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

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    Determination of Morphological and Genetic Diversity of Wild Guppy (Poecilia reticulata) in Sri Lanka across the MHC Complex with Special Reference to Class IIB Region
    (Uva Wellassa University of Sri Lanka, 2013) Godagama, G.R.M.N.; Fernando, T.S.R.; Bulumulla, P.B.A.I.K.; Jayamanne, S.C.
    Wild guppies have potential in developing various strains with attractive colour patterns, tail types and tolerance to wide range of environment conditions, resistance to disease conditions due to high immunity. Application of molecular genetic markers, are important to identify diversity among wild guppies which are economically beneficial to ornamental industry and to implement conservation of these valuable genetic resources. Major histocompatibility complex (MHC) genes are highly polymorphic gene family and exon 2 of class II B gene is functionally important in immunity and disease resistance. Hence, in the present study attempts are made to assess the genetic and morphological diversity of wild guppy of Sri Lanka with special reference to immune related MCH class II B gene. A total of 238 wild guppies were collected from 10 regions to represent different agro-ecological zones of the country. The standard length in between 13-24 mm was selected (179 fishes) to collect morphological data and genomic DNA was extracted from muscle tissue using Chelex 100 DNA extraction kit. A PCR based method was used to amplify exone 2 region of candidate gene with forward (5’GTG GAT TTC AGA GAA TAT GCA 3’) and reverse (5’ TGA TTT ATC CAG AGC GGT TTG 3’) primers. Touch down PCR was followed to amplification in the temperature range of 47 to 45 . Selected fish sample consisted of 43.6% of male fishes and 56.4% female fishes. Significant association existed in tail types and colour patterns versus region. Highest variation of tail pattern types was recorded from Buttala region and 40.8% of guppies consisted round tail type. High variation of colour pattern is observed from Badulla region. 58.7% fishes had brownish gray colour pattern and 43% had golden upper body colour pattern. Variation of upper body colour in all regions was similar. PCR protocol was optimized. There was a morphological diversity between wild guppy fishes in different regions of Sri Lanka. Exon II in MHC class IIB region was amplified and optimized PCR protocol for further studies. Sequence variation based on Single Nucleotide Polymorphism (SNPs) and differences of immune response of wild guppy population is yet to be analyzed.
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    Development of Standard DNA Size Markers for Short Tandem Repeat Loci BM1818 and BM1824 of Cattle DNA Typing in Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2013) Nugapola, N.W.N.P.; Bulumulla, P.B.A.I.K.; Fernando, T.S.R.; Illeperuma, R.J.
    Cattle are the most common type of livestock animal raised for meat, dairy, as draft animals and various other purposes. Sri Lankan cattle population mainly consist of indigenous cattle “Sinhala batu cattle”, European pure breeds (Jersey, Friesian, Ayrshire), Indian cattle breeds (Sahiwal, Sindhi, Tharparkar), and their crosses. Identification and traceability of cattle and their products is extremely important for proven of ownership, exploration of pedigree and animal disease control. Traditional identification methods are less effective and associated with many economic, cultural and ethical concerns and are not reliable methods of establishing maternity or paternity of cattle. Deoxyribo Nucleic Acid (DNA) based identification is based on the unique, unalterable, inherited DNA profile of an individual animal as an identifier. Testing the Short Tandem Repeats (STR) of DNA in establishing the identity and parentage is widely used for humans in Sri Lanka. However DNA based identity testing methods are needed to be developed for cattle in order to use it as an accurate method than conventional methods to address problems of cattle identity and traceability. Currently there is no proper procedure of DNA based of identification in Sri Lanka due to the unavailability of standard DNA size markers and there is an urgent need of developing standard DNA size markers for cattle STR (allelic ladders) for accurate genotyping of an individual. Therefore the present study was conducted to develop standard DNA size markers for STR loci BM1818 and BM1824. Methodology Selection of loci Two bovine specific STR markers (BM1818, BM1824) recommended by the Food and Agriculture Organization (FAO) and the International Society for Animal Genetics (ISAG) (FAO, 2011) and previous studies done by Goor and Van de (2011) were