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1. Pcr-Based Diagnosis Of Canine Parvovirus In Dogs

by Farhan Towakal | Prof.Dr.Masoos Rabbani | Prof.Dr.Khushi Muhammad | Prof.Dr.Zafar | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2007Dissertation note: Canine parvovirosis, caused by a haemagglutinating canine parvovirus (CPV), one of the most important acute viral infectious diseases of pups, had been prevalent in the country. In the present study, 40 faecal samples, from clinically suspected cases of parvovirus diseased dogs and stray dogs were collected. The samples were diluted and centrifuged to collect the supernatant. Being a haemagglutinating (HA) virus; the pre-filtered supernatant from all suspected samples was checked for any HA activity using 1% washed chicken erythrocytes. Out of total of 40 samples, 30 samples were found HA positive. All the HA positive samples and samples found negative were processed for extraction of viral DNA with Genomic DNA purification kit. The net isolated DNA samples were subjected to Polymerase Chain Reaction (PCR) by using specific primers (developed against the variable genomic region of the capsid protein of the DNA) of the CPV. One of the selected primer pair designated as (37, 38) amplified the genornic region present in the CPV-2, the other primer pair designated as (39, 40) that amplified the region present in the CPV-2b.Optimization of PCR was done for the molecular level diagnosis of canine parvovirus and this technique was previously not available or standardized in local conditions of Pakistan. With the use of primer pair (37, 38) best results were found on following optimizing conditions like annealing temperature 55°C, primers concentration (reverse and forward) 25pmoles, Magnesium Chloride concentration 2mM, DNA(Template) volume Spl, Taq DNA polymerase(12.5U) and 2001iM of each dNTPs. For the primer pair (39,40) best results were found on following optimizing conditions like annealing temperature 5 5°C, Magnesium Chloride concentration 1.5mM, primers concentration (reverse and forwid) 20pmoles, DNA(Template) volume 6il, Taq DNA polymerase(12.5U), 2001.iM of each dNTPs. The PCR products were analyzed and compared for their banding pattern of 681bp and 427bp amplified by the primer pairs (37, 38) and (39, 40), respectively, against the standard DNA ladder (1 OObp) by using horizontal agarose gel electrophoresis. One of the HJA positive samples was not amplified by the PCR. The results of this study showed that the specificity of PCR reaction as compared to the HA test and the presence of the CPV-2 and 2b in Pakistan as the prevalent pathogenic strains. This study has been a first step for the molecular characterizationldiagnosis of canine parvovirus in local conditions of Pakistan. It is hoped that this study will pave the way for further advanced studies on this topic. Availability: Items available for loan: UVAS Library [Call number: 0972,T] (1).

2. Immune Response Of Buffaloes To Foot And Mouth Disease Virus Vaccine

by Munir Ahmad Tariq | Prof.Dr.Khushi Muhammad | Prof.Dr.Muhammad Akram Muneer | Faculty of Veterinary Sciences.

Material type: book Book; Format: print ; Nature of contents: biography; Literary form: Publisher: 2007Dissertation note: Foot and Mouth Disease (FMD) is a highly contagious infection of cloven-footed animals such as buffalo, cattle, sheep, goats and camels and FMD is characterized by high rise of temperature, salivation, smacking of mouth, vesicular lesion in the buccal cavity, inner flares, coronary band and interdigital spaces, memory glands etc. In Pakistan FMI) disease is caused by "0", "A" or "Asia-i" type of the virus of an Aphthovirus of Picornaviridae. The vaccinal serotypes of FMD virus were characterized as "A", "0" and "Asia-i" by virus neutralization test using imported mono-specific rabbit antiserum. Each of the serotypes multiplied rapidly on monolayer of Baby Hamster Kidney -21 (BHK-21) cells. The BHK-2 I cells were propagated in carrel and roux flasks in MEM 199 containing 10% fetal bovine serum. Heat treated goat serum was equally effective as growth promoter for BHK-21 cell line. The cells rapidly multiplied and formed a monolayer within 72 hours at 37 °C. The cells were harvested using trypsin (0.025%) without affecting the cell viability that was observed by cytometeric as well as by colorimetric assays. The cells were stored in cryogenic containers and revived successfully on 12 months post storage. The FMD virus isolate ("0", "A" and "Asia-i") grew well on the monolayer of BHK-21 cells and produced more than 106, and i04 units of the Tissue Culture Infective Dose-50 (TCID50) on 5th passage, respectively. Each of the virus serotypes was effectively inactivated using 0.12 % formaldehyde, or 0.004 M of Binary Etyhieneimine (BET). The inactivated virus suspension was admixed with either oil base, lanolin or aluminium hydroxide gel and homogenized to get stable vaccine preparation. The adjuvant containing vaccines induced detectable level anti-FMDV-VN antibodies titer in buffalo calves on 19 days post-priming. Oil and gel based FMD vaccines induced detectable geometic mean titer (GMT) of the anti-FMDV-CFT antibodies (2-3 and 7-8) on 19 days post vaccination, respectively. The oil and gel based vaccines induced 1: 64 and 1:80 GMT titer of the anti-FMDV-CFT antibodies on 128 and 64 days post-vaccination, respectively and the titer declined there after as 1: 9 and 1: 3.3 on 258 days post vaccination. From this study it can be concluded that oil based vaccine induces the antibody response in buffalo latter than that of gel adsorbed vaccine. Higher titers of the antibodies are retained for comparably longer period of time by oil based vaccines. Moreover, age of buffaloes, animal species and vaccine storage at 4 C exhibited undetectable effects on the antibody response to the vaccine. The study has indicated that vaccination programs against field infection of FMD in all the domestic cloven footed animal species could be effective way of immunoprophylaxis. Availability: Items available for loan: UVAS Library [Call number: 0973,T] (1).

