1.
Production, Purification And Evaluation Of Anti Tetanus Serum
by Mian Muhammad Khubaib Sattar | Prof. Dr. Tahir Yaqub | Dr | Mr. Muhammad Zubair Shabbir.
Material type: ; Format:
print
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not fiction
Publisher: 2012Dissertation note: To produce anti-tetanus serum, ten female sheep of about 3 years of age are immunized with commercially available tetanus toxoid vaccine (Imatet™, Amson vaccines and Pharma) for eighteen weeks to these experimental animals with two weeks interval. Serum samples of all the sheep were also collected fortnightly and stored at -20 ºC.
ELISA was performed to determine the antibody (IgG) titer of all the test samples. Out of 90 samples, 20 samples showed tetanus antibody (IgG) titer of 100.8 I.U. or more, while 8 samples presented tetanus antibody (IgG) titer of 160.9 IU or more. Out of these 8 samples, 3 samples had tetanus antibody (IgG) titer of 190.9 I.U. or more. Maximum tetanus antibody (IgG) titer was 195.4 I.U. at day 120.
Three samples exhibiting maximum antibody titer (190.9 I.U., 195 I.U. and 195.4 I.U.) were mixed in equal quantities for purification of Immunoglobulins (IgG). A volume of 15 mL of aggregated serum samples was mixed with 15 ml of saturated ammonium sulfate having final concentration of 45 % in the mixture which is continuously stirred at room temperature for 1 hour. Mixture was centrifuged at 10000 rpm for 30 minutes in refrigerated centrifuge machine and dialyzed against 10 changes of PBS at 4 ºC. Desalting is checked with BaCl2 solution.
The purified tetanus immunoglobulins (IgG) were treated with papain to produce Fab Fragments. Then the protein content of the purified tetanus immunoglobulins and Fab fragments was estimated with Bradford protein Assay. BSA standard curve was used to produce a regression equation [Y (OD Value) = 0.218 + 0.033 X (Protein Concentration)] which was used for calculation of the protein contents of the samples.
The purified tetanus immunoglobulins and Fab fragments were tested for purity with SDS-PAGE analysis. Then in vivo toxin neutralization test was performed in mice to check the tetanus toxin neutralization ability of the sera produced.
Availability: Items available for loan: UVAS Library [Call number: 1420,T] (1).
2.
Determination Of The Hepatitis C Virus Genotyping Prevailing In The Hepatitis Suspected Patients In District Mardan,
by Suliman Qadir Afridi | Prof. Dr. Tahir Yaqub | Dr. Fariha Akhtar | Dr. Yasin Tipu.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1469,T] (1).
3.
Efficacy Of Commercial Disinfectants Against The Water Contaminating Bacteria At Commercial Broiler Farms
by Mian Muhammad Salman | Dr. Aftab Ahmad Anjum | Pfor. Dr | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Water is an important constituent for poultry. Poor hygienic conditions of water are health hazard for poultry. Many outbreaks are caused by consuming poor quality water.. Fifty water samples from different broiler farms in and around Lahore were collected from drinkers in sterile containers. Bacterial load was assessed using total viable count and coliform count. The counts were above the threshold level (50cfu/ml for coliform and 100cfu/ml for total viable count) showing that water used at poultry farms was of low microbiological quality. Five Disinfectants PHMB20% (.75ml/lit, 1.5ml/lit, 3.0ml/lit), PHMB11% (1.5ml/lit, 3.0ml/lit, 6ml/lit), 0.2% chlorine dioxide (0.1ml/lit , 0.2ml/lit, 0.4ml/lit) Glutral 9.8%(1.5ml/lit, 3.0ml/lit, 6ml/lit), organic acid(1.5ml/lit, 3.0ml/lit, 6ml/lit) were used and they resulted in log reduction of TVC by PHMB20% (5.83±4.36, 6.14±3.98, 9.35± 0.68), PHMB11% (9.42±0.21), 0.2% chlorine dioxide (2.45±0.97, 3.19±0.73, 6.33±0.80 ) Glutral 9.8%(6.87±1.00, 9.73±1.00,9.73±1.00), organic acid(4.75±1.21, 6.62±1.26, 6.90±1.15).PHMB20%,PHMB11%, Glutral 9.8% and organic acid were effective at normal dose, while 0.25 chlorine dioxide was effective at normal dose against at normal dose. Log reduction in Coliform count at half, normal and double dose by PHMB20% (6.52±3.33, 6.96±2.46, 7.96±0.98), PHMB11% (7.89±1.01), 0.2% chlorine dioxide (3.65±0.73, 5.08±0.98, 6.27±0.97) Glutral 9.8%(8.48±0.99), organic acid(5.18±1.21, 5.93±1.26,6.46±1.15±) . PHMB20%, PHMB11%, 9.8% Glutral, organic acid were effective against coliform bacteria at half dose while 0.2% chlorine dioxide was effective at normal dose. Glutraldehyde was effective at normal dose amongst all disinfectants against Total viable bacteria and coli form bacteria.