selected for this study. Sample collection and DNA extraction Blood samples (n=38) and tissue samples (n=12) were randomly collected from unrelated animals from different areas of the country according to the recommendations provided by FAO 100 extraction protocol described by Phillips (2009) with minor modifications. DNA from tissue samples was extracted by ChargeSwitch Forensic DNA Purification Kit (Invitrogen™, Life Technologies Corporation) according to the kit’s manual. PCR amplification PCR amplification of extracted DNA samples was carried out with 5 µL of genomic DNA in a 50 µL reaction volume, consisted of 5 µL of 10X STR buffer (DreamTaq Green Buffer - Thermo Scientific), 5 µL of each dNTP (Guangzhou Geneshun Biotech Ltd.), 5 µL of primer (FAO, 2011 and Goor and Van de 2011) (Integrated DNA Technology), 0.30 µL of Taq DNA polymerase (DreamTaq-Thermo scientific) and 30 µL of sterilized distilled water. The PCR reaction was carried out in a Veriti 96 well thermal cycler (Applied Biosystem, USA) under the following PCR cycling conditions (Sodhi et al., 2006): 2 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at annealing temperature (56 to 62 °C) of each primer, 1 min at 72 °C, and final extension of 10 min at 72 °C. Subsequent to PCR, the products were subjected to 3 % w/v agarose gel electrophoresis to verify the success of the PCR reaction. Then the PCR products were subjected to 7 % denaturing polyacrylamide gel electrophoresis. Amplified products were detected using DNA Silver Staining procedure described by Bassam et al. (1991) and Promega technical manual. Allelic ladder construction Allelic ladders were constructed by two methods as follows (1) DNA samples that can contribute to maximum number of polymorphic alleles to each locus were selected, pooled and amplified by PCR reaction (100 µL reaction volume), (2) PCR products that can contribute to maximum number of polymorphic alleles to each locus were selected and pooled to create the allelic ladders. Results and Discussion According to the Table 1, allelic range of the BM1818 locus was from allele 11 to 21. The highest frequency was shown by allele No.18 at a percentage of 35.0 percent. Allele 11 had the lowest frequency with 1.3 percent. When considering BM1824 locus, allelic range was from allele 11 to 19. The highest frequency was shown by allele No.13 at a percentage of 34.1 percent. Two alleles (11 and 18) showed the lowest allelic frequency of 1.2 percent.
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    DNA Fingerprinting of Thunnus obesus and Thunnus albacores Fish Species for Proper Identification in Large Scale Fish Processing Industry
    (Uva Wellassa University of Sri Lanka, 2016) Perera, D.R.C.; Gunathilaka, P.A.D.H.N.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Bulumulla, P.B.A.I.K.
    Detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. The effects of species substitution are far-reaching and include economic fraud, health hazards, and illegal trade of protected species. In Sri Lanka tuna fish industry is a rapid developing field. However, the species identification prior to the processing is achieved through morphological characteristics, which is not a reliable method. Therefore, the aim of this study was to develop a diagnostic method by combining Polymerase Chain Reaction with Restriction digestion to differentiate Thunnus obesus (bigeye tuna) and Thunnus albacores (yellowfin tuna) species in order to facilitate the fish processing industries and fish exporters by developing the test for species confirmation. Deoxy ribonucleic acid (DNA) extracted from muscle tissues of T obesus and T albacores were analyzed. DNA was amplified using primers flanking a region of cytochrome b gene of 558 by and digested using two restriction endonucleases, EcoNI and Scat A product having band sizes of 187 by and 371 by was observed from T albacores after digesting with EcoNI. The digestive product by Scal resulted 215 by and 343 by band sizes for both T albacores and T obesus. The polymorphism of DNA profiles obtained by restriction digestion was used to differentiate the T albacores and T obesus species. Therefore, the current study carries a reliable approach to identify and distinguish T obesus and T albacores from the other tuna species. Keywords: Tuna species, DNA extraction, Polymerase chain reaction, Restriction Enzyme digestion
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    Evaluation of Escherichia coli, Salmonella serovars and Staphylococcus aureus Contamination of Retail Chicken Meat in Badulla District, Sri Lanka
    (Uva Wellassa University of Sri Lanka, 2013) Madurangi, G.H.P.; Chandrasena, G.; Fernando, T.S.R.; Wjesundara, W.M.N.M.; Bulumulla, P.B.A.I.K.
