1.
Isolation Characterization And Growth Optimization Of Starch Hydrolyzing Fungi From Soil Of Livestock Farms
by Saba Sana | Prof. Dr. Aftab ahmad anjum | Dr. Muhammad Nawaz | Prof. Dr.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1898,T] (1).
2.
Screening And Characterization Of Phytase And Bile Salt Hydrolases Producing Probiotic Lactobacilli Isolated
by Madiha arif | Dr. Muhammad Nawaz | Prof. Dr. Khushi muhammad.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2001,T] (1).
3.
Isolation And Characterization Of Antibiotic Resistant Lactobacilli From Fermented Food Products
by Shahgull | Dr. Muhammad Nawaz | Prof. Dr | Prof. Dr. Aftab anjum.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2002,T] (1).
4.
Occurrence Of Bacterial Contaminants In Poultry Meals And Their Antibiotic Resistance Pattern
by Nayyab Tariq (2009-VA-207) | Prof. Dr. Aftab Ahmad Anjum | Dr. Muhammad Nawaz | Dr. Muhammad Nasir.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Poultry is the second largest industry after textile industry in Pakistan. Its consumption
rate is very high as compared to other animal protein sources, as it is cheaper as compared to red
meat. To fulfill increasing demand of poultry, poultry production quality must be improved.
Many factors affect poultry production. One factor is feeding process. Efficiency of poultry
production depends mainly on feeding process which influences both the quality and quantity of
the poultry production (Grepay 2009). The rearing of poultry birds on commercial level requires
use of bulk quantities of poultry feed. Poultry feed costs 60-70% of total cost for production
(Sahraei et al. 2012). The main purpose to increase poultry production is to fulfill nutritional
requirements of human population that largely rely on poultry and poultry by products as a
source of protein(Obi and Ozugbo 2007).
Poultry feeds are food materials designed to contain all necessary feed ingredients for
proper growth, meat and egg production in birds (Obi and Ozugbo 2007). It is a mixture of
various components including plant proteins (cereals and by products, grains etc), animal byproducts,
fats, vitamins and minerals (Ravindran 2013). The major component of poultry feed is
protein which is the key component of eggs and meat. Protein sources in poultry feed are of
plant, marine and animal origin. Plant proteins may lack some of the essential amino acids, thus
are incomplete protein. Proteins of animal origin are better growth promoter than protein of plant
origin, but their safety is a concern. Among plant based proteins, soybean and canola meal are
produced in higher amounts worldwide (Alali et al. 2011). The animal protein sources include
poultry, fish, meat bone and poultry by products meal. Poultry meal is derived from clean tissues
Introduction
2
of slaughtered poultry including bone after the moisture and fat have been extracted in the
rendering process. It may contain whole birds excluding feathers (Anonymus 2014). Among all
protein based meals, poultry meals and poultry by products meal are of superior quality and
provide higher protein content than plant, marine and meat based meals (Samli et al. 2006).
Quality of animal feed has gained importance worldwide. The feeds are found to be
associated with infectious or non-infectious hazards, thus influence human health (Sherazi et al.
2015). Poultry feed can act as carrier of animal and human pathogens (Aliyu et al. 2012). Poultry
feed can get contaminated at any point of harvesting, processing, storage or dispersal of feed.
Primary mode of poultry feed contamination is by dust, soil, water and insects. Poultry meals can
be another source of feed contamination. Poultry meals are added in feed as a source of protein.
Feeds of animal origin like poultry meals are richer in nutrients and water as compared to feed of
plant origin thus are found to have higher microbial load, facilitating the multiplication of
bacteria (Kukier and Kwiatek 2011). Inclusion of contaminated meals in feed increases microbial
load of poultry feed. The contamination of poultry feed not only influences appearance and
nutritional value of feed, but also affects animals and human who consumes it (Maciorowski et
al. 2007). The profitability of poultry production can be greatly affected due to the frequency of
feed contamination and the detrimental effects of the aflatoxins on performance of chickens
(Anjum et al. 2011). Poultry feeds have been implicated in several poultry diseases of viral
(Avian Influenza, Newcastle disease), bacterial (Salmonellosis, Infectious Coryza) and fungal
origin. Many human diseases like Traveler’s Diarrhea and Salmonella Paratyphoid fever have
been associated with consumption of poultry birds that contracted infections from poultry feed
(Obi and Ozugbo 2007).