Availability: Items available for loan: UVAS Library [Call number: 1482,T] (1).
4.
Sources Of Salmonella Contamination In Poultry Meat During Processing And Its Resistance To Antibioties
by Atif Masood Ahmad Khan | Prof. Dr. Tahir Yaqub | Prof. Dr. Khushi Muhammad | Veterinary and Animal.
Material type: ; Format:
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drama
Publisher: 2013Dissertation note: The unhealthy birds which are slaughtered at poultry retail shops may be transferring the pathogen to the healthy meat via butcher's block, clothe (used for cleaning carcass), weighing scale, table, knife and drum in which they are bled after slaughter. The tools which are used in butchers' chicken sale point ; different objects in their shop; the feed they give to birds and bird droppings ; all were analyzed and not surprising they were found heavily contaminated with Salmonellae. Salmonellae is an enteric organism and at chicken sale points contamination to objects through birds' intestine is not much surprising. Therefore a strong need to push the pressure on government to devise laws and set standards for clean premises at chicken sale points. Extensive and irrational use of antimicrobials in human and veterinary sector in the treatment, prophylaxis and as feed additive have made this organism resistant to many of the commonly used antimicrobials.These resistant organisms are being transferred to human body due to the consumption of contaminated poultry meat. Therefore the Salmonellae in humans show resistance to many antibiotics. It is assumed that Salmonellae can transfer resistant genes via bacterial conjugation, transformation and transduction.As a result it is becoming resistant to many antimicrobials. Therefore a strong check on irrational use of antibiotics is needed.
The purpose of current study was to estimate the prevalence of antimicrobial resistant Salmonellae at chicken sale points in Lahore city. In current study 250 samples of 8 different types were collected from different poultry meat sale points in Lahore city.The selection of sale points was random. The samples included50 samples of each poultry feed and bird droppings. 150swab samples of butcher wooden blocks, table, drum( in which the birds are slaughterd), butcher's balance, knife , cloth (used to clean block, knife, table and chicken meat) were also collected from different chicken meat sale pointsin Lahore city.The Samples were analyzed for the presence of Salmonellae by culturing and biochemical tests.The percentage of Salmonellae positive samples inwooden block, weighing balance, cloth, birds' droppings, drum, bird feed, knife and table surface was 44, 24, 36 ,16, 32, 8, 28, 20 respectively.Overall prevalence of Salmonellae was 23.2 %. The isolated Salmonellae were then checked for antimicrobial resistance against 18 antimicrobials by using disk diffusion method. All the Salmonellae isolates were resistant to atleast four antimicrobials. 49 different antimicrobial resistance patterns were found.
Availability: Items available for loan: UVAS Library [Call number: 1563,T] (1).
5.
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: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1564,T] (1).
6.
Isolation And Characterization Of Auxin Producing Bacterial Strains From Plant Rhizosphere
by Kanwal Aziz | Dr. Jawad Nazir | Dr | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
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drama
Publisher: 2013Dissertation note: Auxins are a class of plant hormones or plant growth substances. Auxins have a cardinal role in the regulation of many growth and developmental processes in the plant. Soil samples were serially diluted and screened for auxin production by Salkowski method. Isolates that have the ability to produce auxin were identified by culture characters, morphology, and bio-chemical profile. From 150 isolates, 04 bacteria were selected (AUX-36, AUX-53, AUX-137, and AUX-142). The bacteria were identified by following the flowcharts described in “Berges Mannual of Determinative Bacteriology”, 9th addition. These isolates were identified as Bacillus megaterium, Escherichia coli, Klebsiella pneumonia and Bacillus marinus respectively. Next, different physical and chemical parameters for growth of bacteria and auxin biosynthesis were optimized. For the optimization of bacterial growth OD values of the culture broth (at wavelength 600nm) was taken by spectrophotometer. To estimate the amount of auxin (ìg/ml) produced in the culture broth; a standard curve (concentration of auxin ìg/ml at x-axis and OD value at y-axis) was prepared by using commercially available auxin.
