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PRODUCTION, PURIFICATION AND CHARACTERIZATION OF CELLULASES FROM WILD AND MUTANT STRAINS OF ASPERGILLUS SPECIES
No full textMicrobes are ubiquitous tiny organisms/biomachines which have enormous potential to \ud
produce many kind of industrially important products (enzymes, metabolites, phenolics, \ud
antibiotics etc) when feeding upon specific medium. This study is about the exploration \ud
of native aspergillus members for the production of cellulase enzyme and to get enhanced \ud
production through mutation. \ud
All the isolates assessed for the production ability of CMCase (carboxymethyl cellulase) \ud
and taxonomically identified as A.niveus, A. fumigatus, A.versicolor, A. niger, A. wentti, \ud
A. terreus, A. nidulance, A.flavus. Optimized medium for selected Aspergillus versicolor \ud
(Gen Bank Accession KF905652) was 0.5% CMC, 4 pH, 0.075% peptone, 0.1% tween \ud
80, 1.5% NaNO3, 0.05% CaCl2 at 120 hrs and 30°C. CMCase purified by 40% \ud
ammonium sulfate followed by gel permeation chromatography with 11 folds of \ud
purification and molecular weight was 59 kDaltons. Km and Vmax determined as 1.134 \ud
mg/ml and 1435 U/ml/minute. CMCase maximum reactivity attained by 50mM Citrate \ud
Phosphate buffer, 0.5% CMC, pH 4 and 30°C for 30minutes.Storage stability at -18°C, \ud
4°C and 25°C retained 69%, 44% and 12% activities at 25th day respectively. \ud
KIBGE-IB37MT was a mutant of Aspergillus versicolor (KIBGE-IB37) the optimum \ud
medium was 0.5% CMC, pH 4, 0.1% peptone, 0.1% Tween 80, 120 hrs and 30°C. \ud
CMCase purified up to 12.26 folds with 59 kDaltons molecular weight. CMCase maxima \ud
obtained by 25mM Citrate Phosphate buffer at 30°C for 30 minutes while Km and Vmax \ud
were 1.13 mg/ml and 1435 U/ml/minute respectively. Shelf life at -18°C, 4°C and 25°C \ud
was estimated as the retention of 70, 63 and 49 % activities respectively
Toxicopathological Effects of Concurrent Feeding of Organophosphate and Arsenic and its Attenuation with Polyphenolic Grape Seed Extract in Broiler Chicks
No full textDuring last many decades there is frightening increase in the use of insecticides which is a dangerous situation for the humans mainly due to the residual effect of the pesticides in crops/vegetables. Chlorpyrifos (CPF), an organophosphate pesticide is being extensively used in the field of agriculture and veterinary science. In the mammals and birds, the chlorpyrifos intoxication is due to the inhibition of acetylcholine esterase which is responsible for the degradation of the neurotransmitter acetylcholine, and the resultant cholinergic over stimulation leads to the manifestations of neurotoxicity. \ud
In Pakistan, the water quality in many areas is poor and the contamination with arsenic is one of the major causes of deterioration. Arsenicosis is one of the major clinical syndromes present in Pakistan due to drinking arsenic rich water. Polyphenolic grape seed extract (PGE) is an antioxidant nutritive supplement having broad spectrum properties and can be used to ameliorate the effects of arsenic and chlorpyrifos. The present study was planned to document the all possible toxicological effects of organophosphate, arsenic and their amelioration with polyphenolic grape seed extract in broiler chicks. For this purpose, three experiments were conducted. The 1st experiment was conducted only with chlorpyrifos on 120 day-old broiler chicks divided in to 4 equal groups to check the toxicopathological effects. \ud
The groups were allotted according to the doses of the chlorpyrifos 5, 10, 20mg/kg BW and control. The selective doses of chlorpyrifos were given for two weeks and for the rest of experiment the birds were fed without chlorpyrifos, i.e., normal feeding. The clinical signs observed during the experiment were salivation, lacrimation, frequent defecation, gasping, tremors and convulsions. In the present study decreased feed intake in dose dependent manner was observed. The changes in body weight were also dose dependent. The changes in blood parameters induced by chlorpyrifos were noted in the present experiment. A significant decrease in the values of total erythocytic count, hemoglobin, packed cell volume and total leukocytic count as compared to control were observed in treatment groups during the treatment period. A significant increase in aspartate amimotransferase, alanine aminotransferase, total proteins and total albunmin levels were \ud
observed in the treated groups as compared to control during the treatment period. Non- significant results were observed in total globulin as compared to control during the experiment. Significantly lower level of acetyl cholinesterase was observed in blood, serum and plasma during the treatment period. The relative weights of liver, kidney and thymus were significantly higher in treated groups as compared to control. However, significant decrease in relative weight of bursa was observed in treatment groups as compared to control. Non-significant difference in relative weight of intestine, brain and spleen among various groups was observed. Grossly, mild hemorrhages were observed in liver treated with different doses of chlorpyrifos. In kidneys, mild to moderate congestion and swelling were observed in treated groups. Microscopically, tubular epithelial necrosis and detachment of epithelium from basement membrane were noticed. Moderate necrotic changes and vacuolar degeneration in lymphoid follicles were observed in bursa of Fabricius. In thymus, moderate to severe necrosis, congestion and vacuolar degeneration were observed. In spleen, mild to moderate congestion and necrosis were observed. Liver showed mild congestion, degenerative changes, congestion and cytoplasmic vacuolation. Necrosis and disruption of the epithelium of villi were observed in the intestine. Mild to moderate necrotic changes were observed in the brain. \ud