Introduction
3
The poultry industry relies on ready to use poultry feed prepared by feed mills (Arotupin
et al. 2007). Both bacteria and fungi including mycotoxins usually contaminate feed at different
stages of pre or post processing, depending upon the conditions under which it is handled or
stored (D’Mello 2006). Poultry meals mostly get contaminated post rendering process. The
cooking step in rendering process inactivates bacteria, viruses, protozoa, and parasites(Meeker
and Hamilton 2006) . Still presence of contaminants in meals is attributed to post processing
contamination. Many bacterial pathogens reported in feed are Escherichia coli, Erwinia
herbicola, Salmonella spp., Listeria spp., Enterococcus fecalis, Cl. perferingens and Cl.
botulinum (Aliyu et al. 2012; Lateef and Gueguim-Kana 2014) . The contaminated feed results
in excessive activation of immune system and ultimately decreases poultry production and its
profitability (Kukier et al. 2012). In addition to bacterial contaminants, toxigenic fungi have
threatened quality and safety of feed and have caused severe losses to poultry industry in recent
times. Cereals and grains based poultry feed mostly get contaminated with fungi (Kwiatek and
Kukier 2008). Mycotoxin producing fungal genera that are reported in poultry feed are
Aspergillus, Penicillium and Fusarium (Greco et al. 2014).
As Poultry feed is the first step of the food safety chain in "farm-to-fork" model. Contaminated
feed can also serve as a source of antimicrobial resistant bacteria in poultry meat(da Costa et al.
2007). There are many evidences that pathogens in feed are transmitted to humans through
animals and food of animal origin. It can also become source of some human pathogens in
environment. Feed contamination by fungi is responsible for animal mycotoxicoses and through
consumption of contaminated animal food, results in human intoxications (Kukier et al. 2012).
Birds utilizing toxins containing feed are economical loss for farmers and also affects consumer
Introduction
4
health through its residues (Alam et al. 2012). Poultry feeds containing antibiotic resistant
bacteria results in loss of poultry productivity, making treatment of poultry diseases difficult.
Thus quality of animal food directly depends on usage of nutritionally balanced and safe feed.
Among many feed sources used, poultry meals are gaining importance for their higher nutritional
value, but very less work has been done in world particularly in Pakistan to determine
microbiological safety of poultry meals produced. There is the need to determine various quality
parameters which should be followed to ensure production of safe meal. Availability: Items available for loan: UVAS Library [Call number: 2252-T] (1).
5.
Isolation And Characterization Of Antibiotic Resistant Lactic Acid Bacteria From Poultry Gastrointestinal Tract
by Nabeea Saleem (2008-VA-234) | Dr. Muhammad Nawaz | Dr. Aamir Ghafoor | Dr. Aqeel Javeed.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Lactic acid bacteria (LAB) are heterogeneous group of bacteria which are fastidious in
nature. LAB has acquired status as Generally-Recognized-as-Safe (GRAS) status. Over-use and
misuse of antibiotics in veterinary and human clinical setups give rise to antibiotic resistant lactic
acid bacteria in gastrointestinal tracts which act as antibiotic resistance reservoir. Acquired and
transferable antibiotic resistance in lactic acid bacteria is a newly identified safety concern in
poultry. So, it is dire need of time to determine the situation of antibiotic resistance in lactic acid
bacteria of poultry gastrointestinal tract (GIT).
The present study was conducted to isolate lactobacilli from indigenous and broiler
gastrointestinal tract of chicken. For this purpose, chicken feces, cloaca and caecum samples
(n=20 each) were collected from Lahore. Lactic acid bacteria were isolated on MRS medium.
Isolates were identified by phenotypic characteristics including Colonial morphology, Gram
staining and Catalase test. While molecular identification of lactobacillus spp. was done by PCR
at an annealing temperature of 55°C using the primers XB-5 and LbLMA-1 with an expected
product size of 250bp. Minimum inhibitory concentrations of different antibiotics such as
ampicillin, erythromycin, tetracycline, ciprofloxacin, cephradine, cefuroxime, ofloxacin,
levofloxacin were determined by the broth micro dilution method following the EFSA
guidelines. Antibiotic resistance genes, including erythromycin arm (B) and tetracycline Tet (M)
were amplified by polymerase chain reaction.
