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1. Characterization Of Mycoplasma Gallisepticum Isolates And Their Use In The Production Of Indigenous

by Mushtaq Ahmad | Prof. Dr. Masood Rabbani | Prof. Dr | Prof. Dr. Tahir Yaqub.

Material type: book Book; Format: print ; Literary form: drama Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1564,T] (1).

2. Genetic And Evolutionary Characterization Of Pakistani Pigeons And Parrots Through Mitochondrial D-

by Sehrish firyal | Dr. Ali raza awan | Prof, Dr. Aftab | Prof, Dr. Tahir yaqub.

Material type: book Book; Format: print Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1873,T] (1).

3. Isolation And Molecular Identification Of H9N2 Avian Influena Virus From Human In Punjab Province Pakistan

by Abdul ahad | Prof .Dr, Masood rabbani | Prof. Dr. Rana | Prof. Dr. Tahir yaqub.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1926,T] (1).

4. Isolation And Molecular Characterization Of Rotavirus From Calf Diarrhea And Preparation Of Vaccine

by Nadia Mukhtar (2008-VA-718) | Prof. Dr. Tahir Yaqub | Dr. Jawad Nazir | Prof. Dr. Asim Aslam.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: The main contribution of the thesis “Title” is threefold. First, rotavirus was isolated and identified from calf diarrhea samples from 10 districts in Punjab. Second, optimization of molecular diagnostics and genome sequencing was done of the positive bovine rotavirus isolates from Pakistan. And thirdly, the preparation as well as evaluation of killed vaccine against bovine rotavirus isolates was performed. The above three objectives of this study were created due to the distribution of rotavirus all over the world as an enteric pathogen in both human as well as animal species. In developing countries where cases of malnutrition are very common in young children and animals, this virus has a special importance as an etiologic agent. It causes severe diarrhea, when accompanied with severe dehydration, leads to high rate of mortality. Among the rest of the infectious diseases present in calves, neonatal diarrhea is a dire threat as it has a major impact on economic viability. Calf diarrhea is the most important problem in dairy calves that causes more financial losses to the calf producers than any other. Although numerous etiological agents may be implicated, Rotaviral diarrhea is one of the main infections causing calves to scour between five to fourteen days of age. The cattle and buffalo calves’ population in Pakistan is devastatingly affected by the neonatal calf diarrhea due to rotavirus outbreaks. Neonatal calf mortality varies from 8.7 to 64 per cent throughout the world accounting for 84 per cent of the total mortality in the first month of age and is particularly high in the third week. While vaccination is available for the disease, it is being imported in Pakistan from other countries. The importation of the said vaccine thus, leads SUMMARY 117 to extra expenses for the farm managers. As mentioned above one of the aims of this study is to develop an effective vaccine against bovine rotavirus and cut down expenses for farm managers. To fulfill the objectives proposed in this thesis, rectal swabs and fecal samples were collected from public/private sector buffalo and cattle farms from 10 districts of the Punjab: Lahore, Faisalabad, Okara, Sahiwal, Sargodha, Chakwal, Bhakkar, Bahawalnagar, Multan and Bahawalpur. The samples were selected on the basis of agro-ecological zones of the province. As sampling based on agro-ecological zones allow for better data collection for recording incidence rate of the disease. Samples (n=10) from each diarrheic and apparently healthy cattle and buffalo calves from all of the districts were collected. In this way a total of 200 samples from buffalo calves and 200 samples from cattle calves were collected for this study. Antigen of bovine rotavirus was screened from calf feces through Direct Sandwich ELISA. Bovine rotavirus samples were further confirmed through the amplification of the VP4 and VP6 genes through Rt-PCR. Homology and phylogenetic analysis of the sequenced samples was also performed. The data gathered through this analysis was helpful in collecting important data regarding the similarities as well as differences of the bovine rotavirus strain present in Pakistani isolates when compared to local regions as well as international ones. The data is also valuable when it comes to production of effective vaccines again rotavirus. RNA viruses are known to mutate unpredictably and it is safe to assume that a particular vaccine might not work effectively against all strains of a particular virus. That’s why analysis of data pertaining to all possible BRV strains is important for creation of an effective vaccine of import quality in order to help the economy of Pakistan. Rotavirus isolate, after adaptation on MDBK cell line, was further