51.
Physico-Chemical Analysis Of Milk From Different Milch Species (Cow, Buffalo, Camel)
by Tahira Jamil (2015-VA-595) | Dr. Sanaullah Iqbal | Haroon Jamshid Qazi | Dr. Muhammad Tayyab | Muhammad Asif Ali.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2017Dissertation note: SUMMARY
Milk is described as almost a complete food as it contains all the essential nutrients in balanced quantity. Milk is a complete basis of proteins, fats and dietary energy and there are several factors that can effect the composition of milk. Factors such as (seasonal changes, feed, environmental changes, lactation, milking durations) and variations in analytical methods such as (evaluating proteins, fats, total solids, ash and moisture) can also lead to differences in results. According to FAO STAT 2010, despite the fact that Pakistan ranked among top five milk producing countries in the world, no study has been made so far that is composed of complete data based on physico-chemical analysis of milk composition of various species with respect to seasonal changes.
Milk samples were collected from three different species from UVAS Pattoki Campus i.e. cow, buffalo and camel in morning and evening time. The samples were then sent to UVAS Lahore Campus. These samples were analyzed to obtain different compositional parameters of milk which includes LR, fat, protein, SNF, TS, Ash, Moisture, pH, COB and APT. In the present study, the results showed that the LR, fat, SNF, TS, Proteins, ash, moisture and pH showed no signifgicant differences when studied between the groups by independent sample t test. All results were statistically non-significant i-e p>0.05. Whereas when results of each sample were studied individually throughout the year by descriptive statistic, it was found that samples of cow, buffalo and showed high content of fats, SNF, TS and protein during the summer season and lower in winter season. Other parmeters like ash, moisture, pH also had significant change throughout the year. The monthly results were found to be statistical significant at p<0.05. COB and APT were analyzed as soon the samples arrived the laboratory. So no clotting or precipitations were observed in the sample and gave the negative results throughout the year.
Thestudy was helpful in generating yearly data that was used in comparing the physico-chemical variations in morning and evening samples of milk among different milk producing species (cow, buffalo, camel) on the basis of seasonal changes.
Conclusion:
The directive of the current research was to analyze the physico-chemical parameters from the morning and evening samples of milk of three milk producing species (cow, buffalo, acmel). It was concluded from the results that no significant differences were found within groups of each sample. Whereas when the analysis were conducted on monthly basis throughout the year, it was determined that fat content of the samples of cow, buffalo and camel was high during the summer season. There are several reasons for this such as lactation, feed composition, milking timings, seasonal variations. SNF, TS and protein contents were directly related to fat. It was possible to state that when the fat of milk was higher the solid not fat, total solids and protein contents were also higher. However the other contents of milk such as ash, moisture, pH, COB and APT were not significantly affected by these factors.
Limitations:
Diet is also an important factor that could affect the composition of milk. This factor can also be researched along with seasonal changes.
Different geographical regions affect the milk composition of animals. This is also another factor of interest.
Physiochemical changes of sheep, goat and humans can also be analyzed on the basis of seasonal changes.
Milk is described as almost a complete food as it contains all the essential nutrients in balanced quantity. Milk is a complete basis of proteins, fats and dietary energy and there are several factors that can effect the composition of milk. Factors such as (seasonal changes, feed, environmental changes, lactation, milking durations) and variations in analytical methods such as (evaluating proteins, fats, total solids, ash and moisture) can also lead to differences in results. According to FAO STAT 2010, despite the fact that Pakistan ranked among top five milk producing countries in the world, no study has been made so far that is composed of complete data based on physico-chemical analysis of milk composition of various species with respect to seasonal changes.
Milk samples were collected from three different species from UVAS Pattoki Campus i.e. cow, buffalo and camel in morning and evening time. The samples were then sent to UVAS Lahore Campus. These samples were analyzed to obtain different compositional parameters of milk which includes LR, fat, protein, SNF, TS, Ash, Moisture, pH, COB and APT. In the present study, the results showed that the LR, fat, SNF, TS, Proteins, ash, moisture and pH showed no signifgicant differences when studied between the groups by independent sample t test. All results were statistically non-significant i-e p>0.05. Whereas when results of each sample were studied individually throughout the year by descriptive statistic, it was found that samples of cow, buffalo and showed high content of fats, SNF, TS and protein during the summer season and lower in winter season. Other parmeters like ash, moisture, pH also had significant change throughout the year. The monthly results were found to be statistical significant at p<0.05. COB and APT were analyzed as soon the samples arrived the laboratory. So no clotting or precipitations were observed in the sample and gave the negative results throughout the year.
Thestudy was helpful in generating yearly data that was used in comparing the physico-chemical variations in morning and evening samples of milk among different milk producing species (cow, buffalo, camel) on the basis of seasonal changes.
Conclusion:
The directive of the current research was to analyze the physico-chemical parameters from the morning and evening samples of milk of three milk producing species (cow, buffalo, acmel). It was concluded from the results that no significant differences were found within groups of each sample. Whereas when the analysis were conducted on monthly basis throughout the year, it was determined that fat content of the samples of cow, buffalo and camel was high during the summer season. There are several reasons for this such as lactation, feed composition, milking timings, seasonal variations. SNF, TS and protein contents were directly related to fat. It was possible to state that when the fat of milk was higher the solid not fat, total solids and protein contents were also higher. However the other contents of milk such as ash, moisture, pH, COB and APT were not significantly affected by these factors.
