Genotyping and characterization studies on sarcocysts of buffalo origin

Abstract

Sarcocystosis is a common parasitic entity affecting wide range of ruminants with newlinecosmopolitan distribution. Tissue samples from oesophagus, tongue and heart were collected from newlinebuffaloes slaughtered at the local slaughter house of Mathura, organized slaughter houses at Unnao and newlineAligarh alongside those brought for post mortem at Department of Pathology, DUVASU, Mathura. newlineTissue samples were screened for the presence of sarcocysts. The average size of the macroscopic newlinesarcocysts varied from 5.0-29.5 x 3.1-7.3 mm. The cysts were thin walled (2.0-2.5 µm) with average newlinesize of bradyzoite ranging from 15.0-16.5 x 3.5-4.3 µm. The average size of microscopic cysts in the newlineheart varied from 0.2-1.0 x 0.15-0.5 mm. The micro sarcocysts found in heart were also thin walled newlinewith average size of bradyzoite ranging from 9.3-10.8 x 3.0-4.1 µm. Based on morphological features, newlinethe macroscopic sarcocysts were identified as S. fusiformis while the microscopic sarcocysts were newlineidentified as S. levinei. On histopathological examination, both the macroscopic as well as microscopic newlinecysts were found to be having characteristic thin wall structure. Histopathological findings revealed newlinemuscle fiber degeneration with mild degenerative changes like loss of striations of muscle fibers newlinearound all those macrosarcocysts. None of inflammatory cells were observed around the intact cysts. newlineNo hyalinization of muscle fibrils was seen. newline Three DNA isolation protocols were compared to find out the most suitable protocol for DNA newlineisolation from sarcocysts. All the three protocols showed good results with regards to DNA extraction newlineyield as well as DNA purity. However, 18S based PCR was able to detect Sarcocystis spp. DNA from newlinemaximum number of samples isolated with kit with modification. A total of 88 sequences were newlinegenerated in the present study. Wide nucleotide variations were noticed within the studied isolates of newlinevarious Sarcocystis spp. as well as with other isolates across the globe for all the studied genes. Studied newlinesequences of S. fusiformis showed 99.1-100.0%, 99.2-100.0%, 99.2-100.0% and 99.1-100.0% nucleotide newlinehomology within themselves and 44.6-95.5%, 77.9-99.9%, 97.3-99.9% and 98.2-99.8% nucleotide newlinehomology with isolates across the globe for 18S, cox 1, 28S and ITS 1 genes, respectively. Alongside, newlinethe studied sequences of S. buffalonis showed 99.3-99.9 %, 99.8-100.0 %, 99.9-100.0 % and 100.0 % newlinenucleotide homology within themselves for 18S, cox 1, 28S and ITS 1 genes, respectively. They also newlineshowed 97.6-100.0 %, 99.7-99.9 %, 95.4-99.9 % and 93.1-99.8 % nucleotide homology with isolates newlineacross the globe for 18S, cox 1, 28S and ITS 1 genes, respectively. So far as studied sequences of S. newlinelevinei are concerned, they showed 99.8-100.0 %, 99.7-100.0 %, 99.7-99.9 % and 99.8-99.9 % newlinenucleotide homology within themselves for 18S, cox 1, 28S and ITS 1 genes, respectively. However, newlinethey showed 99.6-100.0 %, 99.8-100.0 % and 95.3-99.9 % nucleotide homology with isolates across the newlineglobe for 18S, cox 1 and 28S genes, respectively. So far as phylogenetic analysis of various Sarcocystis newlinespp. is concerned, S. fusiformis was found close to S. cafferi for 18S gene while rest three genes newlineshowed separate clade for S. fusiformis. S. buffalonis was found close to S. hirsuta in phylogenetic trees newlinefor all the four genes. S. levenei was found close to S. cruzi in phylogenetic trees for all the four genes. newlineDespite the occurrence of some genetic variations in the hypervariable regions of the 18S rDNA in newlineSarcocystis spp., restriction sites of the studied enzymes were found conserved amongst all studied newlinesarcocysts. Dra I gave uniform restriction digestion pattern with S. fusiformis. However, it was not able newlineto cut S. buffalonis and S. levinei. Ssp I gave similar sized digestion products with S. fusiformis and S. newlinebuffalonis but failed to digest S. levinei. Fok I was able to digest all the three Sarcocystis spp. giving newlinecharacteristic restriction patterns. Hence, Fok I was found best to differentiate these three Sarcocystis newlinespp. on PCR-RFLP basis. newline