Seroprevalence of Babesia canis Infection in ...

Seroprevalence of Babesia canis Infection in Clinically Healthy Dogs From Western Romania Author(s): Mirela Imre , Róbert Farkas , Marius Ilie , Kálmán Imre , Ionela Hotea , Sorin Morariu , Doru Morar , and Gheorghe D r bu Source: Journal of Parasitology, 99(1):161-163. 2013. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/GE-3129.1 URL: http://www.bioone.org/doi/full/10.1645/GE-3129.1

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J. Parasitol., 99(1), 2013, pp. 161–163 ! American Society of Parasitologists 2013

Seroprevalence of Babesia canis Infection in Clinically Healthy Dogs From Western Romania ´ ˘ abus ˘ ! Department of Mirela Imre, Robert Farkas*, Marius Ilie†, Ka´lmań Imre‡, Ionela Hotea, Sorin Morariu, Doru Morar§, and Gheorghe Dar Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine Timis!oara, Calea Aradului nr. 119, 300645 Timis!oara, Romania; *Department of Parasitology and Zoology, Faculty of Veterinary Science, Szent Istvań University, Budapest, Hungary; ‡Department of Food Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine Timis!oara, Calea Aradului nr. 119, 300645 Timis!oara, Romania; §Department of Internal Medicine, Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine Timis!oara, Calea Aradului nr. 119, 300645 Timis!oara, Romania. †To whom correspondence should be addressed. e-mail: [email protected]

risk parameters should be considered in the development and implementation of effective control strategies (Maia et al., 2007). In Romania, information regarding the occurrence of canine babesiosis is limited to only a few preliminary studies based on evidence of the parasite in a blood smear (Tudor et al., 2008; Ilie et al., 2010) and on serologic examination of a small number of animals (Imre et al., 2011). Additionally, molecular identification of Babesia canis canis and Babesia canis vogeli has been reported in dogs showing specific clinical signs for babesiosis (Ionit!a˘ et al., 2011). The aim of our study is to complete the picture of B. canis infection in dogs living in the Banat Region, the western historical part of Romania, and to identify the possible risk factors associated with infection. Between August 2010 and May 2011, blood samples from 197 randomly selected, clinically healthy dogs living in the Banat Region were collected into sterile vacutainer tubes by cephalic venipuncture. Data recorded were age, gender, breed, ‘keeping’ areas (urban or rural), and life style (companion, hunting, and kennel) of animals. All of the sampled dogs were born in the region and had no history of travelling abroad. The sera samples, obtained by centrifugation at 1,200 g for 10 min, were stored at "20 C until evaluation. The presence of antibodies against B. canis was detected with an indirect fluorescent antibody test (IFAT) using a commercially available antigen kit (Mega Cor Diagnostic GmbH, Horbranz, Austria) according ¨ to the manufacturer’s instructions. All sera were examined at 1:32 (cut-off value), the recommended screening dilution by the manufacturer. Slides were examined by the same reader using a fluorescence microscope (Motic-031, Miotic Incorporation LTD., Wetzlar, Germany). Samples with clearly fluorescent B. canis in the viewing field were considered to be positive following comparison with the positive and negative controls. The data were analyzed using Minitab 16 Statistical Software (Minitab, Inc., State College, Pennsylvania). The risk association for canine babesiosis (age, gender, breed, habitat, and lifestyle) was determined with a binary logistic regression model and was expressed as an odds ratio (OR) with a 95% confidence interval. Differences were considered statistically significant when P-values were less than 0.05. The results are summarized in Table I. Of the 197 clinically healthy dogs studied, 39 (19.8%) were seropositive for B. canis. The infected dogs resided in a variety of areas including villages and cities. Statistical analysis showed significant differences (P ¼ 0.031) of infection between dogs living in rural (28.4%; 19/67) and urban (15.4%; 20/130) areas. The seroprevalence of canine babesiosis was significantly higher in hunting dogs (15/34; 44.1%) as compared to companion (8/65; 12.3%; P ¼ 0.0006) and kennel (P ¼ 0.001; 16/98; 16.3%) dogs. No significant difference (P ¼ 0.481) was found between companion and kennel dogs. The age of the youngest and the oldest seropositive dog was 7 mo and 11 yr, respectively. No association was found between the seroprevalence and the age groups of infected animals. Neither the numbers of seropositive male (22/105; 20.9%) and female (17/92; 18.5%) dogs nor the numbers of infected pure (24/116; 20.6%) and crossbred (15/81;