propagated to determine TCID50 for vaccine preparation purposes. Final dose of the vaccine was adjusted to SUMMARY 118 approximately 3ml, containing 40% culture and 60% adjuvant. Final vaccine contained 1ml of inactivated bovine rotavirus harvested culture, 1.8ml of Montanide ISA 70, 0.2ml of PBS and 0.05% of Thiomersal sodium. Efficacy of the vaccine was checked in rabbits. For vaccine efficacy testing twenty one month old rabbits were procured. Rabbits were reared in individual isolator units in the shed facility of Quality Operations Laboratory, UVAS, Lahore. The collected rabbits were divided into two groups, vaccinated and unvaccinated rabbit groups. Each group had 10 rabbits. One ml of rotavirus vaccine was administered intramuscularly in vaccinated rabbits group. In unvaccinated rabbits group 1ml of normal saline was injected intramuscularly. The second dose of vaccine was administered at 24 days post-vaccination of first dose. The rabbits from both groups were bled at 0, 14, 28 and 42 days post-vaccination. The antibody response of rabbits to rotavirus vaccine was determined through using Antibody detection kit. The rabbits were challenged on day 42 post-vaccination using live field strain of rotavirus having TCID50 1 × 108.5. The rabbits were observed daily up to 14 days post-vaccination for appearance of diarrheic signs. The stool samples of ELISA positive were further confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) at least 14 days post-vaccination. The field trials were conducted at Livestock Production Research Institute, (LPRI) Bahadurnagar, Okara. The field study was done to evaluate the prepared rotavirus vaccine for prevention of neonatal calf diarrhea. For this trial, 100 dams were selected. The dams were divided into two groups and each group consisted of 25 pregnant cows and 25 pregnant buffalos. A total of 50 dams (25 cattle and 25 buffalo) were vaccinated intramuscularly with 3ml of prepared inactivated rotavirus vaccine. The 50 remaining dams (25 cattle and 25 buffalo) were kept unvaccinated. SUMMARY 119 The blood samples were collected for serum separation after 0, 14, 28 and 42 days post vaccination in dams. The antibody titers were measured using antibody detection ELSIA kit. After calving, newborn calves were fed with the colostrum obtained from the vaccinated dams daily for 5 consecutive days. Similarly, the calves from unvaccinated dams were fed on colostrum from their unvaccinated dams. The 5 calves from vaccinated and 5 from the unvaccinated dams were isolated in individual isolators. These calves were challenged orally with 1ml of live field strain of rotavirus having 1 × 108.5 TCID50 and the animals were observed for diarrheic signs for 7 days. All of the collected data was subjected to statistical analysis of (one way) ANOVA and t-test using SPSS. The <0.05 p-value determined the significance of the results through this study. The data collected through this study allowed for the creation of valuable inferences. According to the current results of this study, the prevalence of bovine rotavirus was shown to be 6% in Punjab. This 6% included 40% and 20% from the districts of Lahore and Faisalabad respectively. Keeping these results in mind, it is to be noted that the recorded prevalence percentage from this study is higher than the prevalence of 2% in Lahore according to a previous study done in the country. It is to be noted that while the 6% prevalence of rotavirus in Punjab detected through ELISA is lower than the prevalence of 16.83% which was detected by ELISA in diarrheic calves from pervious researches, the 12% prevalence detected by ELISA in this research is higher than the prevalence of 7.25% detected by ELISA in diarrheic calves from past data. In the present study of this thesis it was observed that the use of killed vaccine for bovines produced more efficient immune response in calves. It also enhanced the clostral rotavirus antibody titers as compared to previous studies where the use of the same strain of modified-live virus in a commercial vaccine administered IM with or without adjuvant did not significantly SUMMARY 120 elevate colostrum antibody titers. The results collected from the present research showed that the average antibody titers in the 25 cattle dams at 0, 14, 28 and 42 days post vaccination were 0%, 57%, 68% and 78% respectively. In a similar manner the average antibody titers in the 25 buffalo dams at 0, 14, 28 and 42 days post vaccination were 0%, 55%, 70% and 82% respectively. These results indicated the protective maternal antibody level against the rotavirus which will be transferred passively to calves. The results indicate that vaccinated dams were able to provide passive immunity to both buffalo and cattle calves in order to provide protection against the deadly virus. Availability: Items available for loan: UVAS Library [Call number: 2570-T] (1).

