Classification of Babesia canis strains in Europe based on polymorphism of the Bc28.1-gene from the Babesia canis Bc28 multigene family
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).
Section snippets
B. canis laboratory isolates from France
Three B. canis isolates (B, 34.01 and G) and B. canis clone A8, which were routinely maintained in in vitro culture (Schetters et al., 1997), were selected in this study because of their already known genetic and phenotypic features. The B. canis A and B isolates were derived from the south-east of France (Drôme department; Uilenberg et al., 1989). They are genetically distinct based on their virulence, antigenic differences and chromosome size-polymorphism (Uilenberg et al., 1989, Schetters et
Polymorphism of the Bc28.1 gene and deduced amino acid sequence from laboratory strains of B. canis
As a first step in the analysis of the Bc28.1 gene polymorphism, the complete genomic sequence of this gene (PCR fragment F5′UTR281&2/R3′UTR281) was determined using the B. canis laboratory strains (A8, B, G and 34.01; Fig. 2). As expected, the Bc28.1 gene from the B. canis A8 clone was strictly identical to sequence n°CS019629. It spanned 852 bp with a 771 bp-length open reading frame (orf; nucleotide region 50–820) predicting a 28 kDa polypeptide of 256 amino acids with a N-terminal signal
Discussion
Genetic variability and antigenic variation are important mechanisms for the survival of Babesia parasites in their vertebrate hosts. This capacity to change the antigenic make-up of the merozoite surface is one of the major impediments of vaccine development, and has been suggested as a possible explanation for the limited efficacy of a commercially available vaccine (Pirodog; Merial) in the field (Bourdoiseau, 2006). This vaccine was developed after the discovery that antigens from
Conflict of interest
The authors have no conflict of interest concerning the work performed in this paper.
Authors contribution
For this work, SR performed the MT1 and MT2-derived molecular typing of european canine blood samples in France; DD performed the development of MT2-derived molecular test in France; LA performed the MT3-derived molecular typing of blood samples in Poland; LC and GB supervised the collect of canine blood samples in Portugal; the collect of canine blood samples from other countries was supervised by TS; AG and BC supervised the work of SR and DD in France; BC and TS drafted the manuscript and
Aknowledgments
This work was supported by grant from MSD Animal Health (Boxmeer, Netherlands). The authors would like to sincerely acknowledge all the veterinarians who agreed to participate in the study and that collected blood samples from dogs in Europe. This work would be impossible without their precious contribution. We also thank Stéphane Delbecq and Karina Moubri-Menage (LBCM-EA4558, France) for their precious help in fruitful discussions and B. canis cultivation. Theo Schetters is Invited Professor
References (48)
- et al.
Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland
Vet. Parasitol.
(2008) Canine babesiosis in France
Vet. Parasitol.
(2006)- et al.
Genetic basis for GPI-anchor merozoite surface antigen polymorphism of Babesia and resulting antigenic diversity
Vet. Parasitol.
(2006) - et al.
Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal
Vet. Parasitol.
(2008) - et al.
Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay
Vet. Parasitol.
(2008) - et al.
Clinicopathological findings in naturally occurring cases of babesiosis caused by large form Babesia from dogs of northeastern Italy
Vet. Parasitol.
(2005) - et al.
PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies
Vet. Parasitol.
(2007) - et al.
Genotypic diversity of merozoite surface antigen 1 of Babesia bovis within an endemic population
Mol. Biochem. Parasitol.
(2010) - et al.
Canine babesiosis in Europe: how many diseases?
Trends Parasitol.
(2012) - et al.
Autochthonous canine babesiosis in The Netherlands
Vet. Parasitol.
(2005)
A subtropical case of human babesiosis
Acta Trop.
Clinical signs, seasonal occurrence and causative agents of canine babesiosis in France: results of a multiregional study
Vet. Parasitol.
Clinico-pathological findings and coagulation disorders in 45 cases of canine babesiosis in Spain
Vet. J.
Vaccines against babesiosis using soluble parasite antigens
Parasitol. Today
Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants
Vet. Parasitol.
Babesiosis in dogs and cats-expanding parasitological and clinical spectra
Vet. Parasitol.
Babesia canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease
Vet. Parasitol.
Clinicopathological findings, molecular detection and characterization of Babesia gibsoni infection in a sick dog from Italy
Vet. Parasitol.
Babesia – a historical overview
Vet. Parasitol.
Frequency and therapy monitoring of canine Babesia spp. infection by high-resolution melting curve quantitative FRET-PCR
Vet. Parasitol.
Structural and functional characterization of Bc28.1, major erythrocyte-binding protein from Babesia canis merozoite surface
J. Biol. Chem.
Detection of a new pathogenic Babesia microti-like species in dogs
Vet. Parasitol.
The clinical course of babesiosis In 76 dogs infected with protozoa parasites Babesia canis canis
Pol. J. Vet. Sci.
Application of the SYBR Green real-time HRM PCR technique in the differentiation of the Babesia canis canis protozoa isolated in the areas of eastern Poland
Parasitol. Res.
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Current address: Unite Molecules de Communication et Adaptation des Micro-organismes (MCAM) UMR 7245CNRS/Museum National d’Histoire Naturelle, Paris, France.