staph vaccine pdf
TRANSCRIPT
Engineered Plasmid DNA Vaccine For Staphylococcus aureus
Dr .M.Muruganandam
2
Engineered Plasmid DNA Vaccine For Staphylococcus aureus
Dr.M.Muruganandam
3
Dedicated to My
Science Teacher Mr. Ramasamy
4
First Edition -2013
ISBN-978-9982-22-418—5
Publisher and Author
Dr.M.Muruganandam,
Email- [email protected]
5
Preface
Still Staph vaccine research is going
on. This book is help to move one step forward
towards staph vaccine Research. This is
prepared based on my lab work. I referred many
previous researcher’s work during this book
preparation, I thank to all of them. During my
lab work time many students assist me, I
sincerely thank to everyone and I thank to Mr.
Das lab Technician for kind help in
Haematological analysis. Finally I thank to Mr.
Stalin Arockiaraj, chairman, SMCET for
providing necessary lab facilities for this work.
M.Muruganandam
6
Contents
Biotherapy
Mutant Strain Vaccine
Mutant Strain plasmid
DNA Vaccine
Engineered plasmid
DNA Vaccine
Heat shock protein
Vaccine
Mixer Staph Vaccine
Recommendations
Bibliography
7
1. Biotherapy
In 2011, Nobel Prize was
awarded in Medical Science for work in
Biotherapy. It is otherwise called
immunotherapy. It helps to boost up immune
system against pathogens. It is helping to control
various diseases. In Biotherapy, various methods
were employed to induce humoral and cell
mediated immunity.
Now Immuno stimulant, Vaccines,
probiotics, micronutrients, etc and various
natural products are used for Biotherapy. It is
designed to repair, stimulate and enhance the
functions of immune system. It boosts up the
activity of T cells, Natural killer cells,
Macrophages, etc and humoral mediated
8
immunity. This is an alternative to other
therapies.
Some beneficial bacteria present in
digestive track which is suggested that these are
constitute non-pathogenic members of
indigenous intestinal micro flora. These are act
as probiotics against various pathogens .These
candidate are able to colonise the gut and act as
antagonistic against pathogens .These harmless
strains producing antibacterial substance may
reduce the use of antibiotics. These probiotics
bacteria act on complex carbohydrates and split
into simpler compounds for absorption. It is also
synthesizing vitamins B complex group, vitamin
K, etc. In the intestinal track some probiotic
bacteria increases appetite and health.
The probiotics bacteria stimulated the
immune system in non-specific way.
9
Researchers tested Lactobacillus as an adjuvant
to an oral vaccine to rotavirus in children and
they confirmed Lactobacillus preparations act as
immunomodulators .The probiotics bacteria
enhance immunity and alter the intestinal
metabolic activity.
The vaccines are biological
preparations .It induce biological memory of
immune cells and it is also used to store
information regarding pathogens in immuno
memory cells. The antigenic portions of the
pathogens are used to prepare vaccines. Now
different type of vaccines were discovered .The
important vaccines are Killed vaccine, Sub unit
vaccine, Live attenuated vaccine, Peptide
vaccine, DNA vaccine, etc. Now various
vaccination methods are employed, the
important methods are injection, topical
10
application, oral drops, eye drops, bath
vaccination, etc. The main components of
vaccine are antigenic part of pathogens,
preservatives,/stabilisers and adjuvant .The main
role of adjuvant is boost up the functions of
immune system. Nowadays various Biological
materials are used as Bio-adjuvant.
DNA vaccines are new generation
vaccines. In Europe, countries mostly use rDNA
based vaccines. In this vaccine, antigenic part of
DNA was identified and isolated from pathogens
and companied with known vector DNA, then it
will insert into harmless microbes. It will culture
and used as vaccine. It gives long-term
immunity compared to other vaccines
.Nowadays cocktail DNA vaccines were
introduced for more than one infectious disease.
11
In our lab trials shows that naked plasmid DNA
and their digested parts acted as good vaccine.
1. Restriction digestion of plasmid DNA
during vaccine preparation.
Nutrients are mainly divided into
micro and macro nutrients. The macronutrients
are carbohydrates, proteins and lipids. Animals
require more macro nutrients for their growth
and maintenances. The micro nutrients are
vitamins and minerals .Many micronutrients are
functions as antioxidants and immunostimulants,
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for eg vitamins C&E. They reduce stress,
especially vitamin C reduce stress. The
micronutrients require very lesser concentrations
in diet. But it is essential for most of the
physiological functions. It is help to boost up
growth and health. The lesser intake or lack of
micronutrient in diet produces disease condition
for eg hypovitaminosis. Sometimes the excess
levels in diet produce diseases foreg hyper
vitaminosis. So we should identify optimum
requirements level for reduce stress and boost up
immune system.
Immune system is stimulated by
various methods that is called Biotherapy. All
these methods help to improve the functions of
immune system and reduce the stress. So if
identify optimum level of one or more methods
13
are helpful to expose maximum potential of
functions of our immune system.
Naked Plasmid DNA vaccine
Plasmid DNA vaccine were
prepared from Naked Bacterial Plasmid DNA.
The Plasmid DNA are self replicating, double
stranded circular DNA molecule present in
bacteria. It is an extra chromosomal DNA
molecule .It always carries one or more gens
responsible for useful characteristics displayed
by the host. They have their own origin of
replication and they replicate independently of
the origins on the host chromosome. The size of
the plasmid ranges from 1kb to 500kb.
Most of the Plasmid DNA exists as
double strand circular DNA ,with both the
strands intact called as covalently closed
circular DNA .If only one strand is intact then
the molecules are described as open circular
14
DNA. Plasmids are used as vectors in
recombinant DNA technology.