In 2nd experiment, effects of concurrent feedings of organophosphate and arsenic were recorded in 150 day-old broiler chicks which were divided in to five equal groups. The groups were allotted different doses of the chlorpyrifos (5mg, 10mg and 20mg/kg BW) and arsenic (50mg/kg BW), i.e., 5mg +arsenic 50mg, 10mg + arsenic 50mg, 20mg + arsenic 50mg, arsenic 50mg/kg BW and control. Clinical signs observed during the second experiment were almost similar as described in experiment one. However, in the treated groups when arsenic was administered in combination with chlorpyrifos, the severity of clinical signs were high in a dose dependent manner as compared to control. Arsenic alone and in combination with chlorpyrifos resulted in reduced feed intake and body weight as compared to control. In the present study, decrease in total erythrocytic count, hematocrit, hemoglobin and total leukocytic count by exposure of arsenic alone or in combination with chlorpyrifos was observed. Increase in total proteins and albumin was observed in high dose group as compared to control. However, non-significant effect \ud
on globulin was observed in this experiment. Increase in the levels of aspartate aminotransferase and alanine aminotransferase were observed in high dosed groups in combination of arsenic and chlorpyrifos as compared to control and other groups. Similar findings were observed in acetyl cholinesterase levels in blood, serum and plasma as in experiment one. The relative weight of kidney was higher in groups fed higher levels of arsenic and chlorpyrifos as compared to control and there was no change observed considerably in arsenic treated group. The increase in the relative weight of intestine was also observed in highest dose combination group as compared to control, while no change was observed considerably in other groups in this experiment. The decrease in the relative weights of bursa and thymus was observed as compared to control and no considerable change was observed in the relative weights of spleen and brain in this experiment. Grossly and microscopically, lesions of similar kind were observed in all organs as in experiment one.\ud
In ameliorative effect of polyphenolic grape seed extract was recorded in last experiment. For this purpose 360 day-old broiler birds were divided in to twelve equal groups. The groups were allotted different doses of the chlorpyrifos (10mg and 20mg/kg BW), arsenic (50mg/kg BW) and polyphenolic grape seed extract (80mg/kg BW), i.e., 10mg, 20mg, 10mg + arsenic 50mg, 20mg + arsenic 50mg, arsenic 50mg, PGE 80mg, PGE 80mg + 10mg, PGE 80mg + 20mg, PGE 80mg + arsenic 50mg, PGE 80mg + 10mg + arsenic 50mg, PGE80mg + 20mg + arsenic 50mg and control. In the present study, the treated groups when given in addition of polyphenolic grape seed extract showed decreased intensity of clinical signs as compared to other groups. The addition of polyphenolic grape seed extract in treatment groups (chlorpyrifos and arsenic) showed improved in feed intake. Over all, considerable increase was observed in erythrocyte indices in polyphenolic grape seed extract treated groups as compared to other groups, also there was considerable decrease in enzymes level except acetyl cholinesterase which was increased in groups treated with polyphenolic grape seed extract. In contrast to first and second experimental findings, increase or decrease in the relative weights of liver, kidney, bursa, brain, intestine, spleen and thymus was observed in the third experiment. This improvement in these parameters about the above said organs indicates the\ud
ameliorating effect of polyphenolic grape seed extract. Grossly and microscopically, the groups treated with polyphenolic grape seed extract showed considerably less degenerative changes as compared to other groups treated with chlorpyrifos alone or in combination with arsenic. \ud
From the findings of the present study it may be concluded that chlorpyrifos and arsenic alone or in combination adversely affect the feed intake, body weight, hematobiochemical, gross and histopathological parameters in broiler chicks which were successfully ameliorated by polyphenolic grape seed extract. So any problem resulting from the arsenic or chlorpyrifos intoxication may be treated and controlled by administering polyphenolic grape seed extra
Efficacy of bio-activated Zn for improving yield and quality of maize
