Development And Optimization Of Multiplex Pcr For The Identification Of A, O And Asia 1 Strains Of FMDV In Pakistan
Material type: Book ; Format:
Publisher: 2010 Dissertation note: Foot and mouth disease (FMD) is highly infectious disease of cattle, buffalo, sheep and goats. It is caused by genus Aphthovirus of Picomaviradae family. FMDV is RNA virus having seven serotypes A, 0, C, Asia I, SAT1, SAT2 and SAT3.
Foot and mouth disease is endemic in Pakistan and causes high economic losses to livestock industry. So priority is to develop quick and efficient methods for detection of FMDV and to limit the spread of disease outbreak. Although CFT, VNT and ELISA are already being used for the diagnosis of FMDV in Pakistan but these diagnostic techniques are time consuming and their specificity and sensitivity is low. Multiplex PCR for the identification of FMDV is very much sensitive and specific, can be done with in three hours after the receipt of samples.
Present study has been designed to optimize multiplex RT-PCR for rapid detection of FMD virus. RNA was extracted from virus stock obtained from QOL, UVAS Lahore and from field samples. After RNA extraction the samples were subjected to synthesize cDNA by the use of Reverse Transcriptase enzyme. After cDNA synthesis PCR reaction was carried out. The amplified products were resolved on 1.5% Agarose Gel. A multiplex RT-PCR strategy was optimized and developed for the detection of virus serotypes A, 0 and Asia l.
Restulst of this study helped to develop an efficient and economical method for rapid detection of FMD virus and also helpful in differential diagnosis from other vesicular diseases.
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Microbial Evaluation Of Raw Meat At Abattoirs And Retail Outlests (Lahore)
Material type: Book ; Format:
Publisher: 2010 Dissertation note: The objective of this study was to evaluate the microbial quality of meat. The present study was planed to determine the aerobic plate count on meat obtained from the abattoirs and local market. A total of 90 meat samples that were collected for determining the microbiological quality of meat. Half of the meat samples (n=45) were collected from various abattoirs and half of the meat samples (n=45) were collected from retail outlets in Lahore City to get an idea of contamination from slaughtering point to retail outlets.
These samples were processed for Aerobic plate counts, E.coli, S.aureus and Salmonella counts. Overall, this study revealed that the level of contamination on meat carcasses was higher in retail meat shops compared to the abattoir. However, the microbial contamination in the abattoir were high if we compare these results to the reports from developing countries like India, Iran and Bangladesh.
Bacterial isolates identified and counted from this study were Staphylococcus aureus (44) out of 90 samples was the most abundant as 48.88%, followed by E. coli (43) 47.77% and Salmonella (26) 28.88%.
Statistical analysis revealed that analysis of variance between various abattoir and the retail meat shops for E.coli, Salmonella and S.aureus showed significant differences with some exceptions. E.coli counts were significantly higher (P < 0.05) in the meat shops and abattoirs. For E.coli most of the data were significant at 5% level (P < 0.05) with some exception in case of beef and goat samples taken from abattoirs which were non significant because of the unhygienic environments. Analysis of variance for Salmonella between various abattoir and the retail outlets were significant at 5% level (P < 0.05). For S.aureus between various abattoir and the retail outlets showed non significant at 5% level (P > 0.05) with some exceptions in case of beef abattoir and goat retail outlet samples taken which were significant at 5% level (P < 0.05).
The higher incidence of microbial load in fresh meat obtained in this study might be attributed to unhygienic and improper handling of animals during slaughter, dressing, evisceration, transportation and unhygienic environments at the retail shops. The usual practice of washing the carcass with the same water in which intestines and offal had been washed was considered as one of the predominant reasons for increased microbial counts of the carcasses. A complete ignorance on the part of the meat handlers/ butchers in hygienic handling of carcasses during slaughter and retailing processes might be the main factors for producing meat with high microbial load.
Levels of microbial contamination in Pakistani abattoirs and traditional retail meat shops reflect the hygiene status of meat production in the developing world. Education of the meat retailers' community which runs the traditional meat shops, in terms of the importance of hygienic and sanitary precautions would go a long way towards providing wholesome and safe meat to the consumers.
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Effect Of Differnet Physico Chemical Substances On The Production Peotential Of Phycocyanin From Spirulina and its Characterization
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Publisher: 2010 Dissertation note: Spirulina is a multi-cellular, filamentous Cyanobacterium, belonging to a blue-green alga of Cyanophyta. Spirulina is recently proven in animal experiments to exhibit various biological activities such as lowering plasma cholesterol levels and blood pressure.
The principal phycobiliproteins present in spirulina are phycocyanin and allophycocyanin which are made up of dissimilar ? and ? polypeptide sub units. The fresh biomass was found suitable for phycocyanin extraction. Freezing and thawing of cells was proved the best method for extraction of phycocyanin (0.4mg/ml), as compared to homogenization, hydrochloric acid and sonication. Nitrogen effects phycocyanin production from spirulina.