2. Serum protein profile analysis.
The extraction of plasmid DNA
protocol describes growth of bacterial cell
culture, harvesting and lysis of bacteria and
isolation of plasmid DNA .Various methods are
available for isolation of plasmid DNA,
However the convenient methods is alkaline
lysis method.
15
3. Molecular Weight determination of antibody
raised against Staphylococcus aureus.
In our lab trials, plasmid DNA was
isolated from pathogenic bacteria by alkaline
lysis method and it was purified. It is then
dissolved in double distilled water and
administrated an intramuscular injection or
16
provided oral drops to albino rats. This plasmid
DNA integrates into the Chromosomal DNA and
produce antigenic proteins and it may induce
long term immunity. After two to three weeks
of vaccination, killed pathogens was injected
into albino rat. After one weeks of injection,
blood samples were collected and analysed
which shows elucidated antibody responses and
cellular responses. So it is concluded that this
plasmid DNA can act as immunogen.
In our lab works, plasmid DNA
Vaccine were prepared and tested in various
bacterial pathogens such as Aeromonas
hydrophila, Salmonella typhi E. coli and
Staphylococcus aureus. In these trials, first
maximum immune response was observed in
killed vaccine and second maximum immune
17
responses were observed in plasmid DNA
Vaccine and protein vaccine.
4. Modified method of Counter current Immuno
electrophoresis for antibody analysis during
various vaccine treatments.
However compared to killed and
protein vaccines, plasmid DNA vaccine gives
long term immunity. It can be stored at room
temperature and transport is also easy. So it is
recommended to DNA vaccine preparation for
18
other bacterial infections. Furthermore in our
lab trails we have studied about mutant strain
plasmid DNA vaccine, digested plasmid DNA
vaccines, cocktail plasmid DNA vaccine from
various bacterial pathogens were prepared and
tested.
5. Modified method of Rocket Immuno
electrophorosisis for antibody analysis during
various vaccine treatments.
All these works, the naked plasmid
DNA produce good results. In our current lab
19
works proves that plasmid DNA, various protein
vaccines and killed pathogens were combined
and mixer vaccine was produced, which gives
good results because killed vaccine and protein
vaccines induce immediate immune responses,
and the DNA vaccine produce long term
immunity. So the mixer vaccines produce good
results.
6. Antibody Titre (96 Well) for different
vaccine treatments.
Staphylococcus aureus (staph) is a
bacterial pathogen, which mainly affect the
20
Immuno suppressed or Immuno compromised
patient’s that means, it affect patients who have
already weak immune system. It produces
various diseases in humans.
Staph causes superficial skin lesions such
as boils, styes and furunculosis; more serious
infection such as Pneumonia, mastitis,
meningitis and urinary tract infections and deep
seated infections. It causes food poisoning by
releasing enterotoxins into food and toxic shock
syndrome by release of super antigens into the
blood stream.
Staphylococcus aureus is most offenly
spread to others by contaminated hands and skin.
Mucous membranes is usually an effective
barrier against infection, However if these
barrier’s are breached (e.g. skin damage due to
trauma or mucosal damage due to viral
infection.) S. aureus may gain access founder
21
lying tissues or the blood stream and cause
infection.
Traditionally, Methicillin Resistant
Staphylococcus aureus (MRSA) infections have
been associated with hospitalization or other
health care associated risk factors. In recent
years physicians and other health care providers
have observed an increasing number of people
with MRSA infections who lack traditional
health care – associated risk factors. These
people appear to have community illnesses and
deaths caused by MRSA infection, mainly cause
death at a level higher than HIV infection.
MRSA has become more
prevent as nosocomial pathogens causing severe
infections. MRSA become resistant to beta
lactams antibiotics especially Methicillin,
Cefoxitin and Gentamicin. The mean incidence
of MRSA has drastically increased and become
22
a worldwide problem. The resistance to
Methicillin is due to resistant genes. Recently
MRSA strains become resistant to several
different antibiotics such as penicillin, oxacillin
and erythromycin.
Coins have the possibility to be one
of the potential sites of MRSA has the
opportunity to transfer from nasal to fingers and
nails followed by the transmission of MRSA
through the hand contact with things such as
coins and others .
Still there is no good vaccine for
human use. Vaccine development research
programmes are going on various parts of the
world. Plasmid DNA vaccines were proposed
for Bacterial food borne pathogens, such as
Aeromonas hydrophila, Escherichia coli,
Salmonella typhi, etc.
23
In our lab, Plasmid DNA vaccines were
prepared from naked plasmid DNA. For staph
infections many vaccines were proposed such as
mutant strain vaccine, Heat stress protein
vaccine, plasmid DNA vaccine, Engineered
plasmid DNA vaccine, combined vaccines etc., .
Combined vaccine is a combination of various
staph proteins and DNA. It produced good
results, because proteins are acts as bio -
adjutants.
In this book, five chapters were
discussed regarding various experiments on
staph vaccine development. These works
abstracts are as follows.
The second Chapter describes
about mutant strain staph vaccine preparation. In
this work, pathogens was isolated from patients
sample in a hospital and cultured in our
laboratory. After that mutant strain was
24
developed by using U.V treatments then
prepares killed vaccines form these mutant
strains. These vaccines were tested in albino
rats.
The maximum immune response was
observed in six minutes U.V treated mutant
strain. It is best for produce killed vaccine
against Staphylococcus aureus infections.
In the third chapter, mutant strain’s
plasmid DNA vaccine developments were
discussed. In this work, staphylococcus aureus
mutant strains were produced by using U.V.
radiation and plasmid DNA was isolated from
all these mutant strains. These DNA were used
as Vaccine.