3. Comparative Efficacy Of Passive And Active Immunization During Newcastle Disease (Nd) Outbreak In Broilers

by Mushtaq Ahmad Gondal | Prof.Dr.Irshad Hussain | Prof | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2007Dissertation note: Newcastle disease is an economically important disease of poultry resulting in huge economic losses every year to the poultry farmers in Pakistan. To compare passive immunization and active immunization during outbreak of Newcastle disease a total of 140 chicks at 16th day of age were divided into seven groups (A, B, C, D, E, F and G) containing 20 birds in each. The level of maternal antibody in chicks, was determined by haemagglutination inhibition titres which revealed that it was the highest at one day and decreased with increasing age. Newcastle disease virus gifted from Dr. Shafqat Fatima Rehmani, Director, Poultry Vaccine Center, Karachi, Sindh was pathotyped by using MDT and ICPI. The Embryo Lethal Dose5o was calculated to be 1083h/0. imi and was found highly pathogenic. Infection was induced in birds through administrating 100 ELD5O1O631/0.lml of Velogenic Newcastle disease virus. Birds of group A, this group seved as a negative control. In group B, this group acted as a positive control. Infection was given by using 0.1 ml of 100 ELD50 of Velogenic Newcastle disease virus intranasally at 26 days of age. In group C, at 16th days of age, all birds in this group were vaccinated with Newcastle disease virus vaccine. At 26th day of age, this group was exposed to infection as mentioned above. In group D, at 24th days of age, all birds in this group were vaccinated with Newcastle disease virus vaccine. At 26th day of age, infection was given by using 0.1 ml of 100 ELD50 of VNDV intranasally into individual bird. In group E, infection and vaccination were given simultaneously at 31 day of age. In group F, In this group, infection was given by using 0.lml of 100 ELD50 of VNDV intranasally, at 26 days of age. When Newcastle disease symptoms were noticed, birds were vaccinated with lentogenic strain of Newcastle disease virus vaccine (Lasota, TAD- Germany) by using 0.5mllbird orally. In group G, infection was given by using O.lml of 100 ELD50 of VND.V intranasally, at 26 days of age. When Newcastle disease symptoms were induced, birds were treated with 64 units of anti-NDV-haemagglutination inhibition yolk antibodies. Use of Lasota vaccine and preformed antibodies in yolk help in decreasing economical losses due to outbreak of Newcastle disease in poultry. Availability: Items available for loan: UVAS Library [Call number: 0995,T] (1).

4. 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).