The comparison of % antibiotic resistance pattern between broiler and indigenous
lactobacillus spp. against different antibiotics was analysed for chi-square test using SPSS
……………………………………………………………………………………………Summary
67
version 16.0. The study provided data on antibiotic resistance pattern of transferable resistance
genes in lactic acid bacteria of poultry gut.
Conclusion:
From the present study it is concluded that a high level of resistance was shown by lactobacillus
spp. against tested antibiotics. Lactobacillus spp. were screened by PCR for known resistance
genes and thus were able to determine the presence of erm(B) and tet (M) genes in all
lactobacillus spp. using erm(B) and tet (M)-specific primers. All lactobacillus spp. were also
phenotypically resistant to erythromycin and tetracycline. Thus, the present study indicates that
such erm (B) and Tet (M) genes occur among different LAB genera and species therefore it is
the need of time to study other resistance determinants to ensure the safety of poultry meat and
spread of resistance determinants. Availability: Items available for loan: UVAS Library [Call number: 2282-T] (1).
6.
Isolation And Characterization Of Avian Isolates Of Lactobacilli Species And Their Antisalmonella Activity
by Anum Shaukat (2009-VA-220) | Dr. Muhammad Nawaz | Dr. Jawad Nazir | Prof. Dr. Mansur-Ud-Din Ahmad.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Poultry industry is second largest industry of Pakistan. Poultry industry is cheaper source
of protein and provides jobs for more than 1.5 million people. It is facing several problems due
to microbial diseases. Salmonella is one of the leading causes of diseases in poultry. These are
being treated with antibiotics but misuse and overuse of antibiotics result in antibiotic resistant
strains of microorganisms. We need some alternatives for treatments. Lactobacilli are one of the
alternatives to antibiotics used as probiotics.
It has been found that the Lactobacilli of poultry origin have antimicrobial activity
against Salmonella.
Lactobacilli was isolated from the droppings, cloaca and caecum of rural poultry birds
using deMan Rogosa Sharpe (MRS) medium. The isolates were screened for anti-Salmonella
activity against S. enterica along with their properties to resist low pH and bile acids, antibiotic
sensitivity, auto-aggregation and co-aggregation. The isolates showed anti-salmonella activity
were identified using microscopic characters and biochemical profile. The isolates were
confirmed by PCR using species specific primers and sequencing 16S rRNA gene.
The data was analysed using one-way ANOVA at significance level P value <0.05 by
using the statistical software Graph Pad Prism version 5.3.
The study was conducted on a total of 60 samples including caecal swabs (n=20), cloacal
swabs (n=20) and dropping (n=20) of indigenous poultry. From these samples, five isolates were
Summary
57
selected based upon the tests performed. Isolates namely CLB-41, CLB-45, PDL-13, PDL-26
and PDL-33 showed best results. Further characterization was done by PCR and sequencing.
Availability: Items available for loan: UVAS Library [Call number: 2313-T] (1).
7.
Activity Of Plant Extract Mediated Silver Nanoparticles Against Selective Medically Important Pathogens
by Qurat UL Ain (2010-VA-295) | Dr. Muhammad Nawaz | Dr. Imran Najeeb | Dr. Muhammad Nasir.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Due to increased use of antibiotics different common pathogenic microbes have developed
antibiotic resistance. The problem of antibiotic resistance is growing day by day. Plants are
being used as a valuable source of natural products to maintain the health. So use of plant
extracts is an interesting and valuable way to control this problem. Metal particles have
antibacterial activity. Extract mediated synthesis of metal nanoparticles is a very impressive
way to control the microbial diseases. To synthesize metallic nanoparticles using extracts is a
superior method than any other method. Silver has distinctive properties as antibacterial
agent. Nanoparticles can be synthesized by various methods but green synthesis is
environment friendly and much more important than other methods.
