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Diagnosis Of Paratuberculosis (Johne,S Disease) In Cattle And Buffaloes Through Histopathological Techniques And Polymerase Chain Reaction

by Farhan Anwar Khan | Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof .Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2008Dissertation note: Paratuberculosis, one of the infectious disease, is the emerging cause of poor health, low productivity and finally death due to single infectious agent among dairy and beef yielding animals (cattle and buffaloes) in the World. Mycobacterium avium subsp. paratuberculosis is the most common cause of bovine Johne's disease. The study was conducted in Lahore to compare conventional methods and PCR for the diagnosis of paratuberculosis caused by M avium subspp. paratuberculosis in 300 cattle's and buffalo's tissue samples (150 of each specie), including terminal ileum and mesenteric lymph nodes. Conventional methods included Ziehi-Neelsen's (ZN) acid fast staining and histopathology. For M paratuberculosis insertion sequence IS 900, specific 626 bp fragment, were targeted. The sensitivity and specificity of PCR was found significant in comparison to Ziehl Neelsen staining and histopathology for the diagnosis of paratuberculosis in cattle and buffaloes. Availability: Items available for loan: UVAS Library [Call number: 1011,T] (1).

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Immunohistochemical And Pathomorphological Studies Of Chronic Granulomatous Enteritis (John'S Disease) in Bovines

by Muhammad Shahid | Prof.Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2010Dissertation note: Paratuberculosis, a disease caused by Mycobacterium paratuberculosis is a peril for both livestock and human beings. The present project was designed to study the pathmorphological changes induced by the organism and standardize more reliable diagnostic techniques to identify the M paratuberculosis. Tissue samples from ileurn and mesenteric lymph nodes were randomly collected from 1 50 cattle and buffalo, each in present study that was conducted in Lahore. Gross lesions were recorded on a Performa. The samples were subjected to acid fast staining of smears from pellets after density gradient centrifugation and paraffin embedded tissue sections. All the samples also subjected to polymerase chain reaction and immunohistochemistry. The smears prepared from bacterial pellets of mucosal and cortical scraping of terminal ileum and MLN were stained indicated 11.4 % small intestine and 12.7% lymph nodes of cattle's and 8.7% and 10.7% lymph nodes of buffalo's tissue samples were positive. ZN staining of paraffin embedded tissue showed 8.0 % small intestine and 10% MLN of cattle's and 6.0 % of small intestine and 8.7% MLN in buffalo's tissue samples were positive. On basis of PCR 5.4% intestinal tissue samples and 6.0% MLN of cattle were positive. 3.4% intestinal tissue samples and 07(4.7%) MLN of buffaloes were positive. In buffaloes 4.0% intestinal tissue samples and 6.0% MLN were positive by IHC. In cattle 6.7% intestinal tissue samples and 8.0% MLN tissue samples were positive by IHC. In cattle, 27/150(18.0%) animals showed lesions in both intestine and mesenteric lymph nodes while 5/32 (15.7%) animals showed lesions in lymph nodes only. Out of 27/150(18.0%) intestinal tissue samples, 20/27 (74.1%) samples showed corrugation of the intestinal mucosa while 7/27 (26%) showed diffuse thickness. In buffalo, 24/150 (16.0%) animals showed lesion in both intestine and mesenteric lymph nodes while 2/26 (7.7%) animals showed lesion in lymph nodes only. Out of 24 intestinal tissue samples, 19/24(79.2%) with gross lesion, samples showed corrugation of the intestinal mucosa while 5/24(20.9%) showed diffuse thickness. In histopathology 20/27 samples of cattle showed focal granulomatous lesions while 7/27(26%) samples showed sever infiltration of macrophages and lymphocytes while 28/32(87.5%) lymph nodes showed infiltration of paracortical and cortical region of the lymph nodes with macrophages ,lymphocytes and multinucleated giant cells While 4/32 (12.5%) samples showed moderate infiltration of macrophages. In buffaloes 19/24 (12.7%) samples showed focal granulomatous lesions while 5/24 (20.9%) samples showed sever infiltration of macrophages and lymphocytes while 22/26 (84.7%) lymph nodes showed infiltration of paracortical and cortical region of the lymph nodes with macrophages ,lymphocytes and multinucleated giant cells While 4/26 (15.4%) samples showed moderate infiltration of macrophages. The sensitivity and specificity of immunohistochemical method was found significant in comparison Ziehl-Neelsen staining and histopathology for the diagnosis of paratuberculosis in cattle and buffaloes. Availability: Items available for loan: UVAS Library [Call number: 1078,T] (1).

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Polymerase Chain Reaction And Restriction Fragment Length Polymorphism (Rflp) By Using Ssu-r DNA Amplification for the Species Specific Diagnosis of Trypanosomiasis in Horses

by Naveed Sabir | Prof.Dr.Zafar Iqbal Ch | Dr.Asim Aslam | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2010Dissertation note: In the current research project, a pari-trypanosome polymerase chain reaction (PCR) was optimized by using 18S single sub unit ribosomal DNA amplification and restriction fragment length polymorphism (RFLP) was also optimized and evaluated for the species specific diagnosis of the trypanosomiasis in horses. Blood samples from one hundred (100) suspected horses were collected aseptically from different localities of Lahore. Fresh blood smear was prepared from each sample. After drying and fixing with absolute methanol, the slides were stained with Giemsa stain. Microscopic examination of stained blood smears revealed 8 positive samples out of one hundred (100) suspected horses. Polymerase chain reaction (PCR) was carried out on the same trypanosomiasis suspected blood samples to evaluate its sensitivity. Genomic DNA was extracted by using Genomic DNA Purification Kit (Fermentas mci., USA). The PCR was performed in a 50 tl reaction mixture. The tubes containing PCR mix were subjected to amplification cycles in a thermocycier after adjusting the amplification conditions. After completion of the amplification cycles, the PCR product was characterized by 1.2 % agarose gel electrophoresis along with 100 bp DNA ladder to estimate the size of the PCR product and the gel was photographed with a Polaroid camera. PCR gave a higher percentage of positive cases i.e. 21% as compared to microscopic examination. Semi-nested polymerase chain reaction was carried out on product of the first run amplification by using same reaction mixture and amplification conditions except for template DNA. In case of semi-nested PCR 1 tl of the simple PCR product was used. Semi-nested PCR gave 100% (21/21) results. Restriction fragment length polymorphism (RFLP) analysis was conducted on nested products of the positive samples. A reaction mixture of 20 1iJ was used and samples were incubated over night at 37 °C in an incubator. The restricted products were characterized by 2 % agarose gel electrophoresis along with 100 bp DNA ladder and photographed with Polaroid camera. Restriction fragment length polymorphism (RFLP) analysis of the nested products revealed that none of the species including T. congolense, T. theileri, T. brucei and T. vivax was found in all (2 1%) positive animals having trypanosoma infestation. It can be concluded from current study that a pan-trypanosome polymerase chain reaction is a superior and sensitive test as compared to Giemsa stained blood smear examination. The test can not only be used for early diagnosis of the trypanosomiasis but it can also be used to screen out the carrier animals those act as a reservoir of the infection for the horses and other susceptible animals. The advantage of this test is its sensitivity, universal applicability and the existence various possibilities for restriction enzyme analysis of the amplified region depending on the trypanosome species. Availability: Items available for loan: UVAS Library [Call number: 1079,T] (1).

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