The constant amount of vitamin C
was provided as adjuvant. After 15 days,
pathogens were injected to all the treatments,
including control treatments. After that blood
25
samples were taken for analysis. The maximum
immune response was observed in six minute
treated U.V. strain’s plasmid DNA. So it is
concluded that, this plasmid DNA is suitable for
S. aureus vaccine preparation.
In the fourth Chapter, Engineered plasmid
DNA vaccines preparation for Staphylococcus
aureus was discussed. Plasmid DNA has wide
variety of applications in vaccine research. Here
it is modified and used as vaccines. First plasmid
DNA was isolated form Staphylococcus aureus
and engineered by various restrictions enzymes.
In this work, two experiments were conducted.
In the first experiment, plasmid DNA
was isolated and digested individually by five
restriction enzymes such as ECOR-I, Hind-III,
Pst-I, Bam-I and Hae-III, then digested plasmid
DNA was used as vaccines. In the second
experiment, isolated plasmid DNA was double
26
digested by using these enzymes and used as
vaccines. Albino rats were used as test animals
in all the experiments. In the first experiment,
maximum immune response was observed in
Pst-I and Hae-III digested treatment. In the
second experiment, maximum immune response
was observed in EcoR-I+Hind-III and Hind-III +
Bam H-I digested treatments. So it is concluded
that, compared to two experiments, double
digested treatments are highly suitable for
plasmid DNA vaccine preparation of
Staphylococcus aureus.
Our studies showed that, plasmid
DNA vaccine is one of the best vaccines. So in
this attempt, try to develop a common plasmid
DNA vaccine for staphylococcus aureus,
Salmonella typhi and Escherichia coli. In this
work, four treatments were tested and one
control treatment was also tested. In the first
27
treatment, plasmid DNA was collected from
Salmonella typhi, Escherichia coli and
Staphylococcus aureus. These plasmids DNA
was mixed well and delivers through
intramuscular injection. In the second treatment,
all the plasmid DNA were digested by Bam H-I
enzyme and mixed well then these digested
plasmid DNA was used as vaccine.
In the third treatment, all the plasmid
were digested by Pst-I enzyme and these also
mixed well and used as vaccine. In the fourth
treatment, all plasmids are double digested by
Bam H I and Pst-I enzymes and used as
vaccines.
The maximum immune response
was observed in double digested treatment
compared to other treatments. So it is concluded
that it is best for development a common vaccine
for these bacterial diseases.
28
In the fifth chapter, Heat stress
protein vaccines developments were discussed.
In this study, the pathogens were exposed two
different temperatures such as 050 C and 055 C
with different time intervals. All the treatments
albino rats were used as test animals. The Heat
stress proteins were isolated and used as
vaccines.
The maximum immune response
was observed in 10 minutes exposed pathogen’s
heat stress proteins in both temperature
treatments. However compared to these two
treatments, 055 C treatment with 10 minutes time
exposed organisms produced heat stress proteins
induce maximum immune response, so it is
concluded that these Head stress proteins are
recommended to further staph protein vaccine
development process.
29
Optimization of a heat stress protein
is another study. In this study Staph-pathogens
were exposed to 45oC temperature for 10
minute. It produces heat stress protein then
these were isolated and used as vaccine. Graded
levels of these protein vaccines were tested in
albino rat. The maximum immune responses
were observed in 25µgm protein vaccine
treatment. So this concentration is
recommended to further vaccine development
process.
Staphylococcus aureus produces
toxins. In this next experiment, the toxins
proteins were isolated and inactivated then used
as vaccines. Different quantity of these toxin
vaccines were tested into the albino rat. The
maximum immune response was observed in 15
µgm toxoid vaccine treatments. After this level
treatments the immune response were slowly
30
decreased. So this is recommended for further
vaccine development process.
In the sixth chapter, Mixer vaccine was
discussed. In this study, various vaccines were
prepared, such as killed vaccines, Heat stress
protein vaccine, Toxoid vaccine, plasmid DNA
vaccine and the mixer of all these vaccines were
tested for their immune responses. Albino rats
were used as experimental animal. The
maximum immune responses were observed in
killed vaccine.
The second maximum immune
response was observed in mixer vaccine. The
remaining vaccine treatments have low immune
responses compared to these two vaccine
treatments. The mixer vaccine is good for their
longer immunity aspects compared to other
vaccines. So it is recommended for further
vaccine preparation process.
31
Finally, based on all these works,
recommendations were pointed out at the end of
this book and all the supporting data for findings
were also provided.
32
2. Mutant Strain Vaccine
Food borne diseases are the major
problem in the worldwide. Around 250 different
foods borne diseases have been described.
Bacteria are the causative agents of two thirds of
food borne disease out breaks. Staphylococcus
aureus is a leading cause of gastroenteritis
resulting from consumption of contaminated
food. The symptoms of food borne diseases are
very widely, depending on the etiological agents.
The common symptoms of food borne disease
are Diarrhea and Vomiting.
The S.aureus causes disease
when they get inside the host, because they can’t
penetrate the skin. So they are associated with
wounds, cuts needle pricks, etc., once inside the
host, they stick to host tissues and produce
33
toxins. In this study, first try to produce mutant
strain by using U.V. radiation and select best
mutant for prepare killed vaccine against
S.aureus infection.
This pathogen was collected from the
patient’s sample in the hospital and then
cultured. The pathogen was confirmed by
regular microbiological and Biochemical tests.
After that, the pathogen was subjected to
mutations under U.V. treatment at various time
intervals such as 0, 2, 4, 6 and 8 minutes and the
colonies were isolated and cultured separately.
All these cells are purified and formalin killed
by using 0.5% formalin at 40c during overnight
treatment than used as whole cell killed vaccine.
Samples 1-5 in the figures shows
pathogen exposure time such as 0,2,4,6 and 8
minutes respectively.
34
Fig-1: Mutant Strains Vaccines influence on
RBC Counts (millions) in Albino rat.