    Food safety is a global challenge for most developing countries. Food borne diseases are mainly caused by E.coli, Salmonella and S. aureus. And foods originated from animal are more susceptible for spoiling. Thus, the present study aimed at evaluating the Escherichia coli, Salmonella serovars and Staphylococcus aureus contamination in retail chicken meat from Badulla district to analyze the microbiological quality of the retail chicken meat in Badulla District. Twenty retail shops were randomly selected from seven secretary divisions in Badulla district. Two whole chicken samples were collected from each retail shop and transferred to the laboratory under refrigerated condition. 25 g of chicken meat samples from different cuts (breast, back, thigh, wings and whole) were taken. Each meat sample was pre enriched with 225 ml of buffered peptone water and placed in incubator at 37 for 24 hours. Loops full of pre enriched samples were streaked on Eosin Methylene Blue agar, Brilliant Green agar and Manitol Salt agar to isolate of E.coli, Salmonella and S.aureus respectively. Inoculated plates were incubated at 37 for 24 hours. Presumptive colonies on each agar plate, sub cultured on nutrient agar plates and incubated at 37 for another 24 hours. Presumptively positive colonies of E.coli, Salmonella on nutrient agar plates were bio-chemically confirmed with Simmons Citrate agar and S.aureus by catalase test. Prevalence of Salmonella in thigh, breast, back and wing cuts were 28.92 %, 20.48 %, 19.28% and 13.25 % respectively. Prevalence of Salmonella in whole chicken sample was 18.07%. No significance association was observed for the prevalence of Salmonella with different chicken meat cuts (P >0.05). Prevalence of Escherichia coli in thigh, breast, back and wing cuts were 18.99%, 26.58%, 26.58% and 11.39% respectively. Prevalence of Escherichia coli in whole chicken sample was 16.46%. There was a significance association between chicken part and the prevalence of Escherichia coli in retail chicken meat in Badulla District. Contamination rates of S .aureus in different cuts of retail chicken meat were thigh (20.99%), breast (25.93%), back (24.69%) and wing (11.11%). A significant association was observed in prevalence of S. aureus in different cuts of chicken carcass taken from the retail outlets of Badulla district (P<0.05). The highest occurrence of Salmonella was reported in Badulla division (19.28%). Incidences of Escherichia coli (24.05%) and S. aureus (18.52%) were significantly high in Bandarawela division. The findings of this study are vital to the public health risk of the country and emphasis the need of developed programme to assure the quality and safety of poultry meat at retail market
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    Expression of a Rabies Virus Specific Antigen by Cloning the Glycoprotein Gene into Escherichia con Expression System
    (Uva Wellassa University of Sri Lanka, 2016) Sewwandi, H.S.; Rodrigo, W.W.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Bandara, K.G.W.W.; Wijesundara, R.R.M.K.K.; Bulumulla, P.B.A.I.K.
