Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt

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• 作者：Bj?rn Gjerde ; Mosaad Hilali ; Sahar Abdel Mawgood
• 关键词：Sarcocystis fusiformis ; Water buffalo ; Bubalus bubalis ; Cats ; Egypt ; Phylogeny ; 18S rRNA ; 28S rRNA ; ITS1 ; cox1 ; Sarcocystis cafferi
• 刊名：Parasitology Research
• 出版年：2015
• 出版时间：September 2015
• 年：2015
• 卷：114
• 期：9
• 页码：3401-3413
• 全文大小：770 KB
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• 作者单位：Bj?rn Gjerde (1)
  Mosaad Hilali (2)
  Sahar Abdel Mawgood (2)
  
  1. Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, P.O. Box 8146 Dep., 0033, Oslo, Norway
  2. Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biomedicine
  Medical Microbiology
  Microbiology
  Immunology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-1955

文摘

A total of 33 macroscopically visible (3-1?×--?mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total?of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-9.9?%. In comparison, the sequence identity was 98.8-9.0?% among eight complete 18S rRNA gene sequences (1,873-,879-bp long), 98.1-00?% among 28 complete ITS1 sequences (853-64-bp long) and 97.4-9.6?% among five partial 28S rRNA gene sequences (1,607-,622?bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93?% sequence identity at cox1 versus interleaved sequences and 98.7-9.1?% sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-0?days post-infection (dpi) and were euthanized 15?dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.