Comparison of Immunogenic Effect of Three Oil Adjuvant Vaccines against Haemorrhagic Septicaemia in Cattle and Buffalo

Haemorrhagic Septicaemia is one of the most common, fatal and acute bacterial diseases of livestock which causes mortality above 70% and is caused by Pasteurella multocida. The only satisfactory and practical method of control and prevention is timely vaccination of all the healthy and in contact animals. Different types of vaccines are being used for the immunity against this disease. In this project three oil based vaccines were produced. Two single emulsion vaccines were prepared by utilizing Montanide ISA-50 and liquid paraffin with lanolin where as one double emulsion with the help of Montanide ISA-206 was prepared. In house quality control testing and safety testing was performed on swiss albino mice. For immune titre IHA was performed by collecting serum from each and every animal including control animals. The comparison of IHA was done via statistical analysis by using GMT, Single emulsion vaccine prepared from liquid paraffin with lanolin gave maximum immune titre out of all the three vaccines in large animals and in young calves ISA 206 gave a significant titre.

Bromelain: Methods of Extraction, Purification and Therapeutic Applications

Bromelain is a concoction of sulfhydryl proteolytic enzymes. Depending upon the site of extraction it can be regarded as either stem bromelain (SBM) (EC 3.4.22.32) or fruit bromelain (FBM) (EC 3.4.22.33). Bromelain remain enzymatic active over a broad spectrumand endure a range of pH (5.5 to 8.0) and temperature (35.5 to 71 ºC). It is one of the extensively investigated proteolytic enzyme owing to its astonishing applications in various industries. This necessitated employing a strategy that result in highest purified bromelain in less steps and lowest cost. Use of modernistic approach such as membrane filtration, reverse micellar systems, aqueous two phase extraction and chromatographic techniques have shown promise in this regard. Besides its industrial applications, bromelain has been widely utilized as a potential phytomedical compound. Some of its reported actions include inhibition of platelet aggregation, anti-edematous, anti-thrombotic, anti-inflammatory, modulation of cytokines and immunity, skin debridement and fibrinolytic activity. It also assist digestion, enhance absorption of other drugs and is a potential postoperatively agent that promote wound healing and reduce postsurgical discomfort and swelling.

Isolation and Identification of Glucose Oxidase Hyper Producing Strain of Aspergillus niger.

Aims: Glucose oxidase is an enzyme with large scale applications in various industries. It is also used in several diagnostic kits which makes it medically important as well. Our aim was to isolate indigenous glucose oxidase hyper producing strain of Aspergillus niger from different soil samples of Punjab, Pakistan. Study Design: An experimental study. Place and Duration of Study: Institute of Industrial Biotechnology, GC University, Lahore from March 2011 to July 2012. Methodology: Two hundred and seventy nine fungal strains were isolated from soil of different localities of Punjab. Isolates were screened for glucose oxidase production using submerged fermentation. Glucose oxidase hyper producer isolate was identified using morphological and molecular techniques i.e. 18S rDNA. DNA was isolated and amplified using PCR. Gene sequencing was done and homology analysis was studied. Rate of glucose oxidase production was also analysed. Results: Glucose oxidase hyper producing isolate was identified as A. niger A247 strain. This strain gave best reproducible results (145.22 ±0.034 U/g of cell mass) after 72 hrs of fermentation at 30ºC and at a medium pH of 7.2. Conclusion: Our results indicate the natural ability of A. niger to produce Glucose oxidase in large quantity instead of using genetic manipulation techniques.

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s (g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Production of extracellular lipases by Rhizopus oligosporus in a stirred fermentor

The present investigation deals with the kinetics of submerged extracellular lipases fermentation by both wild and mutant strains of Rhizopus oligosporus var. microsporus in a laboratory scale stirred fermentor. Other parameters studied were inoculum size, pH, agitation and rate of aeration. It was found that the growth and lipases production was increased gradually and reached its maximum 9.07± 0.42ª U mL-1 (W) and 42.49 ± 3.91ª U mL-1 (M) after 30h of fermentation for both wild and mutant strain. There is overall increase of 109 percent (W) and 124 percent (M) in the production of extracellular lipases as compared to shake flask. Another significant finding of the present study is that the fermentation period is reduced to 30 h in case of wild and 23 h in case of mutant from 48 h in shake flask studies. The specific productivity of mutant strain (qp = 377.3 U/g cells/h) was several folds higher than wild strain. The specific production rate and growth coefficient revealed the hyperproducibility of extracellular lipases using mutant IIB-63NTG-7.