    Rabies is an infectious disease characterized by dysfunction of the central nervous system caused by Lyssavirus of family Rhabdoviridae. Detection of rabies antibodies are used to confirm if people have been successfully immunized. Currently, these detection methods require lots of expertise and are generally carried out in reference laboratories at a high cost. Therefore, it is vital to develop and standardize simple techniques such as Enzyme Linked Immunosorbent Assay (ELISA) for determining the level of antibodies against rabies virus at a lower cost. Hence, the aim of the present study was to clone rabies virus specific glycoprotein gene into bacterial expression vector for the production of recombinant protein. Initial attempts were made to isolate plasmid DNA of pET-28a (+) vector and pcDNA3. -RVG recombinant plasmid containing previously cloned Rabies Virus Glycoprotein gene (RVG). Both plasmids were successfully digested with BamHI and XhoI restriction enzymes. The purified Rabies Virus Glycoprotein gene was cloned into pET-28a (+) bacterial expression vector. The pET-28a (+)-RVG plasmids were successfully transformed into TOPIOP competent cells through electroporation. Transformants were screened by rapid screening method. Out of 20 colonies 8 were identified as recombinants. Further screening of recombinant colonies will be carried out by digesting with restriction enzymes. Putative correct recombinant construct will be transferred into bacterial expression system for the expression. Keywords: Rabies, Rabies virus glycoprotein gene, Cloning
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    Investigation of Genetic Variation in Bmp4 Gene in Local Indigenous and Jamnapari Crossbred Goats in Damana Veterinary Service Division Sri Lanka
    (Uva Wellassa University of Srilanka, 2011) Wijesena, H.R.; Bulumulla, P.B.A.I.K.; Lokugalappatti, L.G.S.; Ariyarathne, H.B.S.
    Small ruminants, such as goats (Capra hircus), constitute an important livestock resource in most countries and are essential for the livelihood of many farmers (Baker et al., 2003). Application of molecular genetics approaches for the genetic progress of quantitative economic traits such as growth and reproduction in goats is an effective way of increasing their production as these methods could lead to finding of genetic markers useful for improved selection. Molecular genetics approaches have been used in the world for goat production in the recent past, and these strategies are yet to be established in Sri Lanka since they require high knowledge and capital investments. Therefore, this study was conducted as a preliminary step for the application of molecular genetics approaches in selection of goats for improved production in Sri Lanka. Single Stranded confirmation Polymorphism (SSCP) analysis is one such powerful genetic screening method to identify the sequence variation in Polymerase Chain Reaction (PCR) amplified products. In the present study, we investigated the PCR-SSCP genetic variation in the intron 2 of Bone Morphogenetic Protein 4 (BMP4) gene, which plays a major role in growth and reproduction. The study was focused on Local types (LT) and Jamnapari crossbred (JC) goats in Damana Veterinary Service (VS) division in the Ampara district of Sri Lanka.
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    Optimization of a Polymerase Chain Reaction Based Technique to Detect Genetically Modified Foods
    (Uva Wellassa University of Sri Lanka, 2016) Thalwattal, T.G.V.N.; Rodrigo, W.W.P.; Achala, H.H.K.; Withana, W.T.G.S.L.; Athapaththu, A.M.M.H.; Bulumulla, P.B.A.I.K.
    Genetically Modified foods are an important outcome in the genetic improvement procedures in plants. Nowadays it has become a significant problem regarding authentication of such foods since non-labelled genetically modified foods are existing in the market. The aim of this study was to optimize a Polymerase Chain Reaction (PCR) based technique to detect Cauliflower Mossaic Virus 35S promoter and Nopaline Synthase terminator, which are intentionally introduced to various crops to create genetically modified foods. Three pairs of primers were used for PCR amplification. Chloroplast tRNA primers were used to amplify chloroplast DNA with 571 bp amplicon length to prove the presence of plant origin DNA. Cauliflower Mossaic Virus 35S and Nopaline Synthase forward and reverse primers with 243 and 118 by amplicon length were respectively used to detect promoter and terminator regions. PCR optimized condition for CaMV 35S promoter (annealing condition- 56 °C, 40 sec.) and NOS terminator (annealing condition- 62 °C, 30 sec.) was carried out in 30 cycles each. Fresh and processed food samples (10 each) were collected from super markets and were analyzed in triplicates. During the analysis of post PCR products using 1% agarose gel, four food samples including corn, biscuit, corn flakes and processed potato samples were detected positive for promoter and terminator regions while a processed cereal mixture was detected as positive only for the terminator region. None of the foods were labelled as GM and it indicates that non labelled genetically modified foods are presence in the market. Therefore, this method could be used as simple and reliable assay for screening of unauthorized genetically modified crops and the processed food products. Keywords: Genetically modified foods, Cauliflower mossaic virus 35S promoter, Nopaline synthase terminator, Polymerase chain reaction
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    Preliminary Studies on the Fowl Semen Dilution and Effect to the Egg Fertility
    (Uva Wellassa University of Sri Lanka, 2013) Vidanage, L.V.B.; Bulumulla, P.B.A.I.K.; Kulakurasuriya, M.S.; Bandara, N.M.S.N.