ABSTRACT: Serum samples from 197 clinically healthy dogs residing in the Banat Region, the western historical part of Romania, were assayed by an indirect fluorescent antibody test for the presence of anti-Babesia canis antibodies. Overall, the seroprevalence was 19.8% (39/197). The percent of seropositive dogs in rural areas (28.4%; 19/67) was significantly higher (P , 0.05) compared to dogs living in urban areas (15.4%; 20/130). Seroprevalence of B. canis infection in hunting dogs was also found to be significantly higher (P , 0.05) compared to canines with other lifestyles, but no significant difference was found between companion and kennel dogs. The statistical analysis showed that no significant differences (P . 0.05) were present between the seroprevalence of infected animals associated with age, gender, or breed. The hunting lifestyle was the only factor (OR ¼ 4.57; 95% CI ¼ 2.1–10.2; P ¼ 0.002) positively associated with seroprevalence in dogs and can be considered the risk factor in the acquisition of infection. Also, the results of the present survey indicate that infection with B. canis in dogs is common in the sampling area and that it is an important pathogen for the local canine population.

Babesia canis, the large species of Babesia (Apicomplexa: Piroplasmida), is an intracellular hemoprotozoan of dogs and wild canids that causes varying degrees of hemolytic anemia, hyperthermia, hemoglobinuria, and several variations and complications of canine babesiosis (Solano-Gallego and Baneth, 2011). The parasite is transmitted by Dermacentor reticulatus ticks (Ixodidae) in temperate regions of Europe. It has been reported that the geographical expansion of canine babesiosis has been associated with travelling of dogs from, and to, endemic areas. The spread of the vector tick species related to the climatic and environmental changes is also important (Cassini et al., 2009; Menn et al., 2010; Solano-Gallego and Baneth, 2011). Microscopic detection of babesiae in the red cells is still the only viable method available to veterinarians from areas where the disease is present. However, the diagnosis in chronically infected and carrier dogs remains a significant challenge due to very low, often intermittent parasitemia that may lead to an incorrect diagnosis in infected animals. Serology is a more reliable method for detection of occult infections in less endemic areas. Several serological surveys of canine babesiosis have been reported in many parts of the world (Irwin, 2009; Solano-Gallego and Baneth, 2011). The data varied significantly, depending on the tested population and the study design as well as on the specificity and sensitivity of methods used. The indirect fluorescent antibody test (IFAT) is the most widely used, and the most specific screening technique, for detection of chronic and inconspicuous infections and is considered to be highly sensitive and moderately specific (Dantas-Torres and Figueredo, 2006; Irwin, 2009; Menn et al., 2010). The seroepidemiological survey plays an important role in the evaluation of the current status of the disease and in assessing the possible risk factors associated with acquiring infection. Moreover, the DOI: 10.1645/GE-3129.1 161

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TABLE 1. Data of seroepidemiological survey on canine babesiosis in 197 randomly selected healthy dogs in the Banat Region, Romania. Individual animal and epidemiological data

No. of animals

No. of positive samples

Prevalence (%)

72 45 51 29

13 10 9 7

18.1 22.2 17.6 24.1

92 105

17 22

18.5 20.9

116 81

24 15

20.6 18.5

130 67

20 19

15.4 28.4

65 34 98

8 15 16

12.3 44.1 16.3

Age #2 years .2 to #4 years .4 to #7 years .7 years Gender Females Males Breed Purebred Crossbred Habitat Urban Rural Lifestyle Companion Hunting Kennel