Melia azedarach have many active components due to which its every part has some
antimicrobial characteristics. Its extract mediated AgNPs are very important in this regard.
Similarly Albizia procera extracts has potential to kill pathogenic microbes. That’s why these
two plants were selected to synthesize nanoparticles. Fresh leaves were collected extracts
were prepared. Silver nitrate solution was prepared and then this solution was allowed to
react with each extract separately.
After the formation, nanoparticles were separated by centrifugation. Then their
characterization was done by UV visible spectroscopy and FTIR. After characterization well
diffusion assay was performed to check antimicrobial activity of these nanoparticles against
selected medically important pathogens. It was found that the antimicrobial activity of extract
mediated nanoparticles was better than the antimicrobial activity of plant extract alone.
Summary
51
In the present work it is explored that nanoparticles formed from extract of Albizia
procera has a greater antimicrobial activity against important medical pathogens. In short it
was proven that nanoparticles can control the pathogens in a better way as compared to the
extract alone. And this result that nanoparticles mediated particles extract of Albizia procera
has suggested many other new facts. Albizia procera’s properties are needed to be explored
more. Availability: Items available for loan: UVAS Library [Call number: 2507-T] (1).
8.
Antimicrobial Activity Of Selected Plant Extracts Against Streptococcus Mutans Isolated From Dental Caries
by Iqra Shaukat (2010-VA-287) | Dr. Imran Najeeb | Dr. Muhammad Nawaz.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Streptococcus mutans is gram positive bacteria and present in cocci and chain form. It is
facultative anaerobes, non-motile, catalase negative and non-spore forming, lactic acid bacteria
and normally found in oral cavity (Rao and Austin 2014). S.mutans involve in endocarditis, dental
caries, have ability to adhere to the cardiac tissue and cause chronic infective endocarditis. Caries
is caused of tooth decay and tooth loss in adult and school aged children. Many microorganisms
can cause dental caries namely S. mutans, S.sanguis, s.mitis, S.salivarius and S. sobrinus but
S.mutans have major role in developing of dental caries. Dental caries is major problem around
the globe in these days. Generally it treated with antibiotics. But now a day’s due to increase in
antibiotic resistance, recently plant extracts and plants parts are being in use as treatment and oral
hygiene. It is therefore, present research was designed to analyze the plant extract which having
antimicrobial activity against oral bacteria and have been used traditionally for cleaning the teeth.
Azadirachta indica (Neem), Acacia nilotica (Kikar), Pongamia pinnata (Sukhchein) and
Salvadora persica (Peelu) were used to check the activity against S. mutans. Aqueous and
methanolic extracts of bark were prepared of selected plants. Extract potential against S. mutans
was checked through well diffusion and minimum inhibitory concentration (MIC) assay. Dental
caries samples were collected from different hospitals from Lahore. Twenty five samples were
processed to isolate S.mutans. Out of twenty five samples twenty isolates of S.mutans were isolated
which showed resistance against bacitracin, confirmed by disk method.. Biochemical tests such
as hemolysis test and sugar fermentation tests were also done for the confirmation of S. mutans.
Furthermore, antibiotic sensitive test was performed to check the sensitive pattern of S. mutans All
S. mutans were resistant to oxacillin, cefmetazole and cephaloridine, and sensitive to streptomycin
Summary
50
and gentamycin. S. mutans showed high level of resistance to ceftriaxone (90%), cefixim (90%),
chloramphenicol (65%) and vancomycin (60%), and intermediate level of resistance to
sulfamethoxazole/Trimethoprim (40%) and ciprofloxacin (30%), and low level of resistance to
amoxicillin (25%), delfopristin (25%) ampicillin (20%), fusidic acid (20%) and linezolid (5%).
MIC value for aqueous extract for Salvadora persica, Azadirachta indica, Acacia nilotica,
Pongamia pinnata are 24-48, 3-48,0,180-12 and >48 mg/ml respectively, and MIC value for
methanolic extract are 0.09-12, 3->48, 0.376-6 and 1.5-24 mg/ml respectively. This in vitro study
gives us natural antimicrobial plants which can help us to control dental caries and endodontic
infections. The effects of these extracts might be beneficial if incorporated in tooth paste, mouth
rinses and dental products to reduce plaque and dental caries. Availability: Items available for loan: UVAS Library [Call number: 2508-T] (1).