No full textZinc (Zn) is an important micronutrient and its adequate supply is considered indispensable for growth, development and normal functioning of plants. In plants, it plays a significant role in photosynthesis, DNA replication, cell division, membrane permeability and integrity, protein synthesis and enzymatic activity, specifically in carbonic anhydrase. Zinc is equally important for human diet and its deficiency affects the immune system, disrupts normal cell growth and reproductive system, and causes skin disorders. Being a co-factor for more than 100 enzymes in human beings, it also provides protection against cancer. Strategies must be employed to increase Zn contents of cereal grains to overcome the Zn deficiency in human beings. Recent reports depict that more than 70% of Pakistani soils are categorized as zinc deficient due to Zn deficient parent material, high soil pH, high calcareousness, more salts and waterlogged conditions and the deficiency passes on to crops as well. Decent amount of zinc is believed to be indispensable for growth and development of maize which is one of the important cereal crops of Pakistan. It is crucial to increase bioavailability of Zn in maize. Among the principal sources used for this purpose, ZnSO4, containing 33% of Zn, is commonly used, but only 4-8 % of the total applied zinc is available to plants while other gets fixed into soil. Furthermore, being expensive, it has economic implications for farmer's community. Contrarily, zinc oxide (ZnO) is a cheaper and insoluble source which contains 80% of Zn. Bio-activation of insoluble source (ZnO) could be a cost effective method to improve Zn availability from it. Keeping in view the above said problem, the present study was conducted to formulate and evaluate bio-activated zinc for improving yield and quality of maize. For bio-activation purpose, several zinc solubilizing bacteria were isolated from rhizosphere of maize grown soil and quantified on the basis of zinc solubilizing potential and maximum pH reduction in broth medium by the bacterial isolates, finally selecting ten potential zinc solubilizing bacteria. The selected bacterial isolates, capable of solubilizing ZnO, were further screened for their plant growth promoting activity under axenic conditions. Out of ten bacterial isolates, AZ6 was selected for further experiments on the basis of maximum zinc solublization potential, pH reduction and improved growth of maize seedlings. The selected bacterium was later identified as Bacillus sp. AZ6 (Accession No. KT221633), on 16S rRNA gene sequence analysis. The Bacillus sp. AZ6 was characterized for its plant growth promoting attributes. The results implied that Bacillus sp. AZ6 had 1aminocyclopropane-1-carboxylate deaminase activity and produced siderophores for the biocontrol purpose. Auxins production was also observed by inoculation of Bacillus sp. AZ6 in the presence and absence of L-tryptophan. Bacillus sp. AZ6 also has the ability to produce organic acids like cinamic, ferulic, caffeic, chlorggenic , syrirgic and gallic acids, which were detected on HPLC. These acids solubilized the insoluble source of zinc (ZnO) by lowering pH of broth media. Bacillus sp. AZ6 was used with the organic material (grinded orange peel) to bio-activate the insoluble ZnO with different formulations. Efficient formulations (BOZ1, BOZ2, BOZ3, & BOZ4) were evaluated for temporal release of zinc. With the application of bio-activated zinc formulations, zinc bioavailability was increased significantly as compared to available form (ZnSO4), on the 60th day of incubation. BOZ4 was the most efficient among all for the whole sampling duration (0, 12th, 24th, 36th, 48th, 60th and 72th day). Different combinations varied in their potential for enhancing Zn bioavailability in soil and they were further evaluated in pot and field trials. The pot experiment was conducted to evaluate and compare the different formulations of bio\ud
activated zinc with the ZnSO4 on maize crop. Data were analyzed following completely randomized design. Sole application of insoluble source of zinc (ZnO) did not influence the growth, physiological, yield and quality parameters of maize whereas ZnSO4 improved these parameters significantly. Inoculations with zinc solubilizing bacteria also promoted these parameters in most of the cases as compared to control (without zinc) but results were at par with the control. Application of bio-activated zinc formulations had a significant effect on growth (fresh and dry root and shoot biomass), physiology (photosynthetic rate, transpiration rate, stomatal conductance, chlorophyll contents and carbonic anhydrase activity), yield and quality (crude protein, crude fiber, ash, oil contents, dry matter and grain zinc concentration) of maize as compared to ZnSO4 in pot conditions. Among the bio-activated zinc formulations, application of BOZ4 and BOZ3 significantly promoted most of parameters as compared to available form of zinc (ZnSO4). BOZ4 improved grain yield 11% more as compared to ZnSO4. The results of pot trial were confirmed under field conditions by conducting experiments on maize in two seasons (Field trial I in March, Field trial II in July). The results implied that growth, physiology, yield and quality parameters were significantly improved by BOZ4 and BOZ3 formulations as compared to ZnSO4. Data were analyzed following randomized complete block design for field experiments. BOZ4 improved the grain yield in field trial I and II by 10% and 12% respectively as compared to ZnSO4. The combined use of organic material enriched with zinc source (ZnO) and zinc solubilizing bacteria seems to be an effective approach, yet, cost effective, less time consuming and environmental friendly as compared to other zinc sources. Concluding, bio-activation of ZnO is an effective strategy for economical supply of Zn for improving yield and quality of maize, ultimately the farmer's community can get the maximum profit from their limited resources in addition to biofortification of crop produce with respect to Zn