The optimum conditions for growth and auxin production by AUX-36 was found to be pH 7, 0.98% osmotic pressure at 37 °C after 72 hours of incubation. If the medium is supplemented with 0.1 and 1.0% glucose, sucrose and peptone then it increased the bacterial growth which ultimately increased the auxin concentration in the broth medium. The growth and auxin
Production by AUX-53 was 0.98% NaCl concentration at 37 °C after 72 hours of incubation. The optimum pH was found to be 7 but it showed good growth at acidic as well as alkaline pH. The addition of glucose and sucrose in the growth medium increased the growth as well as auxin production. The optimum conditions for the growth of AUX-137 were as follows: pH=7, 0.98% osmotic pressure, temperature 37 °C. However the isolate had good growth at 28 °C and 2% NaCl concentration as well. The bacterial cell density and auxin increased with incubation time up to 72 hours. The isolate produced highest concentration of auxin under the same conditions. Similarly, the cell density and auxin increased with the increasing concentration of glucose in the growth medium. Sucrose increased the auxin only in the culture filtrate. While the bacteria AUX-142 showed highest growth as well as auxin production at 42 °C after 72 hours of incubation. The optimum pH and osmotic pressure was found to be 7 and 2% respectively. The cell density and concentration of auxin increased with the increasing concentration of peptone in the growth medium. Addition of tryptophan (1-2%) increased the auxin concentration in the culture supernatant of all isolates.
Next, the seed germination test and plant pot experiment were performed of selected isolates to observe the effect of bacterial inoculation on wheat plants. In seed germination test treatment of seeds with AUX-36, AUX-53 and AUX-142 significantly increased the root length and number of root hairs as compared to non-treated seeds. In plant pot experiment comparison of various growth parameters of inoculated plants with non-inoculated plants revealed the improvement in plant growth by bacterial inoculation.
Availability: Items available for loan: UVAS Library [Call number: 1684,T] (1).
7.
Isolation And Characterization Of Phytase Producing Microrganism From Soil
by Ghazal Aziz | Dr. Aftab Ahmad Anjum | Prof. Dr | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
print
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not fiction
Publisher: 2013Dissertation note: Phytase is an enzyme of great importance because it is added as a biofertilizer to soil and added in animal feed to increase the uptake of inorganic phosphorous. Phytase production is the property of plant growth promoting rhizobacteria (PGPR) that harbor in rhizosphere part of the soil. These phytase producing bacteria can be utilized as biofertilizers as and can increase the soil fertility and crop production.
Soil samples were collected and screened for the production of phytase (an extracellular) enzyme on phytase screening media (PSM). Six bacterial isolates (PHY02, PHY03, PHY06, PHY07, PHY12, and PHY30) showed distinguished clear zones (> 6mm) on PSM. Isolates were identified as Lactobacillus casei PHY02, Enterobactor intermedius PHY03, Bacillus badius PHY06, Escherichia coli PHY07, Shigella sonnei PHY12, and Klebsiella pneumonia PHY30.
Effect of physical parameters (temperatures, pH and osmotic pressure) on growth and enzyme production by selected isolates was determined. Optimum growth and production of phytae by PHY02, PHY03, PHY06, PHY07, PHY12, and PHY30 (27, 9, 19, 40, 32, and 19 IU, respectively) was at 37°C. PHY07 showed highest enzyme production, followed by PHY30 and PHY02. Isolate PHY06 showed similar growth and enzyme activity at 37°C and 42°C but it was significantly reduced at low temperature. Effect of pH on phytase production on selected isolates indicates that all isolates produces maximum amount of phytase at pH 6.5. At pH 6.5 enzyme units released by PHY02, PHY03, PHY06, PHY07,
PHY12, and PHY30, were 26, 15, 19, 41, 19, and 32 IU, respectively. Production of enzyme decreased with the increase in osmotic pressure. PHY02, PHY03, PHY06, PHY07, PHY12, and PHY30 showed optimum enzyme production (27, 15, 17, 41, 18, and 32 IU, respectively) at 1 % NaCl in PSM (Figure 1C).