Different concentrations of nitrogen spirulina medium were provided. Among which 1.875g/L spirulina produced phycocyanin (0.412mg/ml). Phosphate effects phycocyanin production from spirulina. Different concentrations of phosphate spirulina medium were provided.Among which 1.5g/L spirulina produced phycocyanin (0.354mg/ml). There is also effect of temperature on phycocyanin production. Spirulina medium 0.192mg/ml at 25oC, 0.390mg/ml at 30oC, 0.184mg/ml at 35oC. There is also effect of light on phycocyanin production. 0.361mg/ml were produce at 1500 Lux.
Molecular weight (66kDa) of phycocyanin was confirmed by SDS-PAGE and explored potential production of phycocyanin from indigenous spirulina.
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Physico-Chemical Growth Requirements And Molecular Characterization Of Indigenous Spirulina
Material type: Book ; Format:
Publisher: 2010 Dissertation note: Spirulina is a microscopic and filamentous cyanobacterium (blue-green alga). It is 60-70% protein by weight and contains a rich source of vitamins, especially vitamin B12 and provitamin A (13-carotene), and minerals, especially iron. One of the few sources of dietary y-linolenic acid (GLA), it also contains a host of other phytochemicals that have otential health benefits. For medical scientists it is gaining more attention as a nutraceutical and source of potential pharmaceuticals. Spirulina has ability to inhibit viral replication, strengthen both the cellular and humoral immunity and cause regression and inhibition of cancers it also has antioxidant property. It also has been receiving increasing interest due to its potential to produce a diverse range of chemicals and biologically active compounds, such as vitamins, carotenoid pigments, proteins, lipids and polysaccharides.
Present study was designed to explore the indigenous spirulina and its mass cultivation by optimizing the physicochemical growth requirements. One hundred and twenty samples were collected from different soils and water reservoirs from three districts (Sargodha, Lahore and Faisalabad) of Punjab. Then spirulina was isolated from collected samples and cultivated under different nutrient, temperature and light regimes to get its maximum bio-mass in our laboratory.
Our results showed that maximum growth of indigenous spirulina was obtained at 30°C and at 1500 lux (light intensity). Nitrogen concentrations (0.625. 1.25 and 1.875 gIl) had no effect on the growth, while phosphate concentrations (0.5, 1.0 and 1.5 gIl) had a minimal and gradual effect on growth as the concentrations were increased. For the confirmation and molecular characterization of indigenous spirulina, DNA was isolated by chioroform-isoamyl alcohol extraction method and its polymerase chain reaction (PCR) was carried out by using specific primer of 16s rDNA gene (CYA1O6F and CYA78IR) and PCR products were run on gel giving an amplicon size of 700 bp.
Now a day in the world people are competing for food supplementation. The spirulina can act as a source of nutraceuticals. This study helps in optimizing the growth of indigenous spirulina. For large scale industrial production its extensive study should be done like physiology, growth, reproduction etc. This will pave an avenue for further nutraceuticals.
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Isolation And Antimicrobial Sensitivity Pattern Of Bacteria Associated With Diarrhea Among Children
Material type: Book ; Format:
; Literary form:
Publisher: 2011 Dissertation note: Diarrheal disease is one of the leading cause of morbidity and mortality among
children in underdeveloped countries. In the present study 140 stool samples, were
collected from cases of children diarrhea and 120 (85.71 %) samples showed bacterial
growth. From these culture positive samples, bacterial pathogens were isolated and
identified as per standard protocols described in Bergey's Manual of Determinative
Bacteriology. Out of 120 stool samples, 163 bacterial isolates were obtained as
Escherichia coli 113 (69.3%), Salmonella enterica 42 (25.8%) and Shigella species 8
(4.9%). From a total of 113 E. coli isolates, 48 (42.5%) were identified as invasive E. coli
and 65 (57.5%) were non-invasive on the basis of binding with the Congo Red dye of the
Medium. Age-wise prevalence of isolates was also analysed as bacterial pathogens were
found more in age group 1 month to 4 years (95.7%), followed by the least isolated from
age group 5 to 8 years (1.84%) and age group 9 to 12 years (2.4%). Antimicrobial
sensitivity profile, was studied by standard Disk diffusion method (Kirby Bauer) for
commonly used antibiotics, showed that all bacterial isolates were more sensitive to
antibiotics amikacin, norfloxacin, ciprofloxacin, imipenem, tazocin and less sensitive to
cephradine, doxycycline, tetracycline and augmentin. The present study findings showed
that although there are a number of causative agents like viruses, bacteria and parasites of
diarrheal disease, bacteria still remain one of the major cause with E. coli, Salmonella
and Shigella being more important bacterial pathogens among pediatric diarrheal patients
in the selected study of four different public sector hospitals in Lahore District.