5. Comparative Genomic Study of Motor Neuron Disease in Horses and Human

by Shakeela Daud (2011-VA-534) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2017Dissertation note: cd not submitted Availability: Items available for loan: UVAS Library [Call number: 2810-T] (1).

6. Genetic Evolution And Development Of Recombinant Vaccine Against Newcastle Disease For Chicken In Pakistan

by Abdul Wajid (2009-VA-705) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2017Dissertation note: Newcastle disease (ND) is one of the most contagious diseases of poultry worldwide. The disease is endemic in Pakistan and recurrent outbreaks have been reported in commercial poultry flocks, domestic pet and migratory birds since 1963 an inception of commercial poultry farming in the country. Disease surveillance is necessary to determine the incidence of the disease as well as to identify the etiological agent of the disease status in the region. The analysis of the field data provides a clue for the higher authorities to take steps for the remedy of the devastating outbreak. A virulent form of Newcastle disease virus caused an outbreak in the northern region of Pakistan during the mid of 2011. The virus was identified as a virulent viscerotropic vvNDV and characterize, belonging to the sub genotype VIIi. However, the virus of this genotype is still circulating in the field though the intensity of the strain to succumb the chickens to cause mortality does not exist. The particular thing in this genotype was its susceptibility to other avian species like pheasants, peafowls, ducks turkeys, peacocks, sparrows and parakeets. As this genotype is circulating since 2011 2016 and still spill over in these avian species. Thus for the last five years (2011-16), 3500 healthy, diseased and dead chickens, pheasants, peacocks, turkeys, peafowls, ducks, sparrows, exotic parakeets, rosy-faced parrots, pigeons, and partridges from 750 different locations s were monitored. Samples were collected from the Northern region of the country Punjab, Khyber Pakhtoonkhawa, Azad Kashmir, including Gilgit,Baltitssan and from Southern region, Karachi, Hyderabad , Mirpursakro and other small cities where the poultry farms are located. The samples were collected by the local veterinarians, poultry Assistants and Animal health practitioners who assist during the surveillance program. Samples were also collected from the farmers who brought their birds for inspection in the lab with the details of the 141 farm. Mostly sampling was done where there was reports of NDV outbreak, tissues were collected usually the trachea, spleen and brain, moreover, the pharyngeal and cloacal swabs not only from the infected birds but also from the healthy birds were collected to assess the virus shedding in the flock. Blood samples were also collected (1% of the birds at farm), for serum collection to assess the immune status of the flock using Haemagglutination Inhibition (HI) test and Enzyme linked immunosorbant assay (ELISA). The Survey Form meet the international standard was filled for each farm for recording the information required to find the diagnostic clue as well as the molecular characterization of the isolates. Pool of five pharyngeal swabs were processed after the passage into 9-day old chicken embryonated eggs and confirming the positive HA test and then confirmed by real time PCR (RT-PCR). In addition, sera were tested against NDV by HI and ELISA tests. The targeted samples were sequenced by complete fusion gene and whole genome using 22 pairs of overlapping primers. The observations indicated that the commercial broiler industry is highly susceptible to virulent NDV and confirmed by data