5. Sero-Prevalence Of Brucellosis In Buffaloes And Cattle Of Swat Valley And Government Livestock Farms,Nwfp

by Azhar Khan | Prof.Dr.Masood Rabbani | Prof.Dr.Azhar | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2008Dissertation note: Brucellosis is an infectious zoonotic disease that is associated with chronic debilitating infections in humans and reproductive failure in domestic animals (Corbell, 1997). The sero-prevalence of brucellosis in buffaloes and cattle were undertaken by collecting samples from animals brought to various slaughterhouses and Private and Govt. farms in Swat valley and Peshawar division by screening through i-LLISA, MRT and RBPT. Out of 850 samples, 600 sera, 200 milk samples were collected along with 50 samples of slaughterhouse worker, butcher and veterinarian for this study. All the serum samples tested through RBPT and I-ELISA showed the overall prevalence 3.67% and 4.33% in the cattle and buffaloes population respectively while the combined prevalence in the cattle, buffaloes and human population through RBPT was 3.38 % and through i-ELISA was 4%. The high rate of brucellosis was recorded through RBPT and i-ELISA in buffaloes ( 4.75%,5.5%) while 0.0% prevalence in male buffaloes through RBPT and iELISA, where as in female buffaloes it was 4.85% through RBPT and through i-ELISA was 5.626%. The comparatively low rate (1.5%) of brucellosis was noted in cattle through the RBPT and 2% through i-ELISA while in female cattle it was 1.587% through the RBPT and through i-ELISA 2.12% with 0.0% in males. Among the serum samples (30) of buffalo and cattle having reproductive disorder were tested through the same tests which showed overall prevalence 16.6%. The prevalence at Cattle Breeding and Dairy farm 1-larichand and Livestock Research and Development Farm (Surrezai was 0.0% through Milk Ring Test and i-ELISA. Also cattle milk samples (110) from private farms in swat valley showed prevalence through Milk Ring Test 0.9% and through i-ELISA prevalence was noted to be 1 .82%. As 50 human serum samples were tested through RBPT and i-ELISA but none of these samples were positive showed that the prevalence of brucellosis in human being is very low, The comparison of RBPT, MRT and i-ELISA (milk and serum) was also analyzed statistically by z-test, the data revealed insignificant results. Availability: Items available for loan: UVAS Library [Call number: 1012,T] (1).

6. Effect Of "In Process Quality Control "Factors On Efficacy Of Bird Flu Vaccine

by Saeed Khan | Prof.Dr.Khushi Muhammad | Prof.DR.Irshad Hussain | Prof.Dr.Muham | Faculty of Veterinary Sciences.

Material type: book Book; Format: print Publisher: 2008Dissertation note: Bird Flu virus was recovered from lungs, trachea, spleen and fecal contents of the infected birds in 10 days old chicken embryos. HA activity and biological titer of the virus improved by serial passages in the 10 days old chicken embryos. This could be due to high rate of mutation of the Bird Flu virus with its successive passages. Formaldehyde and binary ethylenimine (BET) effectively inactivated the virus. However formaldehyde inactivated virus showed mitigation in HA activity during storage. The BET 5mM inactivate the virus with in 16 hours of incubation at ambient temperature (25°C) or 37°C. It has minimal detrimental effect on the HA activity of the virus, even during storage at refrigeration temperature. Bird Flu virus vaccines without adjuvant induced poor antibody response in the vaccinated broilers. The vaccine containing aluminium hydroxide gel induced antibody response that reached at peak level on 1 8 days post priming and decline thereafter. The vaccine containing montanide (oil based vaccine) increased (90.5 GMT) up to 42 days of age. Boosting of the birds primed with gel based Bird Flu virus vaccine improved the production of antibody titer, while boosting of birds primed with oil based Bird Flu vaccine showed undetectable effect. This was due to increasing trend of antibody titer in oil based primed birds. Montanide based vaccines are therefore recommended for broiler, layers and breeders in high risk area of the disease. Vaccines containing decreased infectivity titer induced decrease antibody titer in the vaccinated broilers. Bird Flu virus improved its HA activity and infectivity titer with serial passages in 10 days old chicken embryos. It is worth mentioning that serial passages of the virus tremendously decreased its antigenicity. It is therefore recommended to prepare commercial vaccine fom passage number 1-4, for effective immuno-prophylaxis. The Bird Flu virus (H5N1) mutates very rapidly every time it passes through chicken embryos. It is therefore suggested to grow the virus at least in bio-safety level-ll plus (BSL-II +) laboratories. On account of it high rate of mutation and risk of human health hazards it is suggested that Bird Flu virus (H5N1) may not be used in vaccine production, however other serotypes containing H5 and N antigen other than N1 may be used for production of commercial vaccine. Availability: Items available for loan: UVAS Library [Call number: 1013,T] (1).