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Activity Of Plant Extract Mediated Silver Nanoparticles Against Selective Medically Important Pathogens
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Due to increased use of antibiotics different common pathogenic microbes have developed
antibiotic resistance. The problem of antibiotic resistance is growing day by day. Plants are
being used as a valuable source of natural products to maintain the health. So use of plant
extracts is an interesting and valuable way to control this problem. Metal particles have
antibacterial activity. Extract mediated synthesis of metal nanoparticles is a very impressive
way to control the microbial diseases. To synthesize metallic nanoparticles using extracts is a
superior method than any other method. Silver has distinctive properties as antibacterial
agent. Nanoparticles can be synthesized by various methods but green synthesis is
environment friendly and much more important than other methods.
Melia azedarach have many active components due to which its every part has some
antimicrobial characteristics. Its extract mediated AgNPs are very important in this regard.
Similarly Albizia procera extracts has potential to kill pathogenic microbes. That’s why these
two plants were selected to synthesize nanoparticles. Fresh leaves were collected extracts
were prepared. Silver nitrate solution was prepared and then this solution was allowed to
react with each extract separately.
After the formation, nanoparticles were separated by centrifugation. Then their
characterization was done by UV visible spectroscopy and FTIR. After characterization well
diffusion assay was performed to check antimicrobial activity of these nanoparticles against
selected medically important pathogens. It was found that the antimicrobial activity of extract
mediated nanoparticles was better than the antimicrobial activity of plant extract alone.
In the present work it is explored that nanoparticles formed from extract of Albizia
procera has a greater antimicrobial activity against important medical pathogens. In short it
was proven that nanoparticles can control the pathogens in a better way as compared to the
extract alone. And this result that nanoparticles mediated particles extract of Albizia procera
has suggested many other new facts. Albizia procera’s properties are needed to be explored
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Antimicrobial Activity Of Selected Plant Extracts Against Streptococcus Mutans Isolated From Dental Caries
Material type: Book ; Literary form:
Publisher: 2016 Dissertation note: Streptococcus mutans is gram positive bacteria and present in cocci and chain form. It is
facultative anaerobes, non-motile, catalase negative and non-spore forming, lactic acid bacteria
and normally found in oral cavity (Rao and Austin 2014). S.mutans involve in endocarditis, dental
caries, have ability to adhere to the cardiac tissue and cause chronic infective endocarditis. Caries
is caused of tooth decay and tooth loss in adult and school aged children. Many microorganisms
can cause dental caries namely S. mutans, S.sanguis, s.mitis, S.salivarius and S. sobrinus but
S.mutans have major role in developing of dental caries. Dental caries is major problem around
the globe in these days. Generally it treated with antibiotics. But now a day’s due to increase in
antibiotic resistance, recently plant extracts and plants parts are being in use as treatment and oral
hygiene. It is therefore, present research was designed to analyze the plant extract which having
antimicrobial activity against oral bacteria and have been used traditionally for cleaning the teeth.
Azadirachta indica (Neem), Acacia nilotica (Kikar), Pongamia pinnata (Sukhchein) and
Salvadora persica (Peelu) were used to check the activity against S. mutans. Aqueous and
methanolic extracts of bark were prepared of selected plants. Extract potential against S. mutans
was checked through well diffusion and minimum inhibitory concentration (MIC) assay. Dental
caries samples were collected from different hospitals from Lahore. Twenty five samples were
processed to isolate S.mutans. Out of twenty five samples twenty isolates of S.mutans were isolated
which showed resistance against bacitracin, confirmed by disk method.. Biochemical tests such
as hemolysis test and sugar fermentation tests were also done for the confirmation of S. mutans.
Furthermore, antibiotic sensitive test was performed to check the sensitive pattern of S. mutans All
S. mutans were resistant to oxacillin, cefmetazole and cephaloridine, and sensitive to streptomycin
and gentamycin. S. mutans showed high level of resistance to ceftriaxone (90%), cefixim (90%),
chloramphenicol (65%) and vancomycin (60%), and intermediate level of resistance to
sulfamethoxazole/Trimethoprim (40%) and ciprofloxacin (30%), and low level of resistance to
amoxicillin (25%), delfopristin (25%) ampicillin (20%), fusidic acid (20%) and linezolid (5%).
MIC value for aqueous extract for Salvadora persica, Azadirachta indica, Acacia nilotica,
Pongamia pinnata are 24-48, 3-48,0,180-12 and >48 mg/ml respectively, and MIC value for
methanolic extract are 0.09-12, 3->48, 0.376-6 and 1.5-24 mg/ml respectively. This in vitro study
gives us natural antimicrobial plants which can help us to control dental caries and endodontic
infections. The effects of these extracts might be beneficial if incorporated in tooth paste, mouth
rinses and dental products to reduce plaque and dental caries.
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