9.
Evaluation Of Inhibition Activity Of Indigenous Lactobacilli Spp. On Ammonia Emitting Bacteria
by Fatima Sajjad (2010-VA-312) | Dr. Muhammad Nawaz | Prof. Dr. Masood Rabbani | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Poultry is the 2nd largest industry in Pakistan which is growing at an amazing rate of more
than 10% from last few years. It provides jobs for more than 1.5 Million people. Although, it is
growing at excellent pace, it still faces many problems. One of the important problems in poultry
farming is the production of ammonia by urease producing microbes. Ammonia is health hazard
for both poultry and human. Urease producing bacteria which are the major problems in poultry
are Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae and Staphylococcus
aureus in poultry droppings. Probiotics such as Lactobacilli can inhibit the urease producing
bacteria in poultry intestine as well as environment thus mitigate the ammonia emission. Present
study was designed to isolate Lactobacilli from indigenous poultry, screen them for
antimicrobial activity against urease producing microbes and determine their effect on growth of
Proteus mirabilis during co-culture experiments. A total of 71 Lactobacilli isolated were
recovered from 20 samples of droppings (10) and caeca (10) of back-yard poultry on MRS agar
plates. Twenty seven (27) isolates demonstrated antimicrobial activity against ammonia emitting
bacteria by agar spot and well diffusion assays. Seven isolates (FSL19, FSL25, FSL39, FSL45,
FSL51, FSL63 and FSL71), having better antimicrobial activity, were selected for co-culturing
with Proteus mirabilis in nutrient broth. FSL25, FSL45, and FSL51 showed more than 2 log10
reduction of Proteus mirabilis in co-culture experiments. FSL25, FSL45, and FSL51 were
identified as Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus salivarius,
respectively by amplifying and sequencing their partial 16S rDNA. It is concluded that
Lactobacillus plantarum FSL25, Lactobacillus fermentum FSL45 and Lactobacillus salivarius FSL51 may be used to mitigate ammonia emitting bacteria in poultry environment after further
investigations. Availability: Items available for loan: UVAS Library [Call number: 2594-T] (1).
10.
In Vitro Activity Of Selected Biocides Against Fungal Isolates From Production Area Of Pharmaceutical Industry
by Sana Ilyas (2009-VA-238) | Dr. Muhammad Nawaz | Prof. Dr. Aftab Ahmad Anjum | Dr. Muhammad Ovais Omer.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Pakistan pharmaceutical industries have grown to grab their position amongst top ten pharmaceutical industries of Asia Pacific region. These are serving with 80% of pharmaceutical needs. The industry on the other hand faces some challenges in terms of sterile pharmaceutical product manufacturing. The fungal contamination causes spoilage to pharmaceutical products, cosmetics, and food products. The fungal contamination to pharmaceutical products has resulted in direct losses to human health and to economy.
A total of 50 air samples were collected from clean area of a pharmaceutical production unit by exposing sabouraud dextrose agar (SDA) plates by settle plate method (4 hours exposure). Fungal colonies were purified by sub-culturing and later identified macroscopically and microscopically. Selected biocides included isopropyl alcohol (70%), chloroxylenol (20%), chlorhexidine gluconate (20%), and benzalkonium chloride (20%) were used in this study. A 100 μl of spore suspension of each fungal contaminant (1.0 × 106 to 5.0 × 106 spores/mL) was exposed to 9.9 mL of biocide preparation for 15 and 30 minutes while exposure was stopped by adding 1 mL of mixture (spores exposed to biocide) into 9 mL of respective neutralizing agents The enumeration of colonies was started immediately after the growth was visible and expressed as Mean±S.D. and converted to log10. Antifungal activity of biocides was expressed as log10 reduction and different biocides‟ activity was compared using ANOVA technique by graphed prism 5.0 statistical software.