7. Study On Molecular Diagnosis Of Canine Distemper Virus

by Muhammad Zubair Shabbir | Prof.Dr.Masood Rabbani | Prof.Dr.Khushi Muhammad | Prof.dr.Zafar | Faculty of Veterinary Sciences.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2008Dissertation note: Samples from fourty five dogs were submitted to the University diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore from January, 2007 to January 2008 for diagnosis of CDV infection. These dogs presented to referring veterinarians with clinical signs suspicious of CDV infection. Hematological examination (lymphocyte count) was carried out using K-EDTA anti-coagulant added whole blood and RT-PCR tests were performed using biological fluid samples that include plasma, nasal and conjunctival swabs. Only distemper positive dogs by RT-PCR were followed up for subsequent lymphocyte count and prognosis of distemper infection. All the distemper positive dogs were lymphopenic but the degree of severity was variable as the samples were collected from dogs of different ages and phase of the disease. The study revealed that lymphopenia can be used to support presumptive clinical diagnosis but required laboratory procedure for confirmation and animal regain its normal value with the passage of time subjected to recovery. During followed up, two dogs were found to be dead because of CDV infection mixed with secondary bacterial infection in which one exhibited the nervous sign like teeth grinding, ataxia, convulsions and in coordination in body movements. Only ten (22.22%) samples were found positive by RT-PCR using plasma, nasal and conjunctival swabs. CDV RNA was detected in 60% of plasma samples, 70% of nasal and 100% of conjunctival swab sample from lymphopenic dogs whereas the percentage was 13.33, 15,55, and 22.22 from a total of 45 samples. No amplicon of expected length was obtained from normal healthy dogs. On comparison of different fluid samples, the sensitivity of conjunctival swab was found to be highly significant followed by nasal swab and plasma. In conclusion, Lymphopenia is the suggestive of clinical infection of dogs with canine distemper virus ad can help in presumptive diagnosis. It is not necessary that all lymphopenic dogs are distemper posit it requires further laboratory confirmtion. In this context, RT-PCR is test of choice with samples including conjunctival swabs and plasma. Availability: Items available for loan: UVAS Library [Call number: 1034,T] (1).

8. 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).

9. 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).