Regulation of Rice Germin-like Protein 2 Gene Promoter in Response to Some Signaling Molecules and Fungal Infection in Potato
No full textGermins and germin-like proteins (GLPs) characterize a major plant proteins family that participate in several stress-linked and developmental activities. Molecular cloning and genetic evaluation of germins and GLPs have assisted well assessment of their regulation and elevated proficiency in the field of biotechnology. Moreover, the plant signaling molecules have fundamental role in the regulation of growth-linked activities and signaling systems in plants under adverse environmental conditions. The ~1100-bp Oryza sativa (rice) root germin-like protein 2 (OsRGLP2) gene promoter fused to a ?- glucuronidase (GUS) reporter gene was used to generate transgenic plants. Solanum tuberosum var. desiree was selected as an experimental plant for Agrobacterium tumefaciens mediated transformation. The present study was focused to analyse the regulation of OsRGLP2 promoter in response to some plant signaling molecules and fungal infection in transgenic potato. Further, an optimized nodal based regeneration protocol was established for potato. Potato micropropagation is valuable in resolving various difficulties associated with cultivation and productivity and it is a useful approach to generate multiple copies of the genetically modified plants. Callus culturing was optimized at different hormonal concentrations and combinations. It was observed that best (90 %) callus growth was found at 2.5 mg/L 2,4dichlorophenoxyacetic acid (2,4-D) while minimum growth (35 %) was observed at 1.5 mg/L zeatin and 1.0 mg/L 6-benzylaminopurine (BAP) combination. Moreover, Murashige and Skoog basal media supplemented with 1.0 mg/L BAP and 1.5 mg/L indole acetic acid (IAA) exhibited best regeneration potential from callus tissue used as explants. Microtuberization in potato plants is a valuable technique for obtaining germfree seed tubers, healthy plantlets, for germplasm exchange and understanding the physiological and morphological biology of tubers. Furthermore, research was conducted to analyse the effect of variable concentrations of carbon (C) source, nitrogen-based (N) supplementary nutrients, plant growth regulators (PGRs), hydrogen peroxide (H2O2) pre-treatment and different in vitro temperatures to reveal the best microtuberization conditions. Microtuberization productivity (growth percent (%) and fresh weight (mg) data records were statistically significant. After the initial optimization of tissue culture conditions, the Agrobacterium tumefaciens mediated transformation protocol was optimized for potato with the OsRGLP2 gene promoter construct. Transgenic potato plants produced were analysed in response to different plant defense signaling molecules, the relative quantification data records revealed that the OsRGLP2 promoter activity was enhanced by jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), hydrogen peroxide (H2O2) and under salt stress but up to a certain limit of salt concentration. It was observed that the OsRGLP2 promoter regulated differently towards various plant signaling molecules. Moreover, the tuberous samples from transgenic potato plants were analysed for the OsRGLP2 promoter functioning as a result of abiotic stress signals. The quantitative real time PCR (qRTPCR) analysis indicated that the GUS gene transcript accumulation detected in tubers in response to JA, SA, ABA, and H2O2 but the OsRGLP2 promoter activity in tubers was fairly low in comparison to non-tuberous samples. On the basis of quantitative results it was revealed that the OsRGLP2 promoter is linked with plant growth and developmental processes, cell signaling, seed germination, seedling development, regulation of plant water balance and osmotic stress as associated with different plant signaling molecules applied. In addition the OsRGLP2 promoter activity was also evaluated in response to potato specific fungal pathogens that were Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. The qRT-PCR results revealed 4-5 fold increase in promoter activity after every 24 hrs following infection. There was 15 fold increase in OsRGLP2 promoter activity after 72 hrs of Fusarium solani (Mart.) Sacc. treatment and a 12 fold increase was observed with Alternaria solani Sorauer. The data confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Moreover, there was a hyper accumulation of H2O2 in transgenic plants which is a clear signal for the presence of H2O2 responsive regulatory elements in OsRGLP2 promoter responsible for triggering particular defense related genes in the potato genome. The present analysis is valuable for the characterization of a monocot promoter activated under fungal stress in a dicot plant. The OsRGLP2 promoter evidently harbours certain biotic stress responsive cis-acting regulatory factors that function under fungal infection. On the basis of the present study, the data signifies the regulation of OsRGLP2 promoter in response to plant signaling molecules and biotic stress conditions. Therefore, it can be stated that the OsRGLP2 promoter is highly significant for future research studies in valuable crops by cloning several genes linked with abiotic and biotic stress resistance under its control