    Poultry farming is considered as a principle component in livestock sector in Sri Lanka which provides commercial income and considerable contribution to national GDP. The poultry industry depends on the breeding materials obtained from poultry breeding centers and private companies which are few in the country. Presently, natural breeding is practiced as breeding method to produced day old broiler chicks which is costly and needs large number of male birds. Introduction of Artificial Insemination (AI) programmers for broiler breeder farms will be a suitable alternative for natural breeding. Though AI is practiced in some of the poultry breeding centers using undiluted semen, poultry semen dilution is a novel area to Sri Lanka (Niroshan, 2003). Since fowl semen is very thick, highly concentrate and less volume secretion, semen diluent can be used to increase the semen volume which facilitates the efficient utilization of poultry semen (Mcgvern, 2002). Therefore, present study was conducted to develop suitable poultry semen diluent and to compare the efficiency of different breeding methods in poultry. Methodology This study was carried out at Karandagolla NLDB farm and Laboratory analysis was carried out at AI center in Kundesale. Semen samples were collected from 46 weeks old male birds separately and the volume of the semen were measured using a pipette, spermatozoa concentration and semen mortality were evaluated by heamocytometer method (WHO, 1999) and Microscopic test observation (Arthur et al., 1989) respectively as semen quality parameters. Ringers’ solution was selected as the semen diluent (Solution 1) and the second solution was modified by adding carbon source (Fructose) to Tabatabeai et al. (2009) as energy source (Solution 2) and semen dilution was done in 1:1 ration of semen and diluent. After preparation of semen diluents, pH level was measured and spermatozoa concentration and semen mortality and motility were evaluated. Out of two solutions, best solution was selected and used for the semen dilution in the breeding trial. Three (03) different breeding methods as which natural breeding, artificial insemination (without semen dilution- WOD) and artificial insemination with semen dilution were practiced for breeding stock of same breed, age and physiological conditions. Females were age separated ensuring that they are free from semen. Artificial insemination program continued after three days interval and egg collection was done daily. Eggs were incubated after collecting and the fertility on day 18 and after hatching was determined. Hatchability percentage was estimated separately in all breeding methods. Data were analyzed using MINITAB 16 software. Results and Discussion The fowl semen mortality percentage was significantly higher in Ringer’s solution than modified Ringer’s Solution (Figure 1). Tough pH level of diluted semen of modified Ringers solution was 8.2 which are at the upper limit of the tolerable pH of spermatozoa (Bongonoff and Schaffner, 1954). Fructose, mainly used as an energy source for fowl semen may have a higher effect than the semen pH in reducing sperm mortality percentage in modified Ringers solution. Further the motility of the sperms is significantly higher in modified Ringer’s solution diluted semen sample than the Ringer’s solution diluted semen (p<0.05). Out of the tested two solutions, modified Ringer’s solution was used for the semen dilution in the breeding trial.
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    Preliminary Study on Microbial Contamination of Bacon & Bacon by Products
    (Uva Wellassa University of Sri Lanka, 2013) Perera, P.A.L.; Bulumulla, P.B.A.I.K.; Kurukulasuriya, M.S.; Jatarathna, S.H.
