Genetic Diversity and Geographic Population Structure of Bovine Neospora caninum Determined by Microsatellite Genotyping Analysis

Javier Regidor-Cerrillo,Carmen Cuevas,Luis M Ortega-Mora,Francisco Díez-Fuertes,Susana Pedraza-Díaz,Mercedes Mezo,Alicia García-Culebras,Marta González-Warleta,Gereon Schares,Frank Katzer,Dadín P Moore

doi:10.1371/journal.pone.0072678

Abstract

The cyst-forming protozoan parasite Neospora caninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among N . caninum isolates based on microsatellite sequences (MSs). MSs may be suitable molecular markers for inferring the diversity of parasite populations, molecular epidemiology and the basis for phenotypic variations in N . caninum , which have been poorly defined. In this study, we evaluated nine MS markers using a panel of 11 N . caninum -derived reference isolates from around the world and 96 N . caninum bovine clinical samples and one ovine clinical sample collected from four countries on two continents, including Spain, Argentina, Germany and Scotland, over a 10-year period. These markers were used as molecular tools to investigate the genetic diversity, geographic distribution and population structure of N . caninum . Multilocus microsatellite genotyping based on 7 loci demonstrated high levels of genetic diversity in the samples from all of the different countries, with 96 microsatellite multilocus genotypes (MLGs) identified from 108 N . caninum samples. Geographic sub-structuring was present in the country populations according to pairwise F ST. Principal component analysis (PCA) and Neighbor Joining tree topologies also suggested MLG segregation partially associated with geographical origin. An analysis of the MLG relationships, using eBURST, confirmed that the close genetic relationship observed between the Spanish and Argentinean populations may be the result of parasite migration (i.e., the introduction of novel MLGs from Spain to South America) due to cattle movement. The eBURST relationships also revealed genetically different clusters associated with the abortion. The presence of linkage disequilibrium, the co-existence of specific MLGs to individual farms and eBURST MLG relationships suggest a predominant clonal propagation for Spanish N . caninum MLGs in cattle.

Highlights

Neospora caninum is a cyst-forming obligate intracellular parasite that is phylogenetically related to Toxoplasma gondii and has been recognised as a worldwide cause of neuromuscular disease in dogs and a major cause of abortion in cattle [1,2,3]
Two alleles were identified for the MS21 marker, which was excluded from subsequent multilocus genotypes (MLGs)-data analyses
Within the MS7 marker, two types of alleles were clearly defined by a single nucleotide polymorphism (SNP) -2 bp from the TA repetitive motif that results in an additional TA repeat for the two unique alleles identified as 9.1 and 10.1 (Table S2)

Summary

Introduction

Neospora caninum is a cyst-forming obligate intracellular parasite that is phylogenetically related to Toxoplasma gondii and has been recognised as a worldwide cause of neuromuscular disease in dogs and a major cause of abortion in cattle [1,2,3]. Advances in research concerning the N. caninum life cycle have confirmed that canids (dog, coyote, dingo and gray wolf) are intermediate and definitive hosts and that cattle and other ungulates, including sheep, goat, water buffalo, bison and deer, are natural intermediate hosts [1,2,3]. Dogs can become infected by ingesting N. caninum-infected bovine tissues, and oocysts are shed in the faeces after sexual reproduction presumably in intestinal cells, which in turn can orally infect cattle [2,3,4,5,6]. Endogenous vertical transmission is recognised as the main route of transmission in cattle [2,3,9], but it has been suggested that N. caninum infection is not likely to persist in the absence of horizontal transmission [10] and recent extensive serological analyses have demonstrated significant post-natal infection rates in cattle [11]. A wildlife cycle for N. caninum has been described involving white-tailed deer (Odocoileus virginianus) and coyote (Canis latrans) as intermediate and definitive hosts, respectively [12,13]

Methods

Results

Conclusion