All the mutant strains were serially diluted
up to 510 dilution and inject to five sets of
animals (albino rats) including control. Vitamin
C 300 mg was given as adjuvant for each
individual. After 15 days formalin treated cells
were injected to all the animals including control
treatments. The immune responses of all the
strains were analyzed on the basis of
immunological and haematological aspects.
012345
1
2
34
5
35
Fig-2: Mutant Strains Vaccines influence on
WBC Counts (Cells/Cu mm) in Albino rats.
Staphylococci can survive dry surfaces
with in the increasing of transmission. S. aureus
expresses many potential virulence factors such
as surface proteins, and toxins which damage
host tissues; it is inherent and acquired
resistance to antimicrobial agents.
The aim of the present work is to select
best mutant strain for prepared killed vaccine. So
if induce mutation through U.V. treatment leads
to increase efficacy at certain limit. Based on
0
1000
2000
3000
40001
2
34
5
36
immune responses, best mutant strain was
selected for preparation of best vaccine. In the
present experiment, the WBC count is increased,
as the U.V. treatment time increases, it attain
peak value in 6 minutes mutant strain.
Fig-3: Mutant Strains Vaccines influence on
WBC Differential Counts (%) in albino rats.
The maximum lymphocyte and
antibody levels were observed in 6 minutes
treatment. Based on these results, it is concluded
0
20
40
60
801
2
34
5Lym
pol
37
that 6 min. U.V. mutation strain is best for
Killed vaccine preparations.
Fig-4: M.S Vaccines influence on Hb (gm %).
Fig-5: Mutant Strains Vaccines influence on
Packed Cell Volume (%).
05
101520
1
2
34
5
0
10
20
301
2
34
5
38
Fig-6: Mutant Strains Vaccines influence on
Antibody levels.
0
2
4
6
81
2
34
5
39
3. Mutant strain’s plasmid DNA Vaccine
Staphylococcus aureus is an
antibiotic resistant pathogen. So there is an
alternative way to control its infection is vaccine
development for immuno therapy. The major
drawback of conventional approaches is that
large quantities of organism usually required to
isolate sufficient antigens for use in vaccine. The
novel approach in the development of new
vaccine is the use of proteins polysaccharides or
peptide fragments corresponding to specific
antigenic determinants of the infecting agents.
One of the novel and powerful method for
vaccine development is DNA vaccines
development which has several advantages. In
this work, efficacy of Mutant strain plasmid
DNA was tested. Here pathogen was
40
collected from patients sample in a hospital and
it was cultured in our laboratory. For
confirmation, all routine microbiological and
Biochemical tests were done. During the
experiment, five sets of spread plate culture were
prepared and four sets were exposed to U.V.
radiation with different time intervals such as 0,
2, 4, 6 and 8 minutes.
First set was maintained as a control.
After 24 hours, the mutant strains were
observed. Then it was isolated and put into
subculture. The mutant strains plasmid DNA
was isolated by alkaline lysis method. After
isolation, it was dissolved in double distilled
water and this was used as vaccine. In this
experiment, albino rats were used as test animal.
Fifteen albino rats were purchased and put
in five groups with separate cages and
maintained the laboratory at one week for
41
acclamentation, commercial feed were provided
into all groups . First mutant strain’s plasmid
DNA was injected (200µl) through
intramuscular injection to four sets of animals,
physiological saline was injected to control
group.
After one week same injection was
provided as booster dose. After two weeks,
formalin killed pathogens ( 510 ) was injected to
all the group of animals. After 24 hrs and 120
hrs blood samples were collected for
immunological and Haematological analysis.
Antibody titre was done only in 120 hrs samples,
with the help of 96 well titre plates.
Samples 1-5 in figures shows
pathogen exposure time 0,2,4,6 and 8 minutes
respectively
42
`
Fig-7: Mut. Stra. Plasmid DNA Vaccines
influence on RBC count (millions) of Albino
rats.
Fig-8:Mut.Str.plasmid DNA Vaccines influence
on WBC count (cells/cu mm) of Albino rats.
0
2
4
61
2
34
5 Sample-I
Sample-II
0
5000
100001
2
34
5sample-I
sample-II
43
Fig-9: Mutant Strains plasmid DNA Vaccines
influence on Lymphocytes (%) of Albino rats.
Fig-10: Mutant Strains plasmid DNA Vaccines
influence on polymorphs (%) of Albino rats.
020406080
1
2
34
5 sample-I
sample-II
0
20
40
601
2
34
5sample-I
sampe-II
44
Fig-11: Mutant Strains plasmid DNA Vaccines
influence on Eosinophils (%) counts of Albino
rats.
This experiment is aimed to develop a
DNA vaccine for the infection of
Staphylococcus aureus. They can be
manufactured more easily than vaccines
composed of a whole cell vaccine, sub cellular
fraction or recombination protein. The DNA is
very stable and resistant temperature extremes,
consequently the storage, transport and
distribution of DNA based vaccines are more
practical and less expensive.
0
10
20
301
2
34
5sample-I
sample-II
45
Fig-12: Mutant Strains plasmid DNA Vaccines
influence on packed cell volume (%) of Albino
rats.
In this work, the mutation was
induced in S. aureus by using U.V. radiation
with different time exposure hen mutant strains
plasmid DNA was isolated and used as vaccines.
After that, primary immune response was
studied. In our previous lab works, the plasmid
DNA induces maximum humoral responses
compared to other vaccines and it also gives
equal level of immunity of protein vaccines.
0
10
20
30
401
2
34
5sampe-I
sampe-II
46
Fig13: Mutant Strains plasmid DNA Vaccines
influence on Haemoglobin (gm %) of Albino
rats.
