Browsing by Author "Bulumulla, P. B. A. I. K."

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    Gastrointestinal Parasitism in Dairy Cattle in Badulla District: Impact of Age, Feeding Pattern and Water Source
    (Uva Wellassa University of Sri Lanka, 2012) Fernando, T. S. R.; Wijesundera, R. R. M. K. K.; Bulumulla, P. B. A. I. K.
    The Gastrointestinal tract of dairy cattle is inhabited by many species of parasites. The prevalence is variable depending upon different intrinsic and extrinsic; epidemiological and biological factors. The economic losses are in the form of mortality, decreased productivity, reduced weight gain and other means affecting the industry globally. Many cattle in Badulla district are underfed during most of the time of the yearand are not supplied with adequate balanced ration. As a result, the general nutritional status of most of the cattle is in subnormal level, which greatly increases susceptibility to parasitic diseases (Blood et al., 1990). This study examines the occurrence of gastrointestinal parasites in dairy cattle in Badulla district and identifies the significance of age, water source and feeding pattern.
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    Identification of Horse Meat and Beef using a Polymerase Chain Reaction Based Method with Cytochrome b Gene
    (Uva Wellassa University of Sri Lanka, 2013) Munasinghe, M. M. E.; Bulumulla, P. B. A. I. K.; Fernando, T. S. R.; Samaraweera, A. M.; Nanayakkara, A. K.; De Silva, D.P.D.C.; Bandara, K.G.W.W.; Senaratne, S.G.
    Adulteration of meat and meat products are taking place in many parts of the world and it is believed that numerous such incidents have even occurred in Sri Lanka as well. Demanding meat and meat products are being adulterated with cheaper or unconventional meat types (eg. dog, horse or rat meat) with phenotypic similarities. This situation created a scenario where the food analyst from many developing countries needs to pay special attention to identify meat. A recent disclosure in UK about an adulteration of beef with equine meat created a paranoid situation, which drastically affected genuine brands produced by large companies and international food chains (Thomson, 2013). Hence, the adulteration must be prevented to ensure traceability of meat from farm to form. In general, molecular methods facilitate accurate and more reliable analysis of meat adulteration. Compared to nuclear DNA, application of mitochondrial DNA (mtDNA) in meat identification experiments provides many advantages, such as maternal inheritance and ubiquitous natures (Kvist, 2000). Thus, mtDNA can be used when the evidentiary material supply is limited. Among the Mitochondrial genes mitochondrial cytochrome b (Cyt b) gene has often used to detect the source of meat (Farias, et al., 2001). Hence, the aim of this study is to establish qualitative polymerase chain reaction method to detect horse meat adulteration in beef using mitochondrial DNA Cyt b region. Methodology Beef and horse (Equus ferus caballus) meat samples were collected from slaughter house at Dematagoda and from Faculty of Veterinary Medicine and Animal Science, at University of Peradeniya, respectively. Genomic DNA was extracted following the protocol as described in Abdel-Rahman et al. (2009) with modifications. First, 600 mg of tissue was homogenized in 6000 μL STE buffer and 48 μL of 10% SDS and 24 μL of proteinase K (10 mg/mL) was added. Then, the mixture was incubated at 37 °C overnight. After that, DNA was purified by equal and chloroform–isoamylalcohol (24:1), successively. Then, DNA was precipitated by adding chilled ethanol in the presence of 3 M sodium acetate. The resulting pellet was washed with 70% ethanol, air-dried and subsequently dissolved in 80 μL of TE buffer. Species specific primers were designed (horse forward (HF) - ATC ATC ACA GCC CTG GTA GTC GTA CAT, horse reverse (HR) - ATG TGG AGG GTG GGG ATG AGT GCT A, cattle forward (CF) - CAT CGG CAC AAA TTT AGT CG and cattle reverse (CR) - GAG CTA GAA TTA GTA AGA GGG CC) to amplify mitochondrial Cyt b gene of cattle and horse. The PCR products were electrophoresed on 2% agarose gel containing 0.5 µg/mL Ethidium bromide and were visualized and imaged using a UV trans-illuminator (Gel Dox XP+ system, BioRad) and gel documentation system (Image Lab 3.0, BioRad) to distinguish the species origin. Furthermore, to investigate the detection limit of the PCR system, DNA was extracted from 600 mg of beef which was mixed separately with 1%, 5% and 20% of horse meat. Results and Discussion MtDNA evolve at a much faster rate than nuclear DNA. At the same time different regions of the mitochondrial genome would evolve at different rates. Therefore, mtDNA maintain variable regions but with a certain level of conservation. Similarly, mitochondrial Cyt b gene contains both slow and rapid evolving regions with conservative and variable regions. The evolution of the Cyt b gene is prevented due to the functional restrictions in the conservative regions (Farias, et al., 2001). Therefore, Cyt b gene is used to identify horse meat from beef as the sequence variability in Cyt b gene between different species is extremely high.