Mutation of Aspergillus oryzae for improved production of 3, 4-dihydroxy phenyl-L-alanine ( L-DOPA ) from L-tyrosine

A produção de L-DOPA a partir de tirosina pela cepa mutante de Aspergillus orizae UV-7 foi melhorada através de mutação química. Diferentes cepas foram testadas quanto a produção de L-DOPA por fermentação submersa, observando-se que a cepa denominada SI-12 foi a melhor produtora (300 mg de L-DOPA por g de células). A produção de L-DOPA pela cepa mutante a partir de diferentes fontes de carbono foi testada em diferentes fontes de nitrogênio, pH inicial e temperatura. Em pH ótimo (5,0) e temperatura ótima (30ºC), todos os açúcares foram utilizados para formação de biomassa, com um rendimento de L-DOPA de 150 mg.g-1, e produtividade volumétrica máxima e especifica de 125 mg.l.h-1 e 150 mg.g-1.h-1, respectivamente. A velocidade de formação do produto aumentou 3 vezes, sendo esse aumento o maior já relatado na literatura. Para explicar o mecanismo cinético da formação de L-DOPA e a inativação térmica da tirosinase, os parâmetros termodinâmicos foram determinados aplicando-se o modelo de Arrhenius: no caso da cepa mutante, a entalpia de ativação e entropia foram 40kj/mol e 0,076 kj/mol.K para produção de L-DOPA e 116 kj/mol and 0,590 kj/mol.K para inativação térmica, respectivamente. Os valores para formação do produto foram mais baixos e os para desativação do produto foram mais elevados que os valores correspondentes à cultura parental, indicando que a cepa mutante foi termodinamicamente mais resistente à denaturação térmica.

Immunoglobulin E screening of serum and lacrimal fluid: a new dimension in the management of chronic conjunctivitis

The purpose of present study is to differentiate between allergic and environment related chronic conjunctivitis on the basis of the measurement of Immunoglobulin E levels in tears and serum. Forty specimens of tear and serum from drivers were sent for Immunoglobulin E assay. Samples were divided clinically into two categories. Group A included twenty four samples [60%] who were clinically chronic conjunctivitis [chronic conjunctivitis is defined here as conjunctival inflammation of period greater than a year, remissions and relapses, with no obvious cause/ophthalmic disease and patient on or off topical treatment] and group B included sixteen samples [40%], of clinically normal [normal having no ocular symptoms] [control]. Length of service as driver, hours of daytime driving, primary ocular symptom, history of atopy or allergy, tear film break up time [BUT] were emphasized at history and ophthalmic examination. None of the subjects had clinically obvious cause of conjunctivitis. The group-A [patients] were randomly treated with fluoromethalone and artificial tears. The patients were required to record the subjective improvement. Tear Immunoglobulin E values were normal [1-2ku/L] in both A and B groups. Serum Immunoglobulin E was elevated significantly in twenty samples [84%] in group A [clinically chronic conjunctivitis] whereas, it was elevated in six samples [38%] in group B [clinically normal group]. Serum Immunoglobulin E levels were normal in 16% and 62% of group A and B respectively. The recovery was significant with fluoromethalone use at 4 weeks as compared to that of artificial tears but at 8 weeks and 12 weeks, there was no significant difference between artificial tears group and fluoromethalone group. Serum Immunoglobulin E and tear Immunoglobulin E levels can be used to investigate the cases of chronic conjunctivitis as normalcy of tear Immunoglobulin E reasonably excludes local allergy. This enables the clinician to resort to tear replacement therapy rather thanprescribing steroids with accompanying side effects. However, there is no correlation of serum Immunoglobulin E with tear Immunoglobulin E