Total 204 colony forming units (CFU) were identified from filling area (36), solution room (47), and buffers (121). The antifungal activity in terms of log reduction was lowest by isopropyl alcohol at 15 minutes and highest was shown by chlorohexidine gluconate at 30 minutes against
Summary
64
Aspergillus flavus. In case of Aspergillus fumigatus all the biocides presented significant difference of antifungal activity at 15 minutes. The response of Aspergillus niger against different biocides at 15 minutes and 30 minutes was same as was in case of Aspergillus flavus while each biocide‟s antifungal activity was found significantly increased with increase in time of exposure. The similar response of antifungal activity of different biocides at both exposure times was noted against Saccharomyces cerevisiae. The antifungal activity of all biocides against penicillium was found significant different at 15 minutes and 30 minutes exposure time. Similarly, each biocide‟s antifungal activity increased with increase in time of exposure. On overall basis, isopropyl alcohol was found less effective while benzalkonium chloride and chlorohexidine gluconate presented comparatively higher efficacy against fungal isolates. Availability: Items available for loan: UVAS Library [Call number: 2705-T] (1).
11.
Characterization And Antibiotic Resistance Profile Of Listeria Monocytogenes Isolated From Fish And Broiler Meat
by Mohammad Nasar (2015-VA-21) | Dr. Sameera Akhtar | Dr. Muhammad Nawaz) | Prof Dr. Mansur ud Din Ahmad).
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Gram positive rod shaped, non-spore former Listeria monocytogenes is the major food-borne pathogens for humans and animals. It can cause serious foodborne infection. The bacterium is saprophyte and can grow on wide range of temperature (0-45°C). Due to this it contaminates different food products. Consumption of this contaminated food products can cause serious problems in neonates, pregnant women and immunocompromised peoples. Its signs may develop between day one to three months after ingestion of the organism. The neonates can develop septicemia, respiratory diseases and meningitis. The pregnant woman may develop influenza like symptoms, or keep an asymptomatic infection that ends in abortion, premature birth or sepsis in the newborn. Healthy people hardly develop clinical signs but a febrile gastroenteritis syndrome has been reported. No doubt this disease is associated with unhygienic food consumption and is characterized by fever, nausea, diarrhea, headache, abdominal pain and sometime myalgia. These symptoms may be resolved in one to three days Listeria monocytogenes was isolated by conventional methods and suspected colonies were identified by Gram staining and biochemical tests catalase and oxidase test. The DNA was extracted from isolated colonies by 10% chelex method. The isolated strains were confirmed through PCR by targeting prfA gene of 479bp. Antibiotic resistance were also checked for confirmed isolates. A total of 160 (Fish meat n=80, Broiler meat n=80) samples were taken for the present study for screening Listeria monocytogenes. The bacterium was found in 19/80 (23.75%) samples of Fish and 5/80 (6.25%) of broiler meat samples through PCR detection.
Summary
45
Later the confirmed isolates were tested to check the resistance profile of the bacterium to different commonly available antibiotics. For this 24hrs old culture were used. Three to five colonies were picked by sterile loop and transfer to test tube containing 10ml normal saline. To check the turbidity the tube was compared with 0.5 Macfarland standard. Then by sterile cotton swab the bacterium was spread on Mueller Hinton agar. Antibiotics were placed by sterile forceps on agar. The plates were incubated at 37°C for 24hrs. Zones of inhibition were measured in mm by the help of ruler and then compared with staphlycoccus aureus break points in CLSI. The susceptibility result shows that the bacterium was resistant to gentamicin. Mostly L. monocytogenes isolates were susceptible to antibiotics used in this study. This study suggested ampicillin as drug of choice for treatment of listeriosis. Preventive measures should be adopted to avoid the risk of the disease. Availability: Items available for loan: UVAS Library [Call number: 2835-T] (1).
12.
Antiviral Potential Of Gold And Silver Nanoparticles Against Newcastle Disease Virus In-Vitro
by Anam Iftikhar (2011-VA-404) | Dr. Jawad Nazir | Dr. Muhammad Nawaz | Dr. Aqeel Javeed.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Nanotechnology supplies a golden platform, where properties of pure metals are modified and improved by converting them into their nanoparticles, and it is applicable to numerous fields such as diagnostics, antimicrobial agents and drug delivery. Nanoparticles act as bactericidal, antifungal and antiviral agents. Several types of nanoparticles (gold,copper,silver,aluminium,magnesium,zinc and titanium) have been reported in literature out of which are known to have antibacterial properties. Viral diseases present challenging problems worldwide. Newcastle disease (ND) is endemic in Pakistan, and is linked with huge economic disastrous to farmers. Application of antiviral therapy to control the active infection of NDV is very limited. Use of nanotechnology to control active virus infection might be a viable solution to limit the disease in effected flocks.