10. Effect Of Various Stress Factors On The Immune Response(Ph.D)

by Muhammad Yasser Mustafa Butt | Prof.Dr.Muhammad Akram Muneer | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print ; Nature of contents: biography; Literary form: Publisher: 2009Dissertation note: Pakistan has vast population of dogs belonging to different breeds. Most of the dogs have no pedigree record which is a great threat to conservation of different breeds. No study on DNA fingerprinting of dogs has been conducted in Pakistan. DNA fingerprinting of dogs is necessary to overcome the problems like forensic cases, sale & purchase, individual identity in case of fertilization by more than one male and ownership disputes. Microsatellite markers have been proved as an efficient and powerful tool for parentage testing and breed characterization of dogs. In this study, a panel of microsatellite markers, having high polymorphism information content (PlC) values, was developed. Blood samples were taken from cephalic vein of two breeds of dogs (German shepherd and Labrador retriever). DNA was extracted by Inorganic method. Primers of microsatellite markers were optimized for successful amplification conditions in the Bio-Rad thermocycler. Multiplex PCR was performed, for amplification of these microsatellite markers on 46 samples belonging to 20 families. Genotyping analysis was performed for the PCR products of microsatellite markers on non denaturing polyacrylamide gel. These results were analyzed statistically software "POPGENE 3.3 and POWER STAT". Allele frequency, heterozygosity, homozygosity, polymorphism information content (PlC), power of discrimination and power of exclusion of all microsatellite markers were calculated. Average power of discrimination among non parents, average hetrozygosity, average observed homozygosity and average polymorphism information content (PlC) value for all alleles was 0.809, 0.6345, 0.29 13 and 0.724 respectively. Moreover combined power of exclusion reached a significant value of 0.9998. Almost all of the microsatellite markers showed significant variations in both German shepherd and Labrador retriever breeds. Microsatellite "REN41D2Ob" showed maximum variation i.e. 17 alleles and microsatellite"REN49F22b" showed the least variation among all microsatellite markers i.e. 4 alleles. Genotyping results of microsatellite markers were clearly different for two different breeds showing a distinct genetic distance between German shepherd and Labrador retriever breeds. Results of this study lead to development of a panel of microsatellite markers which can be used for parentage analysis and breed characterization of dogs. This was a preliminary study on dogs in Pakistan. This facility can be provided on commercial basis to pet owners and kennel clubs. Moreover this study can become the basis for further research investigations in canines in Pakistan. To evaluate effects of various stress factors on immune response and growth performance of broiler chicks, a total of five experiments using 2000 broiler chicks were conducted. In each experiment, chicks were divided into five groups (A, B, C, D and E), and each group consisted of 80 day-old-chicks. In each experiment, the chicks were exposed to stress factors, such as temperature, stocking density, feed deprivation, water restriction and light. Each chick in groups A, B, C, and E was vaccinated against IBV, NDV, IBDV and HPSV, but chicks in group D were kept as unvaccinated controls. Blood samples from each group were collected on 36 day of age, at 18 hrs for determining TLC, DLC and H/L ratio. The antibody titers of chicks in different groups were analyzed using HI test at days 36th and 56. The cellular response was analyzed by injecting PHA-P in the wattles of bird during post stress period. The effects of each stress on lymphoid organs were determined. The potential to resist virulent NDV challenge and effect of stress factors on body weight gains and FCR of chicks was also determined. In experiment 1, conducted to determine the effect of various temperature ranges on broiler chicks, it was observed that the heat stressed (HS) birds showed non-signilicant dilYerence in TLC values. The HS effect on lymphoid organs indicated that the mean weight of thymus of chicks in group B (0.29±0.02) and C (0.59±0.13) was significantly (P<0.05). lower than those in groups A (4.11±3.26), D (4.50±0.77) and E (4.35±0.21). The mean bursa weight of heat stressed chicks in goups A (0.94±0.59) and B (0.20±0.01) were significantly (P0.05) lower as compared to non- heat stressed chicks in groups D (1.42±0.22) and E (1.33±0.18). The mean spleen weight of groups A (1.21±0.13) and B (1.30±0.11) was significantly (P0.05) lower than groups D (L93±0.16) and E (1.52±0.10) indicating the adverse effect of increased temperature. The FCR valueswere significantly (P0.05) different among groups in 6th1 week and effect of Vitamin C was found significantly (P<0.05) improved than Vitamin E and glucose treatment. At 36 day of age the HI titer was recorded significantly (P<0.05) lower in group A (GMT 61) than B (GMT 144) and C (GMT 109) groups while group E (GMT 186) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 days was 79, 156, 122 and 216, respectively. There was nonsignificant (P>0.05) differences in wattle thickness (cm) among groups A (1.51+0.06), B (1.77±0.26), C (1.2±0.25) and D (1.52±0.22) but increased in group E (1.83+0.08). The mortality was found significantly (P<0.05) higher in groups A (14) and D (40) on challenge with NDV virulent virus. In experiment 2, conducted to determine the effect of various levels of stocking densities on broiler chicks, it was observed that stressed birds had showed non-signilicant (P>0.05) difference in TLC values. The effect on lymphoid organs found that the mean thymLls weight (grn) of chicks in groups A (0.37±0.04) and B (0.74±0.17) were significantly (P<0.05) lower than groups C (1.50±0.35), D (4.43±0.72) and E (4.40±0.23). The mean bursa weight (gm) of groups A (0.33+0.03) and B (0.57+0. 