Prevalence and Molecular Characterization of Diarrheagenic Escherichia coli in Southern Khyber Pakhtunkhawa, Pakistan
No full textDiarrheagenic Escherichia coli (DEC) pathotypes are ranked third among the list of infectious agents, due to high morbidity/mortality rate in developed and developing countries. DEC transmission is linked to the ingestion of contaminated food and water. Fecal wastes from domestic animals, wildlife and humans are added to the water bodies via leakage or surface runoff. Humans also contaminate water bodies by adding poorly treated or untreated sewage effluents, spilling of septic tanks etc. Due to poorly developed sewage system monsoons rains in Pakistan resulted in heavy floods across the country affecting 21 million people in 2010. During the said calamity resulting in 5.3 million medical consultations, where 708, 891 13% were made only for acute diarrhea. \ud
Irrigation of agricultural farms with untreated water results in contamination of food products during harvesting, processing and handling. Foodborne diarrheal outbreaks were reported due to consumption of green leafy vegetables, drinking water and meat sources. Complications of Escherichia coli (E. coli) pathotypes infections are ranging from mild to severe life threatening diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. This study was conducted to assess the prevalence and molecular characterization of Diarrheagenic E. coli in Southern Khyber Pakhtunkhawa, Pakistan. Diarrheagenic E. coli pathotypes were isolated from diarrheal stool specimens, vegetables, meat and water sources collected for present study (2010-2012). DEC strains were isolated using (1) MacConkey agar, (2) Eosin Methylene blue agar (EMB), (3) Sorbitol MacConkey agar and (4) Cefixime Tellurite Sorbitol MacConkey Agar. Furthermore, E. coil strains were identified through biochemical tests. Colorless, nonsorbitol fermenting E. coli strains on Sorbitol MacConkey agar were confirmed as E. coli O157:H7 using DrySpot E. coli O157:H7 agglutination Kit (Oxoid, UK). Diarrheagenic E. coli pathotypes were also tested for antibiotic resistance using Kirby-Bauer disc diffusion method. Characteristics virulence factors DEC pathotypes i.e. (1) stx1, (2) stx2, (3) stx2c, (4) eae, (5) tir, (6) hlyA, (7) est (8) elt and (9) bfpA were detected using Multiplex PCR. Amplification conditions comprised of 94°C for 6 min, followed by 35 cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min. \ud
Amplicons were analyzed by electrophoresis on agarose 1.5% w?v gels using standard \ud
conditions, followed by staining with ethidium bromide. (1) Water, (2) Meat and (3) Vegetable samples collected during the present study were processed for the isolation and identification of diarrheagenic E. coli pathotypes using differential media, as described above. Multiplex PCR was used the detection of virulence genes i.e. stx1, stx2, stx2c, eae, tir, hlyA, bfpA, heat stable toxin (ST) and heat labile toxin (LT). Amplification conditions comprised of 94°C for 6 min, followed by 35 cycles of 94°C for 50 s, 57°C for 40 s and 72°C for 50 s, and finally 72°C for 3 min.\ud
Amplicons were analyzed by electrophoresis on agarose 1.5% w? v gels using standard \ud
conditions, followed by staining with ethidium bromide. \ud
Prevalence of diarrheagenic E. coli was found as high as 57% (515/900) during the \ud
present study. Based on stool physiology, 54.4% E. coli strains were isolated from \ud
watery diarrheal samples, compared to 37.6% from mucoid stool and 8% from bloody \ud
diarrheal stool specimens. Of group I, 155 (30%) isolates were confirmed as E. coli \ud
strains. Similarly, number and percentage isolation of DEC strains from each respective \ud
age group is as follows: Group II 137 (26.6%), Group III 59 (11.4%), Group IV 47 \ud
(9.2%), Group V 44 (8.6%), Group VI 42 (8.2%), and Group VII 31 (6%). However, \ud
none of the age groups achieved statistical significance. E. coli strains were obtained \ud
from 40% male patients compared to 60% strains from female patients. The frequency of \ud
E. coli strains recovered different units of hospitals were as; 31.8%, 52%, 3.15% and \ud
12.94% from medical ward, Pediatrics ward, OPD and ICU, respectively. From outdoor \ud
patients 44.5% samples were collected from refugee camps survey. E. coli strains from \ud
age Group I, was 14.1% where identified as E. coli O157:H7, followed by Group II: \ud
12.4%, Group III: 13.5%, Group IV: 10.6%, Group V: 9%, Group VI: 14.2% and Group \ud
VII: 6.4%. During the present study, 11.8% (61/515) strains were isolated during winter \ud
season, 21.3% (110/515) during spring season, 47% (242/515) during summer season \ud
and 19.8% during autumn season. \ud
Samples from pond water were contaminated with 28% (14/50) E. coli strains, where \ud
14% (2/14) were identified as serotype O157:H7. Tap water was found to be free of \ud
serotype O157:H7 contamination. Sewage water samples were contaminated with 62% \ud
E. coli strain, where 16.13% (5/31) were identified as serotype O157:H7. Similarly, 38% \ud
(19/50) irrigation water samples were contaminated with E. coli pathotypes, where \ud
13.8% (3/19) were identified as serotype O157:H7. DEC strains 64 isolated from water \ud
