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Morphologic and Molecular Investigation of Schistosomes from the Mesenteric Vein of Slaughtered Cattle

Md. Shahadat Hossain
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Abu Hadi Noor Ali Khan
Department of Pathology, Bangladesh Agricultural University,Mymensingh 2202, Bangladesh

Anita Rani Dey
Department of Pathology, Bangladesh Agricultural University,Mymensingh 2202, Bangladesh

Umme Kulsum Rima
Department of Pathology, Bangladesh Agricultural University,Mymensingh 2202, Bangladesh

Nurjahan Begum
Department of Parasitology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Anisuzzaman
Department of Pathology, Bangladesh Agricultural University,Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

In cattle, schistosomosis, caused by different species of helminthes under the genus Schistosoma (Trematoda: Schistosomatidae), is a chronic and wasting disease, contributing considerable economic losses through impairment of production. Accurate diagnosis of Schistosoma spp.by traditional and molecular methods is the key to its management. A total of 64 cattle mesentery were collected from different slaughter houses of Mymensingh district, Bangladesh. This study has shown that out of 64 cattle examined, 40 were infected with schistosomes species. Tentatively, we identified different species of Schistosoma on the basis of morphologic and morphometric analysis.Up to 1-400 adult Schistosoma indicum or Schistosoma spindale were detected from a positive sample but could not confirm the co-infection state.Histopathological examination revealed cross-section of schistosomes in lymphatics and veins of mesentery. To identify species-specific schistosomes this study developed a new approach where two specific primers (SI16sRNA and SPMit) were designed and used to amplify the genomic DNA of S. indicum and S. spindale on selected eight sample by polymerase chain reaction (PCR). The SPMit primer amplified a fragment of 330bp which was positive for all eight samples (100%) of schistosomes whereas SI16sRNA primer amplified a 606 bp fragment which was positive for three samples (37.5%). Three samples showed mixed infection with both species of S. indicum and S. spindale.In conclusion, Cattle of Mymensingh district were infected/co-infected with schistosomes and can be a cause of ill health, require further observation.

Keywords: Mesentery, Molecular Investigation, PCR, Schistosoma indicum, Schistosoma spindale.

Location: Mymensingh Sadar

Start Date: 00-12-2013

End Date: 00-05-2014

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

To investigate the morphologic and molecular of ova and adult parasites of schistosomes from the mesenteric vein of slaughtered cattle

Methodology:

A total of 64 mesentery with its intestinal part were collected from cattle of different slaughter houses of Mymensingh Sadar. The samples were collected during the period of December, 2013 to May, 2014. Samples collected early in the morning and dispatched to the Parasitology Laboratory, Department of Parasitology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh. The mesentery was separated from its attachments and cut into small pieces and immersed in large glass jars containing normal saline and left undisturbed for 4-5 hours to recover schistosomes. Tissues were squeezed and removed. The saline was therefore, searched for blood flukes. Collected parasites were tentatively identified preparing permanent slide examining under light microscope following the keys and previous description. Side by side, parasites were preserved at -20⁰C by keeping them in a properly labelled vial containing normal saline. Finally samples were transported to the Molecular Biology Laboratory, Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh for PCR amplification and characterization. Schistosomes affected mesenteries and intestines were collected and fixed in 10% buffered neutral formalin. Formalin fixed tissue samples were processed and stained with hematoxyline and eosine.

DNA extraction

For DNA extraction from schistosomes traditional DNA extraction method was followed. Briefly, stored Schistosoma spp. was chopped by using fine head scissors. About 20-30 Schistosoma spp. was chopped in a 1.5ml Eppendorf tubes containing 600μl cell lysis buffer and vortexed to wet the tissue. The mixture was incubated at 56 for 2 hours to lyse the tissue cells, and then cooling it at room temperature. The solution was centrifuged at 5000g for five minute and the supernatant was collected. After that equal volume of PCI was added and vortexed vigorously at high speed for 20 seconds to mix the sample with the PCI and centrifuged at 5000g for one minute. The clear supernatant (top layer) containing the DNA was collected in a micro centrifuge tube. Then 1/10th volume of 5N NaCl was added and mixed. After that 2.5 times ice cool absolute ethanol was added and kept at -20⁰C for 10 minute. The solution was centrifuged at 14000g for 15 minute, the supernatant was discarded and desalted twice with 80% ethanol. The mixture was then centrifuged at 14000g for 15 minute at room temperature and carefully aspirated the 80% ethanol. The DNA pellet was very loose at this point and care was taken to avoid aspirating the pellet into the pipette tips. The pellet was allowed to dry in air for 15 minute, 25 μl of nuclease free water was added and concentration of DNA was measured using spectrophotometry (A260 and A280) and agarose gel electrophoresis. The DNA suspension was labelled and stored at -20⁰C until PCR was carried out. Four set of primers were used to identify the species of Schistosoma using PCR.

PCR

PCR was performed in a total of 25 μl reaction volume consisting of 2x PCR master mix (Promega® Inc, USA), 20pmol of primer in each and 150-300ng of DNA template. A total of 35 cycles of PCR amplification reactions were carried out. The cycling parameter for the PCR identification of two genes in a thermal cycler (Master Cycler Gradient, Eppendorf, Germany) was an initial denaturation at 95⁰C for 5 minute. The PCR identification of SI16sRNA gene in a thermal cycler included the denaturation at 95⁰C for 1 minute, annealing at 52⁰C for 1 minute and extension at 72⁰C for 1 minute. The PCR amplification for the identification of SPMit was the denaturation at 95⁰C for 1 minute, annealing at 54⁰C for 1 minute and extension at 72⁰C for 1 minute. The final extension for PCR was set at 72⁰C for 10 minute and the reactions were held at 4.Finally, the PCR reactions were terminated by adding 3μl 50mM EDTA and PCR products were analyzed by electrophoresis in 1.5% agarose gel, stained with ethidium bromide and examined under UV light using an image documentation system (Cell Biosciences, Alphalmager HP, USA).

Source of Information: IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS); e-ISSN: 2319-2380, p-ISSN: 2319-2372. Volume 8, Issue 4 Ver. III (Apr. 2015), PP 47-53

Article Link (Online Journal): www.iosrjournals.org

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, specific PCR based identification provide powerful resolution and are helpful tool for the investigation of species of S. indicum and S. spindale in cattle. This technique provide a method for the rapid differentiation of different species of Schistosoma that may in combination with biochemistry and molecular biology, help us to understand and take effective control measures against schistosomosis in endemic regions.

Knowledge Management: Journal