available in the laboratory in the survey form. Contrary to that a little is known regarding the maintenance and enzootic trends of vNDV infection level in domestic and wild birds. Poor strategy of the use of vaccines and vaccination as well as the existence of virulent form of NDV in the domestic and pet birds indicate a possibility of the root cause of the ND eruption in the developing countries. A continuous isolation of virulent viruses of the panzootic Newcastle disease virus of sub-genotype VIIi since (2011-2016 from commercial chickens and from various other avian species in the country provide evidence for the existence of epidemiological links intermingling of the strain among them. Therefore, to avoid the huge economical losses in the commercial poultry the second largest industry in Pakistan, their close proximity should be strictly avoided. The mass vaccination of the poultry flocks is in practice in all commercial 142 poultry farms in Pakistan. However, the use and availability of a reliable and standard vaccine, as well as the correct usage of vaccine dose of the live attenuated LaSota vaccine are the key factors to improve their efficacy in the field. Minor outbreaks have been occurring in the field even though a severe outbreak was occurred in 2011-12 collapsed the poultry industry with other pet and wild birds. To minimize the continuity of these minor outbreaks in the field for long time there is a need for more effective vaccine to control the particular genotype of the ND virus. In the present study, DNA vaccine was developed using the SFR-55 NDV strain antigens, in the form of fusion (F) and hemagglutinin-neuroaminidase (HN), namely pcDNA3.1-F and pcDNA3.1-HN. In vitro expression of both genes construct was assessed by reversetranscriptase- PCR (RT-PCR) and western blotting. In the trial an inactivated oil-based emulsion vaccine was prepared using the field strain SFR-55 and compare with the commercial vaccine LaSota strain commonly used by the poultry industry. Birds were divided into six groups, the first two groups were immunized with pcDNA3.1-F and pcDNA3.1-HN alone respectively and third group with was vaccinated with both antigens pcDNA3.1-F+HN. The other two groups were immunized with inactivated (wvSFR-55) and LaSota vaccines as described above, the last group was injected with empty vector as control. The birds were immunized twice at 14 and 21 days of age intramuscularly (DNA vaccine), subcutaneous and eye-drop by inactivated and LaSota vaccines respectively. The birds were challenged with live virulent NDV strain using a dose of 10,000 ELD50/0.1ml per chicken. Results indicate that Inactivated and LaSota vaccines provided high protection (>80%), as compared to pcDNA3.1-F, pcDNA3.1-HN, pcDNA3.1- F+HN gave 70%, 75% and 20% respectively. There was 100% mortality in control chickens. The administration of two vectors expressing F and HN antigens induced high immune response, and provide protection than when used separately. However, the groups immunized with 143 pcDNA3.1-F, pcDNA3.1-F+HN and inactivated vaccine resulted in lower amount of virulent virus shed after challenge when compared to the group immunized with standard LaSota. In summary, the co-administration of both NDV glycoprotein antigens increased protection than use separately. DNA-based vaccine can be used safely to reduce mortality and most importantly lower the risk of virus transmission due to decreased level of virulent virus shedding. Availability: Items available for loan: UVAS Library [Call number: 2910-T] (1).

7. Bacterial Profiling And Development Of Molecular Diagnostic Assays For Detection Of Bacterial Pathogens Associated With Bovine Mastitis

