Phylogenetic congruence of Sarcocystis neurona Dubey et al., 1991 (Apicomplexa: Sarcocystidae) in the United States based on sequence analysis and restriction fragment length polymorphism (RFLP)

Abstract

The objectives of the present study were to assess the genetic diversity, phylogeny and phylogeographical relationships of available Sarcocystis neurona isolates from different localities in the United States. All 13 Sarcocystis isolates from different hosts were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses using two published DNA markers (25/396 and 33/54). The 334 bp sequence of the 25/396 marker of these isolates and Besnoitia darlingi, B. bennetti, Toxoplasma gondii and Neospora caninum were sequenced and compared. Phylogenetic analysis was performed using neighbour-joining (NJ), maximum parsimony (MP) and minimum evolution (ME) methods based on the sequences of the 25/396 marker of the 13 Sarcocystis isolates obtained in this study and sequences of 10 related isolates from GenBank. Phylogenetic trees revealed a close relatedness among S. neurona isolates in the US (nucleotide sequence diversity <5.0%). US isolates formed a monophyletic group and appeared more closely related to each other than to the South American isolates, which formed a separate lineage. NJ and ME trees with Kimura 2-parameter model separated S. neurona into two separate groups: a northern US group and a Southern US group. These findings suggest a correlation between grouping of the isolates and geographical segregation and were consistent with a genetic bottleneck hypothesis during opossum colonisation of North America. These data do not support either the view of S. neurona as a single super-species or its division into multiple subspecies.

Genetic Variation among Isolates of Sarcocystis neurona , the Agent of Protozoal Myeloencephalitis, as Revealed by Amplified Fragment Length Polymorphism Markers

“…These results suggest that the SSU rRNA region has limited value as a marker within S. neurona populations, since there was not enough variation at that level. Also, 25/396 sequence data also demonstrated low nucleotide divergence within S. neurona isolates from horses and opossums (12). The AFLP method has a higher multiplex ratio (i.e., number of loci simultaneously analyzed per experiment) than single-gene analysis.…”

“…Understanding the clinical role of S. neurona genetic variants in causing EPM is greatly hampered by the difficulty involved in their isolation, cultivation, and identification (12). Additionally, the molecular identification and typing of S. neurona associated with equine diseases and that isolated from opossums by current published methods are hampered by a lack of resolution at the inter-or intraspecific level (12). There have been a few studies that used molecular methods for the identification of S. neurona.…”

“…There have been a few studies that used molecular methods for the identification of S. neurona. These included use of random amplified polymorphic DNA (RAPD) (31), sequencing analysis of SSU rRNA (9) and SAG1 genes (10), and restriction analysis of PCR amplicons of a diagnostic marker (12,31). These methods differ in their discriminatory power, reproducibility, and ease of interpretation.…”

An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM)

“…The initial efforts to generate nucleic acid sequence data for S. neurona were conducted in the context of phylogenetic analyses, development of diagnostic probes, and included the use of RAPD assays and sequence analysis of the 18S rRNA locus (Fenger et al, 1994; Granstrom et al, 1994; Dame et al, 1995; Marsh et al, 1999; Tanhauser et al, 1999a, b; Elsheikha et al, 2005a; Elsheikha et al, 2005b; Elsheikha et al, 2006a; Elsheikha et al, 2006b; Elsheikha and Mansfield, 2007; Elsheikha, 2009) to identify species-specific genetic markers. Some of these investigations led to PCR-based tests that detect S. neurona and/or distinguish it from other closely-related species (Fenger et al, 1995; Tanhauser et al, 1999a, b).…”

Molecular characterization of Sarcocystis neurona strains from opossums (Didelphis virginiana) and intermediate hosts from Central California

“…Different types of genetic markers have been used to characterize S. neurona isolates from various hosts. Two of these markers, the first internal transcribed spacer (ITS-1) region in the nuclear ribosomal gene array and the 25/396 marker, have proven useful for making inter- and intraspecific comparisons among S. neurona isolates (Tanhauser et al, 1999; Elsheikha et al, 2005). The S. neurona surface antigen gene snSAG1 has also been assessed as an intraspecific genotyping marker (Hyun et al, 2003; Elsheikha and Mansfield, 2004).…”