Effects of carbon source on both growth and phytase production of isolates showed that PHY03, PHY06, PHY07, PHY12 had significantly higher (P<0.05) cell densities and enzyme production in glucose, while PHY02 and PHY30 had higher enzyme activity at 0.3% lactose.
Nitrogen source in growing media also effects the growth and production of enzyme. PHY02 and PHY12 had better growth and production at 0.1% peptone, while PHY07 and PHY30 had significantly higher phytase level in media modified with peptone but at higher concentration (0.3%). Addition of tryptone in growth medium significantly enhanced the growth and enzyme production by PHY03, and PHY06.
Availability: Items available for loan: UVAS Library [Call number: 1685,T] (1).
8.
Seroprevalence Of Bluetongue In Domestic Animals
by Farid ahmed khan | Prof..Dr. Khushi Muhammad | Ms. Sehrish | Prof. Dr Tahir yaqub.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1925,T] (1).
9.
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: ; Format:
print
; Literary form:
not fiction
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1926,T] (1).
10.
Efficacy Of Surface Disinfectants Against Bacterial Pathogens On Experimentally Contaminated Pathogens
by Sana Ahmed (2009-VA-241) | Dr. Jawad Nazir | Dr. Amir Ghafoor | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Surface disinfection plays a key role in the prevention, control and reduction of many communicable infections as contaminated surfaces are the major source for transmission of microorganisms. Five types of surface cleaning products (Astonish Germ Clear, Cif Floor Cleaner, Dettol Surface Cleaner, Finis Floor Cleaner and Lysol Surface Cleaner) were evaluated for antibacterial activity against three bacterial cultures (E. coli, K. pneumonia, S. aureus) through Quantitative non-porous Surface Carrier Test derived from EN 13697.
Efficacy testing was performed through surface contamination techniques. Glass slides surfaces were artificially contaminated followed by recovery of the bacteria through vortex method both before and after the application of product for 5 minutes. Each of the experiment was repeated thrice and microbicidal effect (ME) values after the application of each product were calculated. Residual antimicrobial activity of the surface cleaning products was measured by applying the working solution of disinfectant on contaminated glass surfaces. Exposure time was given to the test surfaces, after each set exposure time the surfaces were treated to recover the microorganisms. Viable count from the eluant was calculated by serial dilution spread plate method. Each of the experiment was repeated thrice to find out the residual antimicrobial effect of the disinfectant products.
ME values of the Finis Floor Cleaner ranged from 4.03 to 5.14, which was the maximum value among all surface cleaning products used against E. coli. Highest ME value against S. aureus and K. pneumonia was shown by Astonish Germ Clear. The ME values ranged from 4.99 to 5.10 against S. aureus and 5.34 to 6.99 against K. pneumoniae. Finis Floor Cleaner was proved to be of maximum efficiency against E. coli where as Astonish Germ Clear was most effective against Staph. aureus and K. pneumonia. The mean log10 CFU values recovered from disinfectant treated
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surfaces when they were exposed to the environment for different time periods of five minutes, six hours and 24 hours are 5.62 to 7.94, 4.17 to 6.35 and 7.16 to 10.25 respectively. The results indicated that the microbial count was reduced significantly at interaction time of five minutes, then at six hours the count was further reduced by Astonish Germ Clear, Cif Floor Cleaner and Dettol Surface Cleaner i.e. these surface cleaners were able to maintain their antimicrobial activity upto six hours. When the exposure to environment further increased to 24 hours, the microbial count started to increase, hence none of the disinfectants has shown antimicrobial activity upto 24 hours. This indicates that significant microbial count can be achieved within the interaction time of five minutes to six hours. Loss of antimicrobial activity upto 24 hours is probably because the active ingredients of cleaning agents get degraded during long interaction time.
Present study emphasizes that the surface disinfection process must be repeated at regular intervals. Regular and timely use of surface cleaning agents must be considered as a crucial measure in controlling disease transmission rates. Availability: Items available for loan: UVAS Library [Call number: 2294-T] (1).
11.