In this study four groups of nanoparticles i.e. gold, silver, magnetic and gold coated magnetic were evaluated against mesogenic strain of Newcastle disease virus. Chicken embryos were used to propagate the virus and infective amniotic allantoic fluid was collected. Evaluation of nanoparticles in reducing virus infectivity as a measure of tissue culture infective dose (TCID50) was performed. Various dosesof nanoparticles (very low, low, medium, high and very high) were allowed to interact with virus suspension in three ways i.e.pre- treatment, post-treatment and co-treatment methods. Virus infectivity before and after the treatment with nanoparticles was measured and subsequently used to calculate reduction factor (RF). All of the experiment was repeated three times.
It was observed that in pre and post-treatment, silver, gold coated magnetic and magnetic nanoparticles groups the infectivity titerswere efficiently reduced at high dose. While in co-treatment, silver, gold coated magnetic and magnetic nanoparticles groups the virus inactivation rates were relatively higher at low and very low doses.
It is evident from the findings that within the tested nanoparticles, silver, coated magnetic and magnetic nanoparticles have equivalent antiviral properties against NDV. While within various treatment methods co-treatment assay proved to be more effective in reducing virus infectivity than the pre and post treatment group. The results of present study are suggestive of testing antiviral properties of the nanoparticles in vivo conditions.
Availability: Items available for loan: UVAS Library [Call number: 2877-T] (1).
13.
Characterization And Physicochemical Optimization Of Phytases Produced By Indigenous Isolates Of Lactobacillus SPP.
by Aanisa Arif (2011-VA-424) | Dr. Muhammad Nawaz | Prof. Dr. Masood Rabbani | Dr. Sanaullah Iqbal.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Phytate is one of the major organic storage forms of phosphorous of phosphorus in
seeds, cereal, oil and legume; in nature about 75%-80% of total phosphorus is available in
this form. Phosphorus is stored in roots and in seeds and cereals as phytate. Phytases are
responsible for breakdown of phytic acid (phytate) into inorganic monophosphates and
free myo- inositol. Phytases are a class of phophatases which hydrolyze phytic acid into
inorganic phosphate and myo inositol or less phosphorylated phosphates. Monogasteric
animal like poultry, human and fish lack phytase due to which they cannot derive
phosphate from phytate and phosphorus is unavailable to them.
So, present study is designed as a first step in a multi-step project to develop
indigenous phytase producing probiotic lactobacilli from different sources, the
optimization of phytase production and effect of physical & chemical factors on the
phytase stability and activity. Lactobacillus isolated from poultry was checked for phytase
production on Phytase screening media (PSM). Enzyme from the isolates showing activity
were quantified by ammonium molybadate method, the enzyme production were
optimized at different physical and chemical parameters such as temperature (30, 35 &
42°C), pH (4,5,6,7 & 8), osmotic pressure (1%,2% and 4%), aerobic/anaerobic conditions,
carbon (glucose, lactose, sucrose), nitrogen sources (peptone, tryptone & urea) and bile
salts (0.3%,1% and 2). Enzyme was partially purified and characterized by SDS-PAGE.
In present study 20 samples of poultry droppings (SP01-SP10) and fermented food (SY01-
SY10) were processed for isolation of lactobacilli. A total of 90 isolates (PDP01-PDP45;
FYP01-FYP45) were selected from MRS plates. Isolates were preliminary confirmed as
Gram positive rods with Catalase negative. All isolates were further purified and stored in
MRS broth supplemented with 15% glycerol at -20oC. Purified lactobacilli isolates were
screened for phytase production on phytase screening medium and zone o f hydrolysis was
Summary
77
measured in mm. Out of total of 90 isolates 62 isolates showed phytate hydrolysis. Out of
62 isolates, 16 were selected on the basis of retention of hydrolysis zone after cobalt
chloride staining. Out of 16 selected isolates, eight isolates PDP05, PDP09, PDP10,
PDP16, PDP23,PDP24, PDP30 and PDP35 were of poultry origin and eight FYP12,
FYP15, FYP17, FYP21, FYP26, FYP31, FYP38 and FYP42 were of fermented foods.