    Bacon is a type of processed meat produced from the sides, belly or back of a pig. Microbial count of the final product is increased due to various kinds of contamination sources leading to rejection of the product and possible health hazards. This study was conducted to determine the microbial contamination sources under current process of bacon production and to increase the final quality of the product by minimizing the contaminations. Meat samples collected at eight different stages of the processing line (slaughtering, chilling, deboning, brine injection, smoking, freezing, bacon slicing and bacon ends slicing) were examined microbiologically. Further, swab samples were collected from deboning table, cutting board, needle of curing the microbiological examination. After identification of possible contamination sources, workers were advised on proper cleaning and sanitizing of the equipments and contact surfaces, use of freshly prepared ingredients/brine solution for curing purpose and maintenance of proper storage conditions of the raw and processed meat for the prevention of microbial contamination. Same sampling procedure was followed for microbiological evaluation after practicing the above hygienic measures in the processing line. Data were analyzed by two-sample t-test using MINITAB 14 statistical software. Microbial loads of E. coli and S. aureus at slaughtering stage, deboned meat, chilled meat and cured meat were found above standard limits. Microbial count in equipment surfaces were found below the acceptable standard values, accordance with the microbiological limits that are referred under SLS specifications. After practicing hygienic measures in the production line of bacon, microbial counts were reduced significantly compared to the previous microbial counts (P<0.05). Microbial loads of E. coli and S. aureus in all the examined processing stages were lower than the standard. It was identified that poor hygienic conditions and working practices of the handlers is one of the most possible reason for poor microbial quality. In conclusion it can be stated that adherence to proper hygienic measures can reduce the microbial count to acceptable level.
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    Study on Growth and Performance of Three Indigenous Village Chicken Types (Small, Medium and Heavy) at the Central Popultry Research Station, Karandagolla, Kundasale
    (Uva Wellassa University of Sri Lanka, 2010) Mathiyalagan, T.; Bulumulla, P.B.A.I.K.; Gamage, D.V.S.D.S.
    Although Sri Lankan indigenous chicken (Gallus gallus) are providing a valuable protein source, unique genetic resources to the country, proper evaluation and utilization remains at a minimum level. Therefore, the present study was aimed to upgrade indigenous chicken population through selective breeding. Indigenous chicken population available in Central Poultry Reach Station were divided into three groups (small, medium and large) based on the weight. Information on body weights, lengths and heights were obtained from 549 chicks, pedigree of small ecotype, medium ecotype and large ecotype sires and dams were taken from the above farm. Semen was collected from each group of males and collected semen was inseminated to selected female groups. Collected eggs were incubated and two hatches were obtained. Data were collected from two hatched groups chicks, these chicks were identified individually from birth, by using numbered wing-bands, and weighed. Parents and chicks were fed by using commercial diets. Data on day old weight, body weights, lengths and heights at 1, 2, 3, 4, 5, 6, 7 and 8 weeks of age were analyzed by (ANOVA); General Linear Model procedure by using three ecotypes. Ecotype had a significant (P<0.05) effect on overall body weight gain per bird and mean body weight from day old to 8 weeks of age. The highest body weight gain per bird was recorded for large ecotype chicks. Among the ecotypes, small and medium had the smaller body weight gains while large ecotype had larger weight gains. The result from the analysis of variance showed a highly significant (P<0.05) difference on length gain per bird, mean length and height had a significant (P<0.05) effect on overall height gain per bird and mean height gain per bird from day old to 8 weeks of age. Three ecotypes were similar in weight, height and length from day old to 1st week. Ecotypes for 2nd week to 8th week of age weight, height and length were differed. Large ecotype chicks were shown high growth and performance among three ecotypes with regard to height, length and body weight. Key word: Ecotype, Dam, Sire, Heritability, Pedigree