Classification of Babesia canis strains in Europe based on polymorphism of the Bc28.1-gene from the Babesia canis Bc28 multigene family

Highlights

• In this study, we first developed a PCR-RFLP molecular test (designated MT2) based on polymorphism of Bc28.1 gene from Babesia canis.

• The use of Bc28.1-derived MT2 test to analyze the genetic diversity of strains in Europe revealed 3 Bc28.1-derived genotypes (A, B or 34) with an overall predominance of genotype B.

• Our data showed that strains from genotype 34 were restricted to France and a great variation in the distribution of strains from genotypes A and B according longitudinal and latitudinal location in Europe.

• These results are discussed in terms of diversity within Dermacentor reticulatus ticks subpopulations.

• Their significance to the development of efficient vaccines against canine babesiosis in Europe is also discussed.

Abstract

The vast majority of clinical babesiosis cases in dogs in Europe is caused by Babesia canis. Although dogs can be vaccinated, the level of protection is highly variable, which might be due to genetic diversity of B. canis strains. One of the major merozoite surface antigens of B. canis is a protein with a Mr of 28 kDa that belongs to the Bc28 multigene family, that comprises at least two genes, Bc28.1 and a homologous Bc28.2 gene. The two genes are relatively conserved but they are very distinct in their 3′ ends, enabling the design of specific primers. Sequencing of the Bc28.1 genes from 4 genetically distinct B. canis laboratory strains (A8, B, 34.01 and G) revealed 20 mutations at conserved positions of which three allowed the classification of B. canis strains into three main groups (A, B and 34.01/G) by RFLP. This assay was subsequently used to analyze blood samples of 394 dogs suspected of clinical babesiosis from nine countries in Europe. All blood samples were first analyzed with a previously described assay that allowed detection of the different Babesia species that infect dogs. Sixty one percent of the samples contained detectable levels of Babesia DNA. Of these, 98.3% were positive for B. canis, the remaining cases were positive for B. vogeli. Analysis of the Bc28.1 gene, performed on 178 of the B. canis samples, revealed an overall dominance of genotype B (62.4%), followed by genotypes A (37.1%) and 34 (11.8%). Interestingly, a great variation in the geographical distribution and prevalence of the three B. canis genotypes was observed; in the North–East genotype A predominated (72.1% A against 27.9% B), in contrast to the South–West where genotype B predominated (10.3% A against 89.7% B). In the central part of Europe intermediate levels were found (26.0-42.9% A against 74.0-57.1% B, from West to East). Genotype 34 was only identified in France (26.9% among 78 samples) and mostly as co-infection with genotypes A or B (61.9%). A comparative analysis of the classification of 35 B. canis strains in genotypes A and B using a previously described 18SrDNA-derived PCR-RFLP test revealed a partial but no direct correlation with the classification based on polymorphism of the Bc28.1-gene described here.

Introduction

Canine babesiosis is a widespread tick-borne disease caused by haematozoan parasites of the genus Babesia (Irwin, 2009). The severity of the disease depends on various factors such as the Babesia species involved, the age and the immune status of the host (reviewed in Irwin, 2009 and Solano-Gallego and Baneth, 2011). In Europe, Babesia canis is the predominant species associated with clinical babesiosis in dogs, and a survey of clinical signs due to B. canis infection suggested that canine babesiosis can be classified into two groups (Matijatko et al., 2012). Complicated forms, with a high mortality rate (12–20%), were mostly observed in Central Europe, particularly in Hungary and Croatia (Máthé et al., 2006, Matijatko et al., 2010). In contrast, B. canis infection in other countries from Europe (such as Italy, Spain, Portugal, France, Netherland, Poland) seems mostly due to uncomplicated forms associated with low mortality rates (<5%, the lowest being in France around 1%) (Matjila et al., 2005, Furlanello et al., 2005, Bourdoiseau, 2006, Ruiz de Gopegui et al., 2007, Cardoso et al., 2008, Solano-Gallego et al., 2008, Adaszek et al., 2009, René-Martellet et al., 2013). Such difference in the virulence of B. canis strains strongly suggests genetic heterogeneity among B. canis strains.

Genetic heterogeneity of B. canis strains was first demonstrated on the basis of chromosomal profiles of two laboratory strains named A and B (Depoix et al., 2002). These strains, collected from the south of France, had been shown before to be distinct according to their virulence and antigenic make-up (Uilenberg et al., 1989, Schetters et al., 1997). Chromosomal analysis, however, is a time-consuming method that requires in vitro culture of strains, which is inappropriate for the study of strains that are collected from the field. Instead, PCR-based molecular biological tests have been used successfully for such studies (Hadj-Kaddour et al., 2007, Matjila et al., 2009, Lau et al., 2010). Polymerase chain reaction (PCR) amplification of the 18SrDNA gene was used to analyze genetic diversity of B. canis strains in Poland (Adaszek and Winiarczyk, 2008, Adaszek and Winiarczyk, 2010). Two genetically distinct groups (also designated A and B) were identified that were associated with virulence (Adaszek et al., 2009). The 18SrDNA gene is less suited to study genetic diversity of a given species because it is relatively conserved among strains of a given species (Zahler et al., 1998, Zahler et al., 2000a, Zahler et al., 2000b, Carret et al., 1999, Birkenheuer et al., 2003, Jefferies et al., 2007, Duarte et al., 2008, Wang et al., 2010).

In contrast, genes encoding virulence factors such as GPI-anchored merozoite surface antigens (GPI-MSA, that play a critical function in the invasion of red blood cell) belong to multigene families composed of polymorphic genes within a given species. It has been suggested that such variable repertoire has evolved to allow the parasite to evade host immune responses (Florin-Christensen et al., 2002, Carcy et al., 2006, Goo et al., 2012). These genes are currently used as genetic markers to analyze the genetic diversity and prevalence of strains of a given species and/or for the molecular survey of vaccine efficacy and the occurrence of breakthroughs, especially within the bovine Babesia species (Berens et al., 2005, Hadj-Kaddour et al., 2007, Lau et al., 2010). In B. canis, the Bc28.1 gene belongs to such multigene family (Carcy et al., 2005, Carcy et al., 2006). It encodes a 28 kDa GPI-anchored protein located at the surface of merozoite which plays a critical function in the invasion of canine erythrocytes (Yang et al., 2012).

Here we describe the development and use of a PCR-RFLP assay based on the Bc28.1 gene for the evaluation of genetic diversity of B. canis strains in Europe. Polymorphism analysis of Bc28.1 gene was first studied by sequencing this gene of four B. canis laboratory strains originating from France (A8, B, 34.01 and G), known to be genetically and phenotypically distinct with regard to their ability to induce in vitro agglutination of infected red blood cells (iRBC). This assay was used for the analysis of more than 390 blood samples from dogs that were suspected of babesiosis and resided in different countries in Europe. In addition, a study was performed to compare the classification of B. canis strains from Eastern Europe (n = 35) according to the Bc28.1-based PCR-RFLP test with the classification obtained with the 18SrDNA PCR-RFLP test (Adaszek and Winiarczyk, 2008).