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Indigenous Elisa Kit For Toxoplasma Gondii: Optimization Of Antibody Detection Elisa Of Sag 1 Protein As An Antigen In Mouse Model

by Madiha Sana (2013-VA-957) | Dr. Muhammad Imran Rashid | Dr. Haroon Akbar | Dr. Wasim Shehzad.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Toxoplasma is an apicomplexan intracellular parasite which is the cause of toxoplasmosis in man and animals. It occurs by the ingestion of oocyst from feces of cats or by eating raw meat in which cysts are present. It is the one of the major cause of encephalitis and abortion in immuno-compromised animals and humans. As it is difficult to screen out infected live animals from field, it is important to vaccine animals as well as humans for toxoplasma to prevent its transmission from animals to humans and from humans to their off springs. Cloning of surface antigen genes plays an important role in development of vaccine and for serology of T. gondii. Enzyme linked immuno-sorbant assay proves to be a significant tool to estimate the humoral response elicited against expressed recombinant protein in mice. The recombinant protein of SAG1 was collected from Molecular Parasitology Laboratory, University of Veterinary and Animal Sciences, Lahore. In the previous studies, SAG1 sequence was cloned in the expression plasmid and successfully expressed in prokaryotic expression system. In the current study, rSAG1 was quantified by using BCA protein assay through BioWORLD protein quantification kit. In another experiment, the Swiss mice were immunized with 15 μg rSAG1 protein 3 times with 2 weeks intervals. Two groups of mice were formed with five mice in each group. Sera were collected after 2 weeks of each inoculation. For performing ELISA, four different experiments were performed with different concentrations i.e. 5μg/ml, 250μg/ml and 500μg/ml with two different dilutions; 1/50 and 1/20. The O.D. values of concentrations 5μg/ml and 250 μg/ml with two dilution series of 1/20 and1/50 were not observed significant while the antigen coating concentration of 500 μg/ml with 1/50 dilution showed 1:160 titre and with 1/20 dilution showed 1: 1280 titre after the 3rd shot. The O.D values with 500 CHAPTER 6 SUMMARY SUMMARY 36 μg/ml concentration with 1/20 dilution after the 3rd shot were observed significant in the inoculated group as compared to the O.D values of un-inoculated negative group. It is suggested to carry out ELISA with purified rSAG-1 protein and to optimize ELISA to test toxoplasma infected mice. Availability: Items available for loan: UVAS Library [Call number: 2433-T] (1).

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Molecular Diagnosis Of Anaplasmosis In Buffaloes

by Muhammad Salman (2008-VA-135) | Prof. Dr. Khalid Saeed | Dr. Muhammad Imran Rashid | Dr. Wasim Shehzad.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Bovine Anaplasmosis is a tick-borne haemo-rickettsail disease, caused by Anaplasma species transmitted mechanically by flies, biologically by ticks and blood contaminant fomites. It is an economically important tick-borne disease of buffalo in tropical and sub-tropical areas of the world. In current study, we developed and optimized PCR first for detecting Anaplasma at genus level in buffaloes. One hundred (100) blood samples were collected from buffaloes around the Lahore region. The stained thin blood films were examined microscopically and 37% blood samples were found positive for intra-erythrocytic bodies which were then selected for DNA extraction. The DNA was extracted using commercially available kit for eventual use in optimization of PCR for diagnosis of bovine Anaplasmosis. The primers were designed targeting 16S rRNA gene of Anaplasma. For the detection, the PCR product was run in 2% agarose gel stained with ethidium bromide and thirty seven samples showed the amplification band at 179bp. The selected samples were sent for ABI sequencing to Singapore for the accurate detection of the Anaplasma species. The sequencing results were blasted with database of Genbank and we observed homology with Anaplasma phagocytophilum. We found 37% prevalence of Anaplasmosis in buffaloes through PCR. However more studies are required to confirm the species of Anaplasma infecting buffaloes (Bobalus bobalis) by designing species specific primers. Furthermore, additional studies are needed to establish the epidemiology of Anaplasmosis by using molecular tools in different geographical areas of the country for their better control. Availability: Items available for loan: UVAS Library [Call number: 2389-T] (1).

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Anthelmintic Activity Of Withania Coagulans Against Gastrointestinal Nematode Of Sheep In District Killa Saifullah, Baluchistan

by Yousaf Gul (2009-VA-145) | Dr. Muhammad Lateef | Dr. Saadullah Jan | Dr. Muhammad Imran Rashid | Dr. Wasim Shehzad.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: Evaluation of anthelmintic activity of Withania coagulans was studied against GIT nematodes of sheep in district Killa Saifullah Baluchistan. Sheep of the district were screen out for the presence of GIT nematodes. Animal positive for GIT nematodes and having 150+ Egg per Gram (EPG) of feces was included in the drug trial. Animals were treated with extract(s) of locally available herbal plant (withania coagulans) and levamisole. Two types of plant formulations that is crude powder and crude methanole extract were prepared each with various dosages. The effect of both medicinal plant and levamisole was observed on different groups of animals and the results were analyzed with appropriate statistical tool. Eighty animals were randomly divided in to eight groups (10 animals in each group) i.e. A, B1, B2, B3, C1, C2, C3 and D. Animals in group A served as control untreated group. Animals in groups B1, B2 and B3 were treated with crude powder of Withania coagulans at the dose rate of 1, 2 and 3 g/kg body weight respectively. And Animals in groups C1, C2 and C3 were treated with crude methanol extract of Withania coagulans at 33.3, 66.6 and 100mg/kg equivalent dose rate of 1, 2 and 3 g/kg body weight respectively. Animals in group D were given Levamisole at the standard dose rate of 7.5 mg/ kg body weight. Data was analyzed by using SPSS version 20.0; comparative analysis was done by applying ANOVA. P value <0.05 was taken as significant. The analyzed data and the results revealed that Levamisole is still a better anthelmintic against ovine nematodes in district Killa Saifullah Balochistan. Efficacy of levamisole tested for 15 days in-vivo sheep was up to 92%. This efficacy was much higher than the various forms and dosages of medicinal plant. The efficacy of Levamisole was significantly higher (P<0.05) than all forms and dosages of medicinal plant. Group C3 treated with crude methanol extract of Withania coagulans at the dose rate of 10mg/kg equivalent to 3mg/kg showed highest efficacy of the plant that is up to 48%. The efficacy showed by the form of the medicinal plant used in group C3 against ovine GIT nematodes was significantly higher (P<0.05) than all other forms of the plant. Animals in group B1, B2, B3, C1 and C2 showed anthelmintic efficacy of 19.47%, 23.58%, 31.66%, 31.76% and 33.33% from day 0 to day 15th post-treatment. Gastrointenstinal nematodes of sheep have produced anthelmintic resistance against Levamisole at the dose rate of 7.5mg/kg. In previous studies Levamisole had showed efficacy of 99.99%, 99% and 98%. It is therefore recommended that further investigation on huge scale should be passed out concerning a great number of animals, quantities higher than those used in the present study, documentation of active principles, and calibration of dose and toxicity studies for drug development from the herbal plant. Availability: Items available for loan: UVAS Library [Call number: 2688-T] (1).

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