sources were randomly selected for pathogroup specific molecular characterization. \ud
Enterotoxigenic E. coli were identified as 56.25% (36/64) of pathogroups including a \ud
combination of heat stable toxin (ST) and heat labile toxin (LT) gene. Enteropathogenic \ud
E. coli were identified as 28% (18/64) comprising of 72% (13/18) typical \ud
enteropathogenic E. coli and 28% (6/18) atypical E. coli. Only 15.6% (10/64) were \ud
identified as E. coli O157:H7 serotype carrying stx1 and stx2 genes. \ud
Beef samples were contaminated with 58% of (29/50) E. coli strains and 20.9% (6/29) of \ud
these were identified as serotype E. coli O157:H7. Chicken meat samples were \ud
contaminated with 16% (8/50) E. coli strains and none of them was identified as serotype \ud
E. coli O157:H7. Sheep meat samples were contaminated with 21.5% (3/14) serotype E. \ud
coli O157:H7. Goat meat samples were contaminated with 34 % (17/50) E. coli \ud
pathotypes, where only one among 17 (5.9%) was confirmed as serotype E. coli \ud
O157:H7. (1) Mixed salad samples were contaminated with 54% of (27/50) E. coli strains, and 14.8% (4/27) of them were identified as E. coli O157:H7. (2) Cucumber samples were \ud
contaminated with 46% (23/50) E. coli strains and 30.4% (7/23) were confirmed as E. \ud
coli O157:H7. (3) Spinach samples were contaminated with 28% (14/50) E. coli strains \ud
and 28.57% (4/14) were identified as E. coli O157:H7. (4) Lettuce samples were \ud
contaminated with 40% (20/50) E. coli strains and 15% (3/20) were identified as E. coli \ud
O157:H7. STEC isolates (100%) were positive for the presence of stx1, stx2. tir, hly and \ud
eae genes. \ud
Among Shiga toxin, Bacteriophage Insertion (SBI) 100% stx1 bacteriophage was found \ud
to be inserted in yehV gene (both right and left side insertion). stx2 was completely \ud
invaded by the bacteriophages at wrbA site, and similar invasions were observed at sbsB \ud
occupied by stx2C bacteriophage. Out of 300 DEC isolates, randomly selected for antibiogram development, included 150 50%) E. coli from outpatients, 75 (25%) from medical ward and 75 (25%) from pediatrics ward. 94% isolates were found sensitive to imipenem, cefuroxime was the second most effective antibiotic (55%). The maximum resistance (92%) was observed against tetracycline, followed by ampicillin 83% and ciprofloxacin 81%. In case of ?lactam antibiotics, high resistance (78%) was observed against amoxicillin/clavulanic acid. \ud
Sewage water and industrial effluents needs prior treatment before entering into to the \ud
environment. Drinking water needs to be boiled before intake. Firm adherence to the \ud
prescribed drugs can decrease trends in antibiotics resistance.Oral rehydration therapy is strongly recommended to minimize extensive dehydration leading to kidney failure. It is further, recommended that the use of antibiotics in food, animals should follow prudent guidelines, to minimize spread of resistant bacteria. \ud
Keywords: Escherichia coli (E. coli), Diarrheagenic E. coli (DEC), \ud
Enterohaemorrheagic E. coli (EHEC), Diarrhaegenic E. coli (DEC), Shiga Toxin \ud
producing gene (stx
STUDY OF LIFE CYCLE OF PARDOSA SPP. (LYCOSIDAE) AND THEIR PREDATORY EFFICACY ON THE AMERICAN BOLLWORM (HELICOVERPA ARMIGERA)
No full textThe life cycle of Pardosa species viz., P. birmanica, P. leucopalpis and P. oakleyi was studied under controlled conditions. The experiment was conducted in the Arachnolgy laboratory Department of Zoology, Wildlife and Fisheries, GC University Faisalabad. The spiders were reared in clear perplex cages on artificial diet. The diet was a mixture of 100ml soya bean oil, one fresh chicken egg yolk and 5ml honey. The cotton swab was soaked in artificial diet and kept in the cage. The diet was changed after regular intervals (24 hours). The laboratory conditions were adjusted at 29±2C, 70±5 RH and 12:12 L: D photoperiod. After mating ten female spiders were selected for research in separate cages. On hatching newly born spiders-lings were climbed on the back of the female spider for 4 days, after that they were separated from the mother back. After separation a group of seventy spider-lings was randomly selected. And each spider-ling was introduced into a separate cage to prevent cannibalism. The manmade diet was given to these spider-lings under same controlled conditions. Daily observation was noted on each cage to study the developmental stages of Pardosa species viz., P. birmanica, P. leucopalpis and P. oakleyi. The measurements of abdominal, cephalothorax and total length were done with the help of micrometer (both sexes). The measurements were varying in each developing instars. The first molting stage was spent on the mother back; hence it was very difficult to measure. Three selected Pardosa species viz., P. birmanica, P. leucopalpis and P. oakleyi exhibited eight instars to become adult. The number of days spent before each molt was noted. The feeding of manmade diet by the ground spider of Pardosa species was the first sign that the mass rearing of these spiders could be utilized against various insects' pest species. In the second part of the research genus Pardosa species was used as a bio-control agent of the American bollworm (Helicoverpa armigera). The feeding potential of the Pardosa species was determined under controlled conditions against various stages of H. armigera insect pests was evaluated. The analysis of the variance regarding the feeding showed that three selected Pardosa spider species (7th & 8th Instar) had significant difference (P < 0.05) for the predation on 2nd instar of H. armigera. The three Pardosa spider species (2nd to 8th Instar) had a significant difference for the predation on 1st instar of H. armigera