18.5%) dogs differed significantly (P ¼ 0.673 and P ¼ 0.457, respectively) from each other. The seroprevalence of B. canis infection has been demonstrated in dog populations in several geographic regions, which largely varies among and within countries and is influenced by environmental factors, animal behavior, and the population being tested. Compared with our results, generally, a lower seroprevalence of canine babesiosis obtained through IFAT procedure has been recorded in other European countries. Thus, 14.1% was reported in France (Cabannes et al., 2002), 7.3% and 13% in Albania (Lazri et al., 2008; Hamel et al., 2009), and 5.7% in Hungary (Hornok et al., 2006). Elsewhere, the highest prevalence (34%) was obtained by Cassini et al. (2009) in different regions of central and northeastern Italy. The seroprevalence obtained in the present study (19.8%) indicates a relatively high possible exposure to B. canis. However, some seropositive dogs might have a cross-reactive response to different Babesia species or to other protozoan parasites that have been frequently reported by other authors using IFAT for a serological survey of canine babesiosis (Anderson et al., 1980; Yamane et al., 1993; Vercammen et al., 1995). From a clinical and epizootiological point of view, the seropositive animals usually have no, or very mild, clinical signs of the disease after a new infection due to their active immunity against the parasite, but they may also act as reservoirs of the parasite for vector ticks (Martinod et al., 1985; Brandaõ et al., 2003). In our study, the number of the seropositive dogs of the different age groups did not differ significantly from each other. In contrast, previous results reported by various authors indicate the highest seroprevalence in the age group of 3.1–5 yr (Hornok et al., 2006), and of 6 to 10 yr (Cabannes et al., 2002). Moreover, Yamane et al. (1994) found that dogs 3 yr of age, or older, have had a significantly higher risk of being seropositive to B. canis compared with dogs less than 1-yr-old. Similarly, the gender and breed were not associated with the seropositive reactions to B. canis in the dog population tested. These data are in agreement with other studies carried out in Brazil (Costa-Junior ´ et al., 2009), California, United States (Yamane et al., 1994), France (Cabannes et al., 2002), and Italy (Cassini et al., 2009). In contrast, Hornok et al. (2006) found a

significantly higher prevalence of antibodies to B. canis among German shepherd and Komondor pure breeds. The seroprevalence of B. canis infection was significantly higher (P ¼ 0.031) in rural (28.4%) than in urban areas (15.4%). This difference may be related to a reduced treatment for hard ticks in the countryside than in the urban habitats, where animals are more regularly treated with anti-tick products. However, in rural areas, dogs live more freely and can be infested with ticks more often than in cities. The binary logistic regression analysis of the data (OR ¼ 4.57; 95% CI ¼ 2.1–10.2; P ¼ 0.002) showed that hunting lifestyle is a major risk factor for acquiring B. canis infection. Most likely, these dogs are infested more often with hard ticks, especially in their hunting trips when they walk in preferred habitats of the vectors. Moreover, in accordance with results of previous studies, Dermacentor reticulatus, the biological vector of B. canis, is distributed widely in Romania, including in this region (Teodorescu and Popa, 2002; Dar ˘ abus ˘ ! et al., 2003). The results of the serological study on canine babesiosis confirm the occurrence of B. canis infection in the Banat Region of Romania as detected by microscopic examination of babesiae (Ilie et al., 2010). Also, our results provide useful information regarding the current seroprevalence of canine babesiosis in the investigated area, which might be beneficial in developing effective strategies for disease management and control, especially by the small animal practitioners. Nonetheless, for obtaining a complex epidemiological picture, additional studies supported by molecular tools and investigations of tick populations on both domestic and wild animals are still required. This study was supported by the Ministry of Education, Research, Youth and Sport and CNCS-UEFISCDI from Romania, Grant TE_277_No.116/2010. The authors would like to thank the veterinarians, owners, and final-year veterinary students for their assistance in the collection of samples.

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RESEARCH NOTES

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