1 7) were significantly (P0.05) lower than those of groups D (1.76±0.05) and 13(1.33±0.08) indicating that less space effect the bursa development in the chicks. The mean spleen weight (gm) of groups A (1.18±0.07) and B (1.52±0.20) was significantly (P<0.05) lower than group D (2.37±0.28). The FCR values were sign ilicantly (P<0.05) different among groups in 6thi week and there was non-signiflcant (P>0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose. At 36th day of age the I-Il titer was recorded significantly (P<0.05) lower in group A (GMT 67) than groups B (GMT 9.6) and C (GMT 102). The chicks in group D (GMT 07) showed negligible HI titer while group E (GMT 185) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the 1-Il antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease indicating that the titers in the birds were not enough to resist the virulent challenge. The postNDV-challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 86, 121, 132 and 210. There wa non-significant (P0.05) differences in wattle thickness (cm) in groups A (1.44±0.07) and C (1.43±0.10) while group E (1.64±0.31) showed significantly (P0.05) increased wattle thickness. The mortality was found significantly (P0.05) higher in groups A (25) and D (40) on challenge with NDV virulent virus. This indicated that less floor space decreased the immune response of the birds which leads to the infection/death. In experiment 3, effect of feed deprivation at different time intervals on broiler chicks was studied, it was observed that feed deprivation stressed birds had showed non-significant (P>0.05) differences in blood cells population. The effect of feed deprivation on the mean thymus weight (gm) of chicks in group C (0.96±0.29) was adversely affected as the chicks in this group had significantly (P<0.05) lower weight than groups B, 1) and E. The bursa mean weight (gm) of groups A (0.48±0.11) and C (0.52±0.06) was significantly (P0.05) lower than those of groups D (1.40±0.18) and E (1.28±0.11) indicating, that 24 hrs and morning off-feed effect the bursa development in the chicks. The mean splen weight(gm) of groups A (I. 19±0.07) and C (1.21±0.06) vcre significantly (P0.05) lower than groups D and E indicating adverse effect ol24hrs and day off feed on chicks lead to infection. The FCR values were significantly (P0.05) different among groups in 6hhl week and there was significant (P<0.05) differences among groups with treatment of glucose than Vitamin C and Vitamin E treated. groups. At 36th day of age, the HI titer was recorded significantly (P<0.05) lower in group B (GMT 15) than C (GMT 74) and group D (GMT 07) showed negligible HI titer while group E (GMT 140) showed significantly (P<0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and Eat the age of 56 days were 68, 54, 115 and 165. There was non-significant (P0.05) difference in wattle thickness (cm) among groups while group E (I .78±0.06) showed significantly (P<0.05) increased wattle thickness. The mortality was Found significantly (P<0.05) higher in Groups C (12) and D (40) on challenge with NDV virulent virus. This indicated that 24 hrs off feed decreased the immune response of the birds which leads to the infection. In experiment 4, studied the effect of water restriction at different time intervals on broiler chicks, it was observed that water restricted birds had nonsignificant (P0.05) differences in blood cells population except lymphocytes percentage was found higher in groups A (43.7±2.47), C (43.7±1.16) and D (53 .3±1 .30) than group B (39.1±1.06). The effect of water restriction on the mean thymus weight (gm) of chicks in group C (0.60±0.07) was adversely effected as the chicks in this group had significantly (PO.O5) lower weight than group D (4.09±0.70) indicating that increased in the period of water restriction in chicks adversely affected the mean thymus weight and chicks reared on ad-flbituni water had higher mean thymus weight. The mean bursa weight (gui) of group C (0.07±0.02) was significantly (P<0.05) lower as compared to group D (1.37±0.88) indicating that water restriction of 24 hr had affected the bursa development in the chicks. The mean spleen weight (gm) of group C (2.64±1.49) was significantly (P0.05) higher than groups A, B and E. The FCR values were significantly (P<0.05) different among groups in 6th week and there was non-significant (P0.05) difference among groups with treatment of Vitamin C, Vitamin E and glucose treated groups. At 36th day of age the HI titer was recorded significantly (P<0.05) lower in group D (GMT 09) than A (GMT 54) and group E (GMT 109) showed significantly (P0.05) higher HI titer. At the age day 56 (06 days post challenge) the HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and E at the age of 56 clay was 78, 63, 48 and 134. There was non-significant (P0.05) difference in wattle thickness (cm) among groups B and E and group A (1.28±0.08) showed significantly (P0.05) lower wattle thickness. The mortality was found significantly (P<0.05) higher in groups B (14) and C (21) on challenge with NDV virulent virus. This indicated that 18 and 24 hrs water restriction decreased the immune response of the birds. In experiment 5, effect of light stress at various time intervals on broiler chicks was studied. It was observed that light stressed birds had showed non-significant (P>0.05) difference on blood cells population except lymphocytes percentage was found higher in groups D (6 1.4±1.16) than group C. The effect on lymphoid organs studied and the mean thymus weight (gin) old chicks in group B (I .90±0.53) was adversely effected as the chicks in this group had significantly (P0.05) lower mean thymus weight than groups D (4.64±0.74) and E (4.34±0.25) indicating that increase in the period of oil-light in chicks adversely effected the mean thymus weight and chicks reared on 24hr light had higher mean thymus weight. The bursa mean weight (gm) 01' group 13(0.43±0.05) was significantly (P0.05) lower as compared to group D (1.59±0.17). The spleen mean weight (gun) of group D (1.88±0.15) was significantly (P<O.05) higher than group B (1.18±0.08). The FCR values were significantly (P().O5) different among groups in 6th week and there was non significant (P0.O5) difference among groups with treatment of Vitamin C, Vitamin E and glucose. At 36° day of age the HI titer was recorded significantly (P<0.05 lower in group C (GMJ 83) and group D (GMT 06) showed negligible HI titer while group E (GMT 128) showed significantly (P0.05) higher HI titer. At the age day 56 (06 days post challenge) in HI antibody titers in all groups registered a rise except in group D. All the chicks in group D died indicating clinical signs of Newcastle disease. The post challenge GM HI titers recorded in groups A, B, C and B at the age of 56 days was 122, 116. 108 and 133. There was non-significant (P>0.05) difference in wattle thickness (cm) among groups while group B (1.53±0.15) showed significantly (P<0.05) increased wattle thickness. The mortality was found significantly (PO.05) higher in groups A (18) and D (40) on challenge with NDV virulent virus. This indicated that 24 hr off-light decreased the immune response of the birds. Availability: Items available for loan: UVAS Library [Call number: 1080,T] (1).