Detection Of Amantadine Resistant Variants Among Avian Influenza Viruses Subtype H9n2 Isolated In Pakistan
by Sabir Subhan (2009-VA-32) | Prof. Dr. Tahir Yaqub | Dr. Muhammad Zubair Shabbir | Dr. Yasin Tipu.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Avian influenza A virus subtype H9N2 is prevalent in poultry industry of Pakistan. Amantadine is an antiviral drug which is being used as prophylactic measure to control this disease despite the occurrence of resistance against this drug. There is need to monitor the resistance of amantadine in Flu viruses. In this study, we collected 100 samples of broilers birds showing mild to severe respiratory signs. Samples were collected from different locations of Punjab, Pakistan. After initial identification via Hemagglutination test, the molecular identification and confirmation of subtype H9N2 was done by multiplex PCR. To rule out the co-infection of NDV, PCR of NDV was also done. The samples which were pure H9N2 were further processed for the screening of amantadine susceptibility. To do this, titration of viruses was done on MDCK cells in the presence and absence of amantadine at the concentration of 2 ug /ml. The results of TCID5O were compared in the presence and absence of amantadine for each isolate and isolates showing difference of 2 log 10 TCID50/0.1 ml were declared resistant to amantadine as described by Masuda et al. 2000. The results of this study revealed that the viruses circulating in the poultry industry if Pakistan are resistant to this drug as we found that out 10 isolates 4 were resistant to this drug. So, there is need to monitor the usage of this drug in poultry as human cases of H9N2 viruses have been reported and virus was of avian origin. Monitoring is necessary because amantadine is recommended in flu pandemics and this virus possesses the pandemic potential and can cross the species barrier. Availability: Items available for loan: UVAS Library [Call number: 2457-T] (1).
12.
Characterization And Phylogenetic Analysis Of Neuraminidase Gene Of Avian Influenza Virus Subtype H9N2
by Muhammad Abid (2014-VA-502) | Prof. Dr. Tahir Yaqub | Dr. M. Zubair Shabbir | Prof. Dr. Aneela Zameer Durrani.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The H9N2 AIV are endemic in Pakistan since 1998 and causing serious outbreaks in poultry industry leading to increased morbidity and mortality, reduced egg production and reduced weight gain thus causing great economic losses. As these viruses have segmented genome so there is a lots of antigenic shift, antigenic drift and genetic reassortment which results in the production of new AIV subtypes. Besides causing significant losses the poultry industry, the H9N2 AIV pose a significant threat to public health and this issue has been pronounced with the fact that these viruses caused infections in Chinese children in 1999. This primary focus of this study was to characterize and to determine the phylogenetic relationship of the N2 gene of H9N2 AIV prevalent in Pakistan with other H9N2 viruses. A total of 10 H9N2 AIV were isolated from 100 samples and analyzed through serological and molecular tests. N2 gene of three isolates was amplified and sequenced.
The isolates showed 99% homology with the H9N2 AIV recently isolated from Pakistan and their phylogenetic analysis revealed that all belonged to the same G-1 lineage and fell in clusters of more recently and closely related H9N2 viruses. There were some amino acid substitutions in different positions of the NA gene as compared to previous H9N2 viruses of Pakistan and these substitutions were the same to other H9N2 viruses isolated in 2015 from Pakistan.
Due to the mutating nature of the H9N2 AIV there is need for the continuous surveillance and characterization of the prevailing H9N2 avian influenza viruses as these virus have the potential to cause serious outbreaks in poultry and also pose a significant threat to the public health. Availability: Items available for loan: UVAS Library [Call number: 2458-T] (1).
13.
Characterization And Phylogenetic Analysis Of Hemagglutinin Gene Of Avian Influenza Virus Subtype H9n2 Isolated In 2015
by Arslan Mehboob (2009-VA-76) | Prof. Dr. Tahir Yaqub | Dr. Jawad Nazir | Dr. Maryam Javed.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: H9N2 Avian influenza outbreaks has caused great economic losses to poultry industry resulting
in decrease egg production, high morbidity and mortality. Due to different antigenic variants H9
has become problematic. It has the ability to cross species barrier and increase in pathogenicity.
Hemagglutination inhibition (HI) test is employed extensively for subtyping and detection of
antibody titre against the virus. Continuous mutations in the HA gene transforms AIV subtype
H9N2 into more pathogenic virus that may have pandemic potential and can cross species
barrier. Thus, it was necessary to identify various antigenic variants of H9 virus. It was important to
study the HA gene as it plays vital role in viral attachment, release of genetic material and
pathogenicity.