According to the results, it is clear that
the treatment of 6 minutes induce higher WBC
counts of nearly 8,100 cells/cu mm. In the
second sample, the same trend was observed. In
the case of antibody production six minute
Mutant strain also produced maximum level
compared to other, treatments. The maximum
antibody production and WBC Count was
observed in plasmid DNA vaccine treatments.
0
5
10
151
2
34
5sample-I
sample-II
47
Fig-14: Mutant Strains plasmid DNA Vaccines
influence on Antibody levels of Albino rats.
Our previous lab work proved that
plasmid DNA is good for induce immune
response during long term conditions. So in this
work, it is concluded that, 6 minutes U.V.
treated mutant pathogens plasmid DNA is act as
best vaccine.
02468
1
2
34
5
48
4. Engineered plasmid DNA vaccine
Due to antibiotic resistant problem,
pathogen control and prevention is very difficult.
But there is another way to control the infection
by development of therapeutic vaccines for
immunotherapy. DNA vaccine development is
one of the novel powerful methods. It has
several advantages. In addition, DNA vaccines
are a greater interest among researchers around
the world.
In the present study the plasmid
DNA was isolated from S. aureus and digested
by single restriction enzymes and also double
digested, all these treatments are tested in albino
rat. The best treatments are recommended for
new DNA vaccine development against
Staphylococcus aureus.
49
The bacterial pathogen Staphylococcus
aureus was collected from the patient’s samples
from local hospital and confirm through regular
biochemical and microbial tests. The plasmid
DNA was isolated by alkaline lysis method. The
plasmid DNA was digested by different
restriction enzymes and used as vaccine.
Samples 1-6 in the figures shows
various enzyme digested vaccines such as
1.control, 2.EcoR-I enzyme digested vaccine,
3.Hind-III enzyme digested vaccine 4.Pst-I
enzyme digested vaccine 5.BamH-I digested
vaccine, 6.Hae-III digested vaccine.
First treatment is undigested plasmid
DNA another five treatments were single
digested plasmid DNA which are digested by
different enzymes (Table.1). Then remaining
treatment were double digested using
50
combination of two enzymes. The digested
plasmids DNA were provided by intramuscular
Fig 15 – Various enzymes digested DNA
vaccine influences on WBC counts (cells / cu
mm) of Albino rat.
injection. After one week, same dose was given
as booster dose. Then after two weeks, blood
samples were collected for analysis.
0100020003000400050006000
1
2
3
4
5
6
51
Fig 16 – Various enzymes digested DNA
vaccine influences on RBC counts (millions) of
Albino rat.
Fig 17 – Various enzymes digested DNA
vaccine influences on polymorph (%) of Albino
rat.
0
1
2
31
2
3
4
5
6
0
20
40
601
2
3
4
5
6
52
Table 1: Various restriction enzymes and their
restriction sites
S.No Name Source Recognition
Sequence
1. EcoR-1 E. coli RY 13 5’ G
AATTC 3’
2. Bsh – I
(Hae –
III)
Bacillus sphaericus 5’GG CC 3’
3. Pst-I An E. coli strain that
carries the cloned
Pst – I gene from
Providencia stuartii
5’ CTGCAG
3’
4. Bam H–
1
Bacillus
anyloliquefacies H
5’ G
GATCC 3’
5. Hind –
III
Haemophilus
influence Rd
5’.... A
AGCTT...3’
3’....TTGA
A....5’
53
Staphylococcus aurous is a major
cause of hospital and community acquired
infection. It causes serious and fatal diseases.
Stills there are no proper vaccine for these
infections. The research is going on. The whole
cell killed vaccine is commonly used in many
diseases. The next step is preparation of mutant
strain whole cell killed vaccine. Our lab work
stated that the 6 minutes UV treated mutant
strain is best for preparing killed vaccine. If
increase the UV treatment more than 6 minutes
most of the potential of virulence factors may
decreased. Our lab studies proved that the
plasmid DNA alone acts as good vaccine for
Aeromonas hydrophila infection. It induces
maximum immune response.
The potential of virulence was
increased in mutant strain plasmid DNA
vaccine. The maximum antibody production and
54
WBC counts was observed in 6 minutes UV
treated mutant strains plasmid DNA treatment,
which is also act as best vaccine compared to
other plasmid DNA vaccines in Staphylococcus
aureus .
Fig 18 – Various enzymes digested DNA
vaccine influences on Lymphocytes (%) of
Albino rat.
Next level trail is the enzyme
digested plasmid DNA role in immune response.
The single and double digested plasmid DNA
0
20
40
60
801
2
3
4
5
6
55
used as vaccine in the case of Salmonella typhi.
The maximum response was observed in double
digested plasmid DNA treatments. So that, here
various digested plasmid DNA were used as test
vaccines. In the present work, two experimental
trails were conducted. In the first experiment
five restriction enzymes were individually used.
First plasmid was isolated and
digested by these enzymes. The digested
plasmid DNA was used as vaccine. The
maximum immune response was observed in Pst
– I digested treatment and Hae – III digested
treatments. The second maximum immune
response was observed in BamH – I digested
other treatments.
In the second experiment nine
treatments and one control treatments were
tested. All the treatments contain double
56
digested plasmid DNA with various enzyme
combinations.
Fig 19 – Various enzymes digested DNA
vaccine influences on Eosinophils (%) counts of
Albino rat.
The maximum immune response was
observed in EcoR – I + Hind III digested
treatment. The second maximum immune
response was observed in Hind – III + BamH – I
digested treatments. So compare to single
digestion, the double digestion plasmid DNA
012345
1
2
3
4
5
6
57
treatments are suitable for new DNA vaccine
preparation.
Fig 20 – Various enzymes digested DNA
vaccine influences on Haemoglobin (gm %) rat
Fig 21 – Various enzymes digested DNA
vaccine influences on packed cell volume (%) of
Albino rats.