11. Antigenic Characterisation Of H9 Subtype Avian Influenza Viruses Isolated Desi And Zoo Birds

by Farrukh Saleem | Dr.Muhammad Mahmood Mukhtar | Prof.Dr.Azhar | Prof.Dr.Khushi Muhammad | Faculty of Veterinary Sciences.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2009Dissertation note: Avian influenza is a viral infection which affects mainly the respiratory system of birds. The H7N3 subtype influenza viruses were isolated for the first time in 1994 from breeder flock in northern areas of Pakistan. A second wave of avian influenza outbreak was detected in 1999. The causative agent of this outbreak was H9N2. The H9N2 considered as low pathogenic avian influenza (LPAI) virus and continuously circulating in poultry flocks causing enormous economic losses to poultry industry of Pakistan. This showed that avian influenza viruses are present in commercial poultry. Most of the efforts to isolate avian influenza A viruses are from commercial poultry and these isolations are outbreak based. That is why we have mad’ an effon to isolate and identify H9 subtype avian influenza viruses from apparently healthy live desi and zoo birds (Lahore, Pakistan). We have successfully isolate H9 subtype influenza viruses from these birds during our study. As these viruses have RNA genome and their RNA polymerase enzyme lacks proof reading activity which resulted in spontaneous mutation in surface glycoproteins (HA and NA) and reassortment of their genomic segments results in escape from host immune response produced by the vaccine. This is the reason that every year we require a new candidate virus for vaccine preparation. We have made an effort to isolate and identify avian influenza viruses from live desi and zoo birds of Lahore and performed antigenic characterization. In this way, we have been able to know the exact status of avian influenza virus strains present in the desi and zoo birds. We also have seen that the imported vaccine have less interaction with the local strains and gives less protective titer although it gives best titers when we raise antisera against imported vaccine. The local vaccines although gives a little bit less titer when we raise the antisera against these vaccines but their antisera have more interaction with the local H9 subtype antigen so it gives better protective immune response. By this study we have seen that antisera obtained from infected chicken give more antibody titer as compare to antibody raised in the rabbits. Infected chicken antisera are more reactive as compare to rabbit antisera. This shows that our isolates have highest similarity with the currently circulating viruses. All above results helped us to devise a new control strategy against avian influenza viral infections present in these local birds. The antigenic characterization of these avian influenza isolates helped us to see the antigenic differences between the isolates of this study and H9 subtype avian influenza viruses used in vaccines. Therefore, this study clearly suggests that a new local H9 subtype avian influenza virus should be used as vaccinal candidate every year for the effective control of influenza viral infections of poultry. Availability: Items available for loan: UVAS Library [Call number: 1082,T] (1).

12. Potentiation Of Fluoroquinolones By The Use Of Promethazine As Efflux Pump Inhibitor

by Rabia Altaf | Prof.Dr.Muhammad Ashraf | Dr.Sheryar Afzal | Prof.Dr.Khushi Muhammad.