In present study, a sum of four H9 virus samples were isolated. Both serological and
molecular confirmation was done. 200 samples from different areas were collected and properly
labelled. They were then processed for egg inoculation in embryonated eggs. Virus was grown in
embryonated eggs and harvested fluid is then proceeded for confirmatory testing.
Haemagllutination and Haemagllutination Inhibition testing was done. RNA was extracted by
Kit method and cDNA was synthesized. Reverse Transcriptase (RT-PCR) was performed using
specific primer sets and then the amplicon were run on agarose gel. The bands obtained was sent
for sequencing and Phylogenetic analysis was obtained using software and tree was constructed.
Protein analysis was also performed.
The present study enabled us to characterize and construct Phylogenetic tree of HA gene of
currently prevailing H9N2 Avian Influenza isolates in Pakistan. Availability: Items available for loan: UVAS Library [Call number: 2474-T] (1).
14.
Prevalence Of H9n2 In Biotic And Abiotic Factors Post Avian Infleunza Outbreak In Different Districts Of Punjab
by Iqra Mahfooz (2010-VA-301) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor | Dr. Maryam Javed.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Avian influenza H9N2 is not only a potential threat to the poultry industry but it is also a disease of zoonotic importance. In the past it caused very high rate of mortality in the poultry and cause huge economic loses. Present investigations were aimed to find out the uncommon resting points of avian influenza H9N2 virus in the environment. This virus is very dangerous for the poultry industry of the country so it is important to find out the hiding places of virus so that we can stop or control the future outbreaks of virus in the poultry and minimize the economic loss of the country. To rule out the above condition a total of 150 biotic and 200 abiotic samples were collected. Refrigerated samples were processed in the Influenza laboratory. Virus isolation and propagation was done through egg inoculation technique. Presence of virus was confirmed by using Polymerase chain reaction (PCR). The biotic samples (11/150) 7.0 percent reacted positive to HA, HI and also confirmed by PCR. All the abiotic samples were found negative for any evidence of presence of avian influenza virus. This study helped us in understanding the natural reservoirs of avian influenza virus. This study design revealed the hibernation of H9N2 virus in the apparently healthy flock production of broilers. Availability: Items available for loan: UVAS Library [Call number: 2563-T] (1).
15.
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: ; 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
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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
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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.
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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
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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).
16.
PREVALENCE OF MAJOR BACTERIAL AND VIRAL POULTRY DISEASES IN LAHORE DIVISION OF PAKISTAN
by Wasiq mehmood (2009-VA-420) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor | Dr. Yasin Tipu.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Poultry is a huge industry as an emerging agribusiness in Pakistan that contributes a lot in GDP of country but infectious diseases contribute as a major obstacle in profitable production of poultry. There is need to study the most current scenario of major diseases together with all the parameters involved. This can help in making effective preventive measures to minimize the losses. In this study we investigated 1008 cases of poultry diseases received at GP Lab, Lahore. Sick and dead birds were received from different locations of Lahore division during November, 2015 to November, 2016. Whole year was divided in to five seasons as winter (15-Nov to 15-Feb), spring (16-Feb to 14-April), hot summer (15-April to June), hot humid summer (July to 15-Sept) and autumn (16-Sept to 14-Nov). Disease was diagnosed on the basis of flock’s history, postmortem findings, isolation and identification of pathogens using various techniques of bacteriology, virology and molecular biology along with different serological techniques. The result of this study revealed that both bacterial and viral health risks are prevailing in Lahore division of Punjab however bacterial problems are greater in number in comparison of viral infections. Prevalence of E. coli infection (Colibacillosis) was greater than any other disease which could be due to poor disinfection and cleaning of control sheds along with poor management of flocks. Avian influenza and ND shared more than 90% of viral problems throughout the year. Mean prevalence of IBD was found to be 1.46% in recent year whereas CAV and adenoviral infection remained up to negligible extent. Very few cases of CRD and necrotic enteritis were reported. Prevalence of diseases has a strong correlation with seasons with incidence highest in winter and hot summer which could be due to challenging management of flocks because of severe climate conditions. Incidence in spring was found out to be 19.46. Hot humid summer and autumn were found to be least harmful seasons with prevalence of 11.23%
Summary
66
and 7.11% respectively. In order to cope up with health issues, up to date studies on prevailing poultry diseases needs to be done in upcoming years as well. Availability: Items available for loan: UVAS Library [Call number: 2822-T] (1).
