4%). Based on clinical- pathological changes, 38 (44.7%), 34 (40.0%), and 13 (15.3%) dogs were diagnosed with uncomplicated babesiosis, complicated babesiosis with a single organ dysfunction, and complicated babesiosis with multiple-organ-dysfunction syndrome (MODS), respectively. The recovery rate (81.6%, 70.1%, and 38.4%, respectively) was significantly different in these groups. The age of dogs appeared to be a risk factor for both severe disease (mean age of 5.8 yr) and MODS (mean age 6.8 yr). These findings document the common occurrence of CB in Dobrogea, southeastern Romania, and suggest that appropriate control measures need to be taken.