Material type: book Book; Format: print Publisher: 2009Dissertation note: This study was conducted to demonstrate the potentiating effect of promethazine, an efflux pump inhibitor, on the sensitivity of Streptococcus pyogenes, Staphylococcus aureus, and Escherichia coli against fluoroquinolones. The bacteria were isolated from the field and were identified by using different microbiological techniques. The sensitivity of these bacteria was tested against four Fuoroquinolones i.e. ciprofloxacin, levofloxacin, norfioxacin and pefloxacin by using Kirby Bauer method. Diameters of inhibitory zones were measured in millimeters and all the tests were performed in five replicates. The same experiment was revised with the addition of Promethazine in concentrations of 64.tgIml, 128j.tg/ml, 192J1g1m1 and 256.tg/ml to the Petri plates separately. Diameters of inhibitory zones were measured and were compared with the negative control. The diameters of inhibitory zones of Staphylococcus aureus against ciprofloxacin(5 tg), levofloxacin(5 .tg), norfloxacin( 1 Oj.tg) and pefloxacin(5 .tg) alone were 14.6 mm, 20.4mm, 11.2 mm and 13.2mm but in the presence of promethazine in 256ig/ml concentration, the zones were 47.6mm, 39mm, 42.2mm, 35.8mm respectively. The diameters of inhibitory zones of Streptococcus pyogenes against ciprofloxacin(5 j.ig), levofloxacin(5 jig), norfloxacin( 1 Ojig) and pefloxacin(5 jig) alone were 22.4mm, 20.6mm, 15.0mm and 16.8mm but in the presence of promethazine (256j.tg/ml) the diameter of inhibitory zones were 40mm, 41mm, 37.8mm, 41.4mm respectively. The diameters of inhibitory zones of Escherichia coil against ciprofloxacin(5 jig), levofloxacin(5 jig), norfloxacin(lOjig) and pefloxacin(5 jig) alone were 23.2mm, 19.6mm, 20mm and 17mm but in the presence of Promethazine (256gig/ml) the zones were 42mm, 39mm, 43mm, 35mm respectively. The increase in the diameter of inhibitory zones of bacteria against fluoroquinolones measured first in the absence and then in the presence of promethazine was found to be significant with P value less than 0.05. the results also demonstrated that this increase in the diameter of inhibitory zones was related to the increasing dose of promethazine, indicating that the increase in the susceptibility of bacteria for fluoroquinolones was a result of inhihition of bacterial efflux pumps by promethazine. Availability: Items available for loan: UVAS Library [Call number: 1107,T] (1).

13. Identification And Molecular Characterization Of Shiga Toxin Producing

by Khawar Ali Shahzad | Prof.Dr.Khushi Muhammad | Dr.Tahir Yaqub | Mr.Tanveer Hussain | FVS.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2011Dissertation note: Escherichia coli is normal inhabitant of all the animals and human beings. Its non-sorbitol fermenting biotype (SNF) was detectable in buffalo (90 percent), cattle (80 percent) or rarely in sheep (20 percent) and goat (30 percent). However, SNF E. coli were un-detectable in droppings of rural chickens and feces of donkeys. The SNF E. coli was detected in 100, 92, 71 and 80 percent of the market milk samples and 100, 83, 83 and 53 percent beef samples from Multan, Sandha, Wagha and Sheikhupura Roads, of Lahore city, respectively. However, SNF E. coli was not detected from freshly aseptically collected milk and beef samples but was detectable in over all 96 percent of market raw milk and 82 percent market beef samples. White colored colonies of each positive sample on Sorbitol MacConkey's Agar (SMA) contained gram negative, rods with round ends non spore former. Each of the isolates was sorbitol non fermenter, lactose fermenter, indole positive, Methyle Red positive, Voges Prauskaur negative and citrate negative. However, each of such isolates showed green metallic sheen on Eosin Methylene Blue (EMB) agar. Each of the isolate was further characterized using polymerase chain reaction (PCR). Bacterial DNA was extracted easily by boiling method when the isolates were grown on Sorbitol MacConkey's agar or EMB agar at 370C for 12-24 hours. The DNA was recovered when the culture was boiled for 5-10 minutes. The DNA of each sample remained stable on storage at 40C for 48 hours or at -200C for 7 days. The isolated DNA (100 samples) when amplified using universal, Stx1, Stx2 and O157 specific primers showed that 82 percent samples were positive for universal primers, 50 percent for O157, 60 % for Stx1 and 51 percent for Stx2. The filtrate of each isolate when diluted as 1:10 dilution and sterilized by filtration induced cyto-pathogenic effect (CPE) on Vero cell line. It is concluded that SNF E. coli O157 normally exists in intestinal tract of buffalo, cattle, sheep and goat. Counts of SNF E. coli O157 were higher in milk samples as compared to beef samples. More than 80 percent samples of milk or beef were contaminated with SNF E. coli O157. Feces of the animals are presumably main source of SNF E. coli contamination of raw milk and beef. PCR is a quick, reliable, and sensitive technique for confirmation of SNF E. coli O157 in the samples. Availability: Items available for loan: UVAS Library [Call number: 1221,T] (1).

14. Isolation And Molecular Characterization Of Antimicrobial Resistant E-Coli Isolation From Retail Meats

by Ali Ahmed | Prof.Dr.Khushi Muhammad | Dr.Mueen Aslam | Prof.Dr.Masood.

Material type: book Book; Format: print ; Nature of contents: biography; Literary form: Publisher: 2010Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1252,T] (1).



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