by Aqeela Ashraf (2012-VA-388) | Dr. Muhammad Imran | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2017Dissertation note: The livestock sector plays a critical role in strengthening the economy of Pakistan. Control of livestock diseases is the primary objective of government livestock departments. Bovine mastitis is among the most significant diseases of livestock as reported by various field surveys in Pakistan. Despite considerable knowledge about mastitis and its etiology, this disease is still prevalent in many dairy herds; it remains most difficult to eradicate or control. It is an inflammation of mammary gland resulting in decreased milk production, veterinary care costs and culling losses. In animal health improvement, there is a paradigm shift from treatment of clinical illness to disease prevention. Recognition of disease is the foundation of disease control and prevention. California mastitis test and somatic cell counting are the most commonly used methods for diagnosis of bovine mastitis. These methods are unable to identify the causative agent. Detection of pathogen is critically important for better control of mastitis. Microbial culturing and biochemical tests are traditionally used methods for pathogen identification. But, these methods are very time consuming and can only detect viable bacteria from the sample and can lead to false negative results. The progress in molecular methods based on PCR has improved the veterinary diagnostics. For the identification of bovine mastitis pathogens, an economical, rapid and sensitive molecular diagnostic assay was developed using multiplex PCR, detecting the pathogenic species-specific DNA. The target species areS. aureus,E. coli, S. uberis, S. agalactiae, S. dysagalactia, S. haemolyticus, S. epidermidis, S. chromogenes andM. bovis. Multiplex PCR assay was developed for the detection of these significantly important bacterial pathogen causing bovine mastitis. Species specific primers were designed which have the ability to specifically amplify the particular gene in the target species. For this purpose various gene regions were selected for different bacterial species which included 16S rRNA, cpn60, phoA and rdr. Initially monoplex PCRs were optimized individually for each target species. For optimizing multiplex PCR assay, various combinations of individual PCRs with varying concentrations of primers and template DNA were used. The final protocol included all the nine sets of primer pairs, every set targeting a unique mastitic pathogen. Multiplex PCR assay was checked for its specificity and analytic sensitivity was calculated. Mastitic milk samples were collected aseptically from various farms. Initial screening was based on Surf field mastitis test and California mastitis test. Milk samples were cultured on nutrient agar, blood sheep agar and MacConkey’s agar. The bacterial isolates were identified and further sub-cultured in nutrient broth. All the isolates were identified on the basis of 16S rRNA sequencing analysis. The developed multiplex PCR assay was used to detect the target bacterial pathogens from the collected milk samples. Limit of detection of developed assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples. The results obtained by 16S rRNA sequencing, bacterial culture based identification and multiplex PCR assay were compared. Sensitivity and specificity were calculated using latent class analysis, specificity was up to 88% and sensitivity was up to 98% for targeted mastitic pathogens. The developed multiplex PCR detected nine bacterial species in a single reaction. Multiplex PCR assay has also detected the bacterial pathogens in a few culture-negative mastitis milk samples. This is the first multiplex PCR assay which can efficiently detect nine important mastitic bacterial pathogens in a single reaction. The development of multiplex PCR assay is useful in early diagnosis and prevention & control of bovine mastitis. Mycoplasma is often ignored as a major mastitis-causing pathogen due to lack of rapid and accurate diagnostic tools. In this study a LAMP assay was developed for the identification of M. bovis from clinical mastitic milk samples. LAMP primers were designed from three gene regions including uvrC, 16S rRNA and gyrB. Bacterial reference strains and mastitic milk samples positive for M. bovis were collected from Quality Milk Production Services, Cornell University, Ithaca, NY. Bacterial strains were further cultured on Hayflick medium containing 15% horse serum and incubated for up to 7 d at 37°C with CO2 enrichment. DNA was isolated from mastitic milk samples and bacterial culture using Qiagen DNeasy Blood and Tissue Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions with few modifications. PCR and LAMP assay was performed for all the samples obtained. Analytic sensitivity was calculated and the limit of detection was up to 50pg/reaction for LAMP assay which is higher as compared to PCR. Sensitivity and specificity was calculated for each of the three tests. Cohen’s kappa values obtained were 0.940 for uvrC, 0.970 for gyrB and 0.807 for 16S rRNA. All three tests showed a high level of agreement between test results and the true mastitis status, indicated by the receiver operating characteristic (ROC) curve. A robust, sensitive and specific LAMP assay has been developed for the detection of M. bovis from mastitic milk. These novel molecular assays could be helpful for correct and timely identification of bovine mastitic pathogens, which is crucial for the control and treatment of the disease.Molecular diagnostic assayshave been developed in the current study based on multiplex PCR assay and loop-mediated isothermal amplification assay. Availability: Items available for loan: UVAS Library [Call number: 2930-T] (1).

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