Limitations:
Diet is also an important factor that could affect the composition of milk. This factor can also be researched along with seasonal changes.
Different geographical regions affect the milk composition of animals. This is also another factor of interest.
Physiochemical changes of sheep, goat and humans can also be analyzed on the basis of seasonal changes.
Availability: Items available for loan: UVAS Library [Call number: 2884-T] (1).
52.
Production, Purification & Characterization Of Recombinant Thermostable Phytase And Its Biological Evaluation In Broiler Chicks
by Furqan Sabir (2007-VA-524) | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Ali Raza Awan.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Phytate is the principle storage form of phosphorus in plants particularly in cereal grains and legumes. Mono-gastric animals doesn’t have ability to utilize phytate as phosphorus source. The animals release the undigested phytate from body with manure that cause environmental pollution. Phytases are responsible for the hydrolysis of phytate, resulting in availability of free phosphorus for the animal. The present study deals with the production and characterization of recombinant thermostable phytase and its biological evaluation in the broiler chicks. The PCR resulted in the amplification of 1.8 kb phytase gene using the genomic DNA of Thermotoga naphthophila as template. The purified PCR product was ligated in pTZ57R/T and the ligated material was utilized for the transformation of E.coli DH5α cells. The positive clones were selected on the basis of blue white screening. The restriction digestion of plasmid DNA from positive clones using NdeI and Hind III resulted in the release insert from the vector. The purified phytase gene after restriction digestion was ligated into pET21a already restricted with the same restriction enzymes and the expression was analyzed using E.coli BL21 CodonPlus (DEL) cells. SDS-PAGE demonstrated the intra-cellular production of recombinant phytase. The conditions were optimized for the optimal production of recombinant phytase (PHYTN). The maximal production of PHYTN was recorded when the BL21 CodonPlus cells having recombinant pET21a having phytase gene were induced with 1.4 mM IPTG and 6 hours post induction incubation period. The recombinant protein was purified using various chromatographic techniques and the purified protein was utilized for characterization. PHYTN showed optimal activity at 80 °C and pH 6 in sodium acetate buffer. The enzyme was found metal dependent and presence of Fe3+ or Cu2+ showed enhancing effect on PHYTN activity. Thermostability studies demonstrated that PHYTN retains 90% residual
SUMMARY
71
activity when the protein was incubated at 80 °C for 1h in the presence of 1.5 mM Fe3+. The kinetic studies of PHYTN demonstrated km and Vmax values of 50 mM and 2500 μmole/min respectively when sodium phytate was used as substrate. The characterized PHYTN was used for poultry trials to check the efficacy of the enzyme in poultry birds.
The results depicted that PHYTN put significant effect on the bird weight gain, feed intake and feed efficiency ratio. Presence of 1000 IU/kg of PHYTN resulted in the weight gain in 3rd, 4th and 5th week of trials from 504.766 to 533.535 g, 767.933 to 823.733 g and 999.833 to 1120.277 g respectively when compared with the control. The study demonstrated that this recombinant thermostable phytase is suitable for poultry feed industry and its domestic production will contribute the economic availability of PHYTN for the poultry feed industry. Availability: Items available for loan: UVAS Library [Call number: 2870-T] (1).
53.
Study On Polymorphism Of Promoter Region Of Bovine Lactoferrin Gene And Its Relation With Mastitis In Nili Ravi Buffalo
by Muhammad Asim (2012-VA-636) | Dr. Sehrish Firyal | Dr. Muhammad Yasir Zahoor | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Dairy animals in Pakistan and worldwide are facing most persistent and economically affecting
disease mastitis. Only a healthy buffalo can produce good quality milk of physiologically normal
composition. Mammary gland inflammation due to mastitis badly affects the quantity and quality
of milk and this causes big loss to dairy industry. Even province Punjab bears economic losses of
240 million per annum due to mastitis.
Susceptibility and resistance to mastitis is affected by the variation in immunity genes. Among
immunity genes Lactoferrin (LF) have important role in immune defense system and perform
antibacterial, antiviral and anti-inflammatory function. LF is found in most of body fluids like,
milk, blood, tear, saliva, bile and mucous. Polymorphism in promoter region of Lactoferrin gene
is associated with mastitis susceptibility and resistance. For screening of the mastitis
susceptibility and resistance of dairy buffaloes, LF is a potential candidate gene.
The present study was designed for the identification of polymorphism in LF gene associated
with mastitis. Blood samples from 20 Nili Ravi buffalos having clinical and subclinical mastitis
were selected. Blood samples of normal Nili Ravi buffalos were also collected. DNA was
extracted; specific primers for amplification of LF gene were designed with Primer-3 software
by using already reported sequence on NCBI.
Amplification of LF gene performed by PCR and sequenced the amplicon. A comparative
analysis of sequence result was performed by using NCBI BLAST. BioEdit software was used to
perform multiple sequence alignment. Comparison analysis of LF gene promoter region shows
multiple mutations in in clinical and subclinical as compare to reported sequence (accession no.
EF650854) at NCBI. While normal samples sequence results are similar to reference data. These
results show LF is a candidate gene for mastitis resistance. Availability: Items available for loan: UVAS Library [Call number: 2923-T] (1).
54.
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: ; 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
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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).
55.
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: ; 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).
