Production, Purification And Evaluation Of Anti Tetanus Serum
Material type: Book ; Format:
; Literary form:
Publisher: 2012 Dissertation 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.
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Isolation And Characterization Of Auxin Producing Bacterial Strains From Plant Rhizosphere
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Publisher: 2013 Dissertation 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.
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Genetic Characterization Of Pakistani Wild Quails Using Mitochondrial Coi Gene
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Publisher: 2015 Dissertation note: A latest taxonomic tool termed as DNA barcoding is being used to genetically characterize animals. DNA barcoding is helpful in determining evolutionary relationship among species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity. DNA barcode works as an efficient tool in food manufacturing industry, diet investigation, forensics analysis, preventing unlawful trade and felonious poaching. The aim of this study was to develop DNA barcode for genetic characterization of Pakistani wild quail. Pakistani wild quail is important due to its demand for eggs, meat production, experimental purposes and gaming as well. Japanese quail was also included in this study because this quail is excessively produced in Pakistani farms. Japanese quail is present throughout the year and is comparatively bigger in size than wild quail. It has longer lifespan; farmers can easily breed this species in farms. It is suitable in poultry due to better meat yield.
COI gene (500bp) was used as a molecular marker for identification at species level. DNA was extracted from blood samples of ten wild quails (Coturnix coturnix and fifteen japanese quails (Coturnix japonica). Reported bird universal primers were used to amplify COI region from the extracted mtDNA samples using PCR. Amplicon were then sequenced by Sanger sequencing method (Sanger et al. 1977). Forward and reverse DNA Sequences were aligned with the reference sequence using nucleotide BLAST on NCBI to observe the dissimilarity among the sequences. Consensus sequences generated were used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. Japanese quail which is thought to be domesticated from Japan, its Pakistani population showed close relation with sequences
generated in Japan for this particular species. Pakistani wild quail species showed its closest linkage with C. coturnix.
In conclusion, COI barcode proved as an authentic tool for species identification and phylogenetic inference of Pakistani wild and farm grown quails. Wild quail species has been characterized using partial COI gene sequences. This study has provided a specific genetic marker which can differentiate Japanese quail from wild quail at molecular level as most of the time both species are confused with each other. It can be helpful to the farmers and bird fanciers because they can select the birds of their choice correctly. This is the first study reporting DNA barcode of this Pakistani quail species. It would help researchers to study about phylogenetic and taxonomic status and also assist quail fanciers and quail farmers to unaffectedly identify their species of interest in farming. Identification of quail species is also important for conservation of biodiversity as it helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
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Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2
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Publisher: 2015 Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic.
In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine.
Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample
showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects.
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Isolation And Molecular Characterization Of Rotavirus From Calf Diarrhea And Preparation Of Vaccine
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Publisher: 2016 Dissertation 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
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
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.
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
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.
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Genetic Variation In The Promoter Region Of Pro Inflammatory Cytokine Tnf-Α Among Hiv Infected People In Lahore
Material type: Book Publisher: 2017 Dissertation 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.
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