0
5
101
2
3
4
5
6
0102030
1
2
3
4
5
6
58
Fig 22 – Various enzymes digested DNA
vaccine influences on Antibody levels of Albino
rats.
Samples 1-10 in figures shows double
digested vaccines by various Restriction
Enzymes Such as 1. Control, 2 EcoR – I + Pst –
I, 3.EcoR – I + BamH – I, 4. EcoR – I + HaeIII,
5. Hind III + Pst – I, 6. Hind III + BamH – I, 9.
Bam H – I + HaeIII, 10.Eco R – I + Hind III.
02468
101
2
3
4
5
6
59
Fig 23 – Various enzymes digested DNA
vaccine influences on WBC counts (cells / cu
mm) of Albino rats.
Fig 24 – Various enzymes digested DNA
vaccine influences on Lymphocytes (%) of
Albino rats.
0
2000
4000
60001
2
3
4
5
6
7
8
9
10
020406080
1
2
3
4
5
6
7
8
9
10
60
Fig 25 – Various enzymes digested DNA
vaccine influences on polymorph (%) of Albino
rats.
Fig26 – Various enzymes digested DNA
vaccine influences on Eosinophils (%) of Albino
rats.
0
20
40
601
2
3
4
5
6
7
8
9
10
012345
1
2
3
4
5
6
7
8
9
10
61
Fig 27 – Various enzymes digested DNA
vaccine influences on RBC counts (millions) of
Albino rat.
Fig 28 – Various enzymes digested DNA
vaccine influences on Haemoglobin (gm %) of
Albino rats.
01234
1
2
3
4
5
6
7
8
9
10
0
5
101
2
3
4
5
6
7
8
9
10
62
Fig 29 – Various enzymes digested DNA
vaccine influences on packed cell volume (%) of
Albino rats.
Fig 30 – Various enzymes digested DNA
vaccine influences on Antibody levels of Albino
rats.
0
10
20
301
2
3
4
5
6
7
8
9
10
02468
101
2
3
4
5
6
7
8
9
10
63
Naked Plasmid DNA Mixer Vaccine
Many bacterial pathogens act as
food borne pathogenic organisms. In these study
pathogens such as staphylococcus aureus,
salmonella typhi and Escherichia coli were
collected from patients samples at local hospital,
done the entire biochemical and biological test
for confirmation. Then prepared three separates
broth and individually inoculated. After 24
hours, plasmid DNA was separately isolated by
alkaline lysis method. In the first treatment, all
the plasmid DNA were mixed and used as
vaccine. In the second treatment, all the plasmid
DNA were digested by Bam-I restriction
enzymes. Then it was used as vaccine.
In the third treatment, plasmid DNA were
isolated individually and digested by Pst-I
restriction enzyme and these are mixed used as
64
vaccine. In the fourth treatment, plasmid DNA
was isolated from all the pathogen and double
digested by using Bam-I + Pst-I enzymes and
were mixed well and used as vaccine. One
control treatment was used. Albino rats were
used as test animals in all the treatment.
All the vaccines were delivered
through intramuscular injections. After
delivered the test vaccines, two weeks later,
blood samples were collected for analysis.
In this attempt maximum immune
response was observed in double digested
plasmid DNA treatment compared to other
treatments.
Sample 1-5 in figures shows various
plasmid DNA mixer vaccines such as 1.control
2.whole plasmid DNA mixer vaccine.3.BamH-I
digested mixer vaccine 4.Pst-I digested mixer
65
vaccine 5.Double digested Plasmid DNA mixer
vaccine.
Figure 31 – Various enzymes digested Plasmid
DNA mixer vaccine influences on WBC counts
(cells / cu mm) of Albino rats.
02000400060008000
1
2
34
5
020406080
1001
2
34
5poly
lym
66
Figure 32 – Various enzymes digested DNA
vaccine influences on WBC differential counts
(%) of Albino rats.
The second maximum immune response
was observed in undigested plasmid DNA
treatment. The Pst-I digested plasmid DNA
treatment gives better results than Bam-I
digested treatment.
Figure 33 – Various enzymes digested Plasmid
DNA mixer vaccine influences on RBC counts
(millions) of Albino rats.
0
2
4
61
2
34
5
67
The RBC count was more or less same
level in undigested plasmids DNA treatment and
double digested plasmid DNA treatments. The
Pst-I and BamH-I digested plasmid DNA have
similar RBC counts. Higher level of polymorph
observed in digested plasmid DNA treatments,
compare to other treatments.
Figure 34 – Various enzymes digested Plasmid
DNA mixer vaccine influences on Haemoglobin
(gm %) of Albino rats.
The antibody levels were higher in
single and double digested treatments. The
control treatment has lesser antibody levels
0
5
10
151
2
34
5
68
compared to other treatments. In this attempt,
maximum immune response was observed in
double digested treatment. So it is highly
suitable for cocktail plasmid DNA vaccine
preparation
Figure 35– Various enzymes digested Plasmid
DNA mixer vaccine influences on Antibody
levels of Albino rats.
.
02468
101
2
34
5
69
5. Heat Stress Protein Vaccine
New types of vaccine for staph infection
were reported, such as mutant strain vaccine,
Heat stress protein vaccine, plasmid DNA
vaccine, engineered plasmid DNA vaccine, etc.
In this attempt, pathogens are exposed in two
different temperatures with various time
intervals then heat stress proteins were isolated
and used as vaccine. Our main objective is
identification of suitable heat stress protein for
vaccine preparation process for staphylococcus
aureus.