Selected isolates and retention of their zone of hydrolysis after cobalt chloride staining are
given in table 4.4. Phytase activity of selected lactobacilli isolates was checked in
modified MRS broth containing 0.2% sodium phytate at 37°C after 24 hrs. Cell free
supernatant was used as crude source of enzyme. Enzyme activity was determined by
using ammonium molybadate method. Amplification and sequencing of 16Sr DNA
(≈1500bp) was done by using universal primers which revealed PDP10, PDP24 and
FYP38 had >99% similarity with Lactobacillus gallinarum, Lactobacillus reutri and
Lactobacillus fermentum respectively with GenBank accession no. MF980924,
MF980925 and MF980923 respectively.
Phytase production by lactobacilli was optimized at different parameters e.g.
temperature (30, 35 and 42°C), pH (4, 5, 6, 7 and 8), osmotic pressure 1%, 2% and 4%.
The effect of oxygen was determined by growing lactobacilli isolates in aerobic and
anaerobic conditions followed by measuring enzyme activity. PDP10, PDP24 and FYP38
showed the best activity at 35°C (6.86 ± 0.15 IU/ml, 5.12 ± 0.12 IU/ml and 5.65 ± 0.13
IU/ml respectively) at pH 5 (6.86 ± 0.15 IU/ml, 5.12 ± 0.12IU/ml and 5.50 ± 0.13 IU/ml
respectively). Maximum phytase activity was recorded at 1% NaCl 4.78 ± 0.14, 4.18 ±
0.13 and 5.58 ± 0.12 IU/ml respectively) whereas anaerobic conditions were favourable for
the production of phytase by selected isolates.
Effect of carbon, nitrogen sources and bile salts was determined by growing
isolates MRS broth (0.2% sodium phytate) modified with different carbon (glucose,
Summary
78
lactose, sucrose) and nitrogen sources (peptone, tryptone and urea) and bile salts (0.3% ,
1% and 2%) followed by measuring enzyme activity. In this study isolates PDP10, PDP24
and FYP38 exhibited maximum phytase activity in the presence of 2% glucose as
compared to other carbon sources lactose and sucrose (4.36 ± 0.11, 4.38 ± 0.18 and 5.01 ±
0.15 IU/ml respectively). Present study revealed 0.1 % peptone as an optimal source of
nitrogen for PDP10 and PDP24 (4.54 ± 0.13 and 4.23 ± 0.19 IU/ml respectively) while
FYP38 (4.56 ± 0.14 IU/ml) showed best result in presence of 0.1% tryptone. All the
isolates showed maximum phytase activity at 0.3% bile salt concentration as compared to
1% and 2% concentration. Enzyme activity of phytase obtained from PDP10 was not
varied while treating it at different pH (4, 5, 6, 7 and 8) at different intervals of time.
Enzyme activity of phytase obtained from PDP24 was lower at pH 8 for 15, 30, 45 and
60min (3.41± 0.10, 3.40 ± 0.09, 3.42 ± 0.08 and 3.41 ± 0.11IU/ml respectively). Enzyme
activity of phytase obtained from FYP38 was lower down from pH 7 to 8 for 15, 30, 45
and 60min (4.41 ± 0.09, 4.42 ± 0.11, 4.43 ± 0.10, 4.41 ± 0.12 IU/ml respectively) & (4.40
± 0.09, 4.31 ± 0.11, 4.33 ±0.10 and 4.34 ± 0.12 respectively). Enzyme activity was
inhibited at 1mM and 5mM concentrations of Ca2+ while metal ions like Mg2+ and Na2+
addition stimulated the phytase activity. Approximate molecular weight of extracellular
protein precipitated from the cell free supernatant of PDP10, PDP24 and FYP38 was
~50kDa. Availability: Items available for loan: UVAS Library [Call number: 2895-T] (1).