Synthesis, Characterization and Application of Silica based Hybrid Materials
No full textThe development of new functionalized materials with increased performance is growing to meet the regulatory standards in response to public concerns for environment. This dissertation is an effort in this direction and describes the synthesis, characterization, and application of various functionalized silica hybrids keeping the perspective of recent technological advancements. The objective of present research is mostly driven by inadequate control of increasing concentration of toxic pollutants in environmental compartments. The synthesized functionalized hybrids are expected to find progressively their place as economical adsorbents. \ud
The direct method adopted for the synthesis of Mesoporous Silica and its functionalized hybrids offers advantage of providing uniform surface coverage and better control of the amount of incorporated organic moiety. The synthesized products are grouped as Mesoporous Silica (SBA15, MSU-H), Monodispersed ORMOSILS (VO, PO, MO), Functionalized Silica Hybrids (AS, GS, MS, VS, PS, AM, GM, MM, VM, PM), and Polysulfone Mixed Matrix Membranes (PM-15, PM-H, PM-VO, PM-PO, PM-MO). \ud
The resulting hybrid materials are thoroughly characterized to determine the surface and bulk properties and to develop understanding of intricate relationship of structural artifacts with its functionalities as adsorbents. The characteristics Si-O-Si stretching vibration bands at 1047cm-1 for Mesoporous Silica is identified on ATR-FTIR. The peculiar hexagons image is scanned under SEM. Further, the distribution of Si, O, and C element to locate the binding sites on fabricated membranes is clearly demonstrated through Mapping. The surface area of 521-580 m²/g is calculated by BET for Mesoporous Silica. The thermal stability is assessed by TG/DTA analysis. The synthesized Mesoporous Silica based hybrids were subjected to 13C and 1H NMR studies and possible structure is deduced. \ud
The selection of novel adsorbents with multiple and diverse application range is a challenge. In the same spirit, the functionalized silica based hybrids are applied in batch mode for the removal of toxic species prevalent in air (N2, Polycyclic Aromatic Hydrocarbons-PAHs), soil and water (metal cations of Hg, As, Cr, Pb, and nitro-, amino-phenols). Each adsorbent demonstrates the removal efficiency for these pollutants. The qualitative performance scale of each adsorbent is characterized as Excellent for Chromium, Very good for Lead, Good for Arsenic and fairly good for Mercury. A general comparison of silica based hybrids as adsorbents for the removal of PAHs and Phenols highlight the different efficacy extent. Phenols are observed to take the lead by showing more adsorption than PAHs. Larger molecular weight and fused aromaticity likely appear as hindrances to better retention of selected PAHs. \ud
It is concluded that Mesoporous silica better retains the liquid pollutants (metals, phenols) attributed to its larger surface area. Desorption for SBA-15 and MSU-H follows Type IV and Type I hysteresis, respectively. It is reported here that -phenyl and -methacrylate substituted ORMOSILS (PO and MO) did not construct the breakthrough curve and deformed because of non-sustainability under the applied temperature-pressure conditions. \ud
It is generally concluded from the results that basic silica framework offers good adsorptive properties. Further, adsorption in functionalized hybrids is facilitated through binding sites provided by organic moiety. The present study, thus, proposes a decontamination model for pollutants ranging from metal cations to gases. The efficient removal with regeneration capacity offer direct compliance to 3R principles. That is to Reduce (pollutants), Reuse and Recycle (functionalized hybrids/membranes). It is expected that these materials will help to reduce the pollution load on the environment. \ud
The possibility for commercialization of mixed matrix membrane is also important output of this research. The application of these materials in existing industrial processes is another avenue to find niches for applications in advanced waste water treatment technologies with no comprise on improved industrial production and sustainable environment
DETERMINATION OF EFFECTIVE PARAMETERS OF METAMATERIALS WITH SPHERICAL INCLUSION AT GHz FREQUENCIES
No full textThis thesis is devoted to the theoretical description of two component \ud
metamaterials as arrays of spherical inclusions. Metamaterials include both electric and \ud
magnetic phenomena. We study the long wave approximation for electromagnetic \ud
response of the generalized infinite chain and develop Effective Medium Theory for two \ud
types of two component composite metamaterials. One type is composed of a host \ud
dielectric material embedded periodically with spherical inclusions \ud
(iron/copper/cobalt/ferrites) and the other is a 2-D sandwich metamaterial structure in \ud