Staphylococcus aureus was collected from
patients in hospital and confirmed by regular
microbiological and biochemical tests. After that
it was introduced into nutrient medium for 24
hrs, the pathogens were isolated and exposed to
two different temperatures (50 and o55 C) with
70
various time duration such as 0, 10, 20, 30 and
40 minutes. During this time they releases heat
stress proteins for their stress resistant process
which were isolated by Acetone precipitation
method and these proteins were used as
vaccines. These proteins were mixed with 0.5ml
of physiological saline and injected as
intramuscular injection to every albino rat, then
blood samples were collected and analysed by
using routine haematological tests for their
cellular immune responses.
In the present study, two experiments
were conducted. In the first experiment,
pathogens were exposed to 50oc, and then heat
stress proteins were isolated and used as vaccine.
The maximum total WBC count, polymorphic
cells, haemoglobin, packed cell volume and
RBC Counts were observed in 10 minutes
71
exposed pathogen’s heat stress protein treatment,
compared to all other treatments.
The total WBC count was increased
in 10 minutes treatment and slowly decreased in
remaining higher dose treatments. So compared
to all these treatments, 10 minutes exposed
pathogen producing cells induce maximum
responses .In the second treatments, Pathogens
were exposes to 50oc and their heat stress
proteins were isolated and used as vaccines.
Here maximum total WBC counts (7600 cells/cu
mm). There observed in 10 minutes treatments.
After 10 minutes treatments the total WBC
count was slowly decreased.
Samples 1-5 in figures shows pathogen
exposure time 0, 10, 20, 30, and 40 minutes
respectively.
72
Fig: 36-Hsp vaccine influence on Total WBC
counts (cells/cu mm) of albino rats.
.
Fig: 37-Hsp vaccine influence on Total RBC
counts (millions) of albino rat.
0
2000
4000
6000
80001
2
34
5Temp-50
Temp-55
0
1
2
3
41
2
34
5Temp-50
Temp-55
73
The maximum platelets count was also
observed in 10 minutes treatments. The total
RBC count, differential WBC count and packed
cell volume levels are more or less not much
changed. So 10 minutes treatment produced heat
stress proteins industries maximum immune
responses. Compared to both temperature
treatments, 55oC produce highest immune
response.
Fig:38-Hsp vaccine influence on polymorph
counts (%) of albino rat.
45
50
55
601
2
34
5Temp-50
Temp-55
74
Generally, microorganisms, release
proteins during stress conditions for their
survival. If rising the environmental
temperature, microbes releases some heat stress
proteins. It is isolated and used as protein
vaccine against the infection. For staphylococcus
aureus still vaccines development research for
human is going on. Many researchers proposed
various types vaccines.
Fig:39-Hsp vaccine influence on Lymphocyte
counts(%) of albino rats.
01020304050
1
2
34
5Temp-50
Temp-55
75
Fig: 40-Hsp vaccine influence on Eosinophils
counts (%) of albino rat
Fig41. Hsp vaccine influence on Haemoglobin
(gm %) of albino rats.
0
0.5
1
1.5
21
2
34
5Temp-50
Temp-55
02468
101
2
34
5Temp-50
Temp-55
76
Fig:42-Hsp vaccine influence on packed cell
volume (%) of albino rats.
Fig:43-Hsp vaccine influence on platelets
counts of albino rats.
0
10
20
301
2
34
5Temp-50
Temp-55
01234
1
2
34
5Temp-50
Temp-55
77
In this study, pathogens were
exposed to two different temperature treatments
with four exposure timings. The maximum
immune responses were observed in 10 minutes
treatments in both temperature treatments.
However compared to 050 C treatment, 055 C
temperature exposure treatment produce more
immune responses, especially 10 minutes time
exposure at 055 C temperature treatment
producing Heat stress proteins induce highest
immune responses compared to other treatments.
So it is concluded that these heat stress
proteins are highly suitable for vaccine
preparations and also useful to new bio-
adjuvant preparation for staph DNA vaccines.
78
Optimization work-I
The next step work is optimization
process. Here graded level of Hsp vaccine was
provided and immune responses were studied in
albino rats. Pathogen was collected from
patient’s samples in hospital tests were carried
out for confirmation, and then introduced in
Nutrient medium.
Toxic proteins were isolated from
broth culture medium by using Acetone
precipitation method and inactivated by 0.5%
formalin at 40 C temperatures with overnight
incubation. Graded level of these toxoid proteins
were provided through intramuscular injection to
different group of albino rats .At the end of the
experiment, blood samples were collected and
analysed for their cellular immune responses.
79
Samples 1-5 in figures show Graded
level of Toxoid protein vaccine such as 1.
Control, 2. 15µgm Toxoid Protein vaccine ,3.
30µgm Toxoid Protein vaccine, 4. 45 µgm
Toxoid Protein vaccine ,and 5. 60µgmToxoid
Protein vaccine.
Fig-44.Graded level of Toxoid protein vaccine
influence on RBC counts (millions) in Albino
rats.
Staph toxin were isolated,
inactivated and provided various quantities to
2.8
2.9
3
3.11
2
34
5
80
albino rats for tested their immune responses.
Fig-45.Graded level of Toxoid protein vaccine
influence on WBC counts (cells/cu mm) in
Albino rats
Fig-46.Graded level of Toxoid protein vaccine
influence on WBC differential counts (%) in
Albino rats.
0
2000
4000
6000
80001
2
34
5
0
20
40
601
2
34
5 LYM
POLY
EOIS
81
Fig-47.Graded level of Toxoid protein vaccine
influence on Haemoglobin (gm %) contents in
Albino rats.
If increases the level of toxoid
proteins, the immune responses was slowly
decreased. The maximum total WBC counts,
lymphocyte counts, packed cell volume and
Haemoglobin levels were observed in 15µgm
toxoid protein vaccine treatments. After that,
higher concentration of toxoid protein vaccine
leads to lesser immune responses.
0
5
10
151
2
34
5
82
Fig-48.Graded level of Toxoid protein vaccine
influence on packed cell volume (%) in Albino
rats.