17.
Genetic Variation In The Promoter Region Of Pro Inflammatory Cytokine Tnf-Α Among Hiv Infected People In Lahore
by Shahid Nawaz (2015-VA-437) | Prof. Dr. Tahir Yaqub | Dr. Arfan Ahmed | Dr. Asif Nadeem.
Material type: Publisher: 2017Dissertation note: Despite of the fact that HIV is one of the most studied virus of the present time, so far there is no legitimate cure for the treatment of HIV AIDS, and in most cases AIDS is often fatal. Immune response has always been vital to cope with any disease. In context of immune response TNF-α is mainly released by the macrophages as a pro inflammatory cytokine. Studies have shown that HIV infected persons have higher concentration of TNF-α released. A particular single nucleotide polymorphism (SNP) is observed at -308 position of the promoter region of TNF- α gene due to which TNF is categorized into TNF1 and TNF2 allele. TNF2 allele is associated with higher concentration of TNF- α which in turn is associated with HIV infection. In order to know the particular association in the population of Lahore we designed this study. The hypothesis was that SNP at -308 position of the TNF- α may be associated with HIV infection. Methodology was designed as follows: 15 HIV positive samples and 15 HIV negative samples were taken and categorized into group A and B respectively. Whole Blood samples were taken from the patients and subjected to DNA extraction using commercially available kit (Favorgen blood DNA mini extraction kit). Specific primers were used to amplify the particular region (-308) of TNF- α through PCR. A small amount of PCR products were subjected to restriction enzyme Ncol. Sequencing was done and the results were analyzed using specific bioinformatics tools such as NCBI. -308 region of the TNF-alpha was analyzed for the SNPs (TNF1 and TNF2). Collected data was analyzed through SPSS for association studies. Outcome could be as follows: The study is designed to identify SNPs at -308 position of the promoter region of TNF-α gene. It will enable us to understand the possible association between TNF- α and HIV AIDS. It will be the first ever study regarding TNF- α and AIDS in Pakistan. Availability: Items available for loan: UVAS Library [Call number: 2833-T] (1).
18.
Comparative Efficacy Of Pre And Post Exposure Prophylaxis Using Indigenous Rabies Vaccine By Im Route
by Kaneez Fatima (2010-VA-202) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor Bajwa | Dr. Maryam Javed.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2017Dissertation note: Rabies is a viral zoonosis that is known to be present in more than 150 countries, including Pakistan. It is a very serious health problem especially in countries with limited resources and poor awareness. It has significant economic impact and almost 100 % mortality if not properly managed.Dogs are responsible for up to 99% of all rabies transmissions to humans. Rabies is vaccine preventable viral disease. The vaccine is very expensive and a significant factor in patient’s compliance in Pakistan especially in rural areas where the main problem exist i.e. more stray dogs and increased probability of being bitten. Availability of a cheap indigenously produced effective vaccine can be very helpful in reducing the cost and overall improvement of the rabies problem in Pakistan.
We randomly selected a total of 50 patients visiting IPH. Among them 10 of pre-exposure prophylaxis and 40 for post-exposure prophylaxis. Twenty patients of the post-exposure group were children and twenty were adults. The NIH-anti rabies vaccine was administered intramuscularly to the persons visiting the IPH for pre and post exposure prophylaxis using Essen regimen. For pre exposure patients three doses on day 0,7,28 was given and for post exposure patients five doses on day 0, 3, 7, 14, and 28 was administered. The 3ml blood was collected on day 0, 28, 60 and 90 following vaccination. Serum was examined by ELISA Kit (Bio Rad Platelia rabies II Kit) for protective antibody titer.
Pakistan is importing anti-rabies vaccine which is much costly, and sometimes unavailability is a serious concern for patients. In the present study we concluded that indigenous rabies vaccine was very effective and protective levels of rabies antibody titers was detected following vaccination in all patients of the study. By widespread utilization of this vaccine we can reduce demand of imported vaccine, thus lessen the economic burden.
Availability: Items available for loan: UVAS Library [Call number: 2875-T] (1).