which the central material contains a chain of infinitely long metallic cylinders and the \ud
lateral slabs are embedded with spherical metallic inclusions. In this thesis the microwave \ud
frequency range 0 to 5 GHz has only been considered. The metamaterial media / \ud
structures are being considered in the thesis as artificial ferrites with their own effective \ud
complex dielectric and magnetic constants. So, the metamaterial media/ structures \ud
presented in the thesis are considered as perfect crystals with their own dispersive \ud
properties in the microwave frequency range. The lattice constant of the crystal is equal \ud
to the constant of the unit cell of metamaterial under consideration. \ud
The expressions for effective permeability tensors of the considered metamaterial \ud
medium have been obtained in microwave approximation. Using Effective Medium \ud
Theory (EMT) and Effective Medium Approximation we obtain expressions for \ud
effective saturation magnetization of the considered metamaterial medium. The effective \ud
magnetic properties for the case of the microwave propagation transverse to bias and in \ud
the direction of bias have also been obtained. Our polycrystalline metaferrites designed \ud
as metamaterial exhibited the Ultra-Low Refractive Index (ULI) phenomenon. In our \ud
thesis we have also performed an analytical modeling and numerical analysis of the \ud
effective electromagnetic response of the proposed metamaterial medium. The analytical \ud
modeling has been done using two different modified mathematical models of the \ud
complex relative permittivity and permeability for unbounded composite medium with \ud
spherical inclusions. Numerical simulations of the above mentioned parameters have \ud
been made using S-parameters obtained from Finite-Difference Time-Domain technique. \ud
The numerical simulation has been carried out for different radii of the inclusions. \ud
Remarkable coincidence between analytical and numerical results is found. The \ud
enhancement in effective relative permittivity and refractive index of the considered \ud
composite has been observed. The generalization has been done by the approaches of S- \ud
and T-matrices. The analytically obtained results are compared with the numerically \ud
calculated ones. \ud
All of the numerical simulations presented in the thesis have been done with the \ud
help of the free Meep FDTD software package while analytical modeling has been \ud
carried out using MATLAB software. \ud
Recommendations for the practical application of presented artificial material \ud
for designing microwave patch antennas have also been discussed
Development, Modification and Applications of Metal Oxides and \ud its Combinations for Bio-Applications in Separation Science
No full textSeparation science, nanotechnology and MS tools play their role coherently to address the issues in phosphoproteomics. The current research work is a contribution towards enrichment tools using nanomaterials. Material synthesis, their characterisation, application in phosphoproteomics develops a comparative perception of interactions, enrichment methodology and current value of metal oxides in the separation field. The role of metal oxides like ceria is tested to enhance the selectivity of iron and tin oxides. Comparative studies are performed to describe the difference between transition metal oxides (zirconia and titania) and lanthanide metal oxides (lanthanum oxide and ceria). Effect of carbon base material (graphene) on the efficiency of NiO as an enrichment material is checked. Using the principle mechanism of metal oxide affinity chromatography (MOAC), phosphopeptides are separated from biological fluids like egg yolk, non-fat milk and human serum. Casein phosphopeptides extracted from non-fat milk are chosen as source of ?- and ?-casein. Phosvitin and lipovittellin are the phosphoproteins extracted from egg yolk. Serum samples are applied after digestion to enrich phosphopeptides by the nanocomposites. Selectivity, sensitivity and comparison to individual metal oxides and graphene are recorded by using the standard protein mixtures. ?-casein digest spiked in bovine serum albumin (BSA), serum or cell extract is used as mixtures in selectivity study. All nanocomposites are tested for their performance in selective enrichment of phosphopeptides. Ceria nanocomposites have advantage of de-phosphorylation which helps to detect the state of phosphorylation. In comparison to magnetic iron oxide, ceria-iron oxide shows preference in selectivity. Ceria-tin oxide nanocomposite has better efficiency as compared to SnO2. Four alumina nanocomposites are produced to define the role of f-orbitals in the enrichment and to address the loss of mono and multi-phosphopeptides by transition metal oxides. It also strengthens the advantage of nanocomposites over individual metal oxides. Valuable features of these nanocomposites are the simplicity of enrichment protocol. NiO grafted on graphene nano-foams alter the surface chemistry which influences the phosphopeptides enrichment. MALDI-MS is adopted as detection tool for the enriched species. Data acquisition is performed thorough online searching with Swiss Prot. Parameters are chosen on the basis of sample nature. Phosphosite Plus and Phosida are explored for the phosphorylation sites in case of serum analysis