The total RBC count has more or
less equal level in all the treatments. The lesser
amount platelets counts were observed in 15µgm
toxoid vaccine treatments. The immune
response of 60µgm toxoid vaccine treatment was
similar to control treatment. Protein vaccines
usually induce immune responses quickly. In
staph vaccine Heat stress protein vaccine (HSP)
was reported.
26
27
28
291
2
34
5
83
Fig-49.Graded level of Toxoid protein vaccine
influence on platelets counts of Albino rats.
. The HSP vaccine and Toxin
protein vaccine are also act as Boiadjuvents.
But there is lack of study in the optimization
process.
In this study, graded level of toxoid
protein vaccine was provided to albino rats
maximum immune response was observed in
15µgm toxoid protein vaccine. If go to higher
doses leads to low immune response that is why
00.5
11.5
22.5
1
2
34
5
84
these concentrates act as saturation point for this
vaccine. So it is concluded that this 15µgm is
recommended to further protein vaccine
development process for staph infection.
Optimization-II
In this study graded level of staph –Hsp
vaccine was provided to albino rats for
optimization study. Here the pathogen was
collected from patient sample and conforms by
routine tests then they were introduced in
Nutrient medium, 24hours later, they were
collected and exposed to 450C temperature for
10 minutes. They produced heat shock protein
for their survival.
Sample 1-5 in the figures shows
Graded level of staph Hsp vaccine such as
1.Control 2. 12.5µgm Heat Stress Protein
vaccine 3. 25µgm Heat Stress Protein vaccine 4
85
37.5 µgm Heat Stress Protein vaccine and 5.50
µgm Heat Stress Protein vaccine.
Fig-50.Graded levels of Staph-Hsp vaccine
influence on Total RBC counts (millions) in
Albino rats.
These proteins were isolated by
Acetone precipitation method, and then these
were used as vaccine. Graded levels of these
proteins were mixed with 0.5ml of physiological
saline and injected as intramuscular injection to
different group of albino rats. After one week
blood samples were collected and analysed for
study the cellular immune responses.
0
1
2
3
41
2
34
5
86
Fig- 51 .Graded levels of Staph-Hsp vaccine
influence on Total WBC counts (cells/cu mm)in
Albino rats.
Fig- 52.Graded levels of Staph-Hsp vaccine
influence on WBC differentials counts(%) in
Albino rats.
0
2000
4000
60001
2
34
5
0204060
1
2
34
5POLY
LYM
EOIS
87
Fig-53.Graded levels of Staph-Hsp vaccine
influence on Haemoglobin (gm %) content of
Albino rats.
Fig-54.Graded levels of Staph-Hsp vaccine
influence on packed cell volume (%) in Albino
rats.
0
5
10
151
2
34
5
010203040
1
2
34
5
88
Fig-55.Graded levels of Staph-Hsp vaccine
influence on platelets counts in Albino rats.
In this study, the maximum amount
of total WBC count, packed cell volume and
haemoglobin were observed in 25µgm protein
treatment. So it is concluded that these level is
suitable for further process in staph protein
vaccine development.
00.5
11.5
22.5
1
2
34
5
89
6. Mixer staph vaccine
Staphylococcus aureus spread through
contaminated food. Many vaccines were
proposed for food borne diseases, such as
Genomic DNA vaccine for common food borne
diseases, DNA vaccine for E.coli , Engineered
DNA vaccines for Typhoid , cocktail plasmid
DNA vaccine for common food borne diseases.
For staph infection many vaccines also
proposed. Heat stress protein vaccine, Mutant
strain vaccine, plasmid DNA vaccine etc., but
there is no work for comparison for various
vaccines efficacy. So the aim of this work is
comparison of four different vaccines efficacy
and finally finds out which one is induce
maximum immune response in albino rat .In this
Study, Pathogens were collected from patient’s
sample in a hospital, Regular microbiological
90
and Biochemical tests were carried out for
confirmation. Then they were introduced in
nutrient medium, it was maintained in laboratory
.After that various vaccines were prepared and
tested for their efficacy. These vaccines are
killed vaccine, Heat stress protein vaccine (HSP)
or Heat shock protein vaccine, plasmid DNA
vaccine, Toxoid vaccine and mixer of all these
four vaccines. For killed vaccine preparation,
cells were isolated from the broth and
inactivated by 0.5% formalin at 40 C in
overnight incubation.
For Heat stress protein vaccine
preparation, pathogens were exposed to 050 C
temperature for 10 minutes then heat stress
proteins were isolated by acetone precipitation
method. During plasmid DNA preparation,
whole plasmid DNA was isolated by alkaline
lysis method and used as vaccine. For mixer
91
vaccine preparation, all these vaccines
preparations were equally mixed and used as
vaccine. All these vaccines were provided at
equal quantity by intramuscular injection to
different groups of albino rats. At the end of the
experiment, blood samples were collected and
analyzed for the cellular immune response.
Samples 1-6 in figures shows
various vaccines such as 1.Toxoid vaccine 2.
Hsp vaccine 3.Killed vaccine 4. Plasmid DNA
vaccine 5.Mixer vaccine and 6. Control.
Fig-56-Various staph vaccines influence on
total RBC counts (millions) in albino rats.
01234
1
2
3
4
5
6
92
Fig-57-Various staph vaccines influence on total
WBC counts (cells /cu mm) in albino rats
Fig-58-Various staph vaccines influence on
WBC differential counts (%) in albino rats.
020004000
60008000
1
2
3
4
5
6
0
20
40
601
2
3
4
5
6
LYM
POLY
93
Fig-59-Various staph vaccines influence on
Haemoglobin (gm %) in albino rats.
Fig-60-Various staph vaccines influence on
packed cell volume (%) in albino rats
0
5
10
151
2
3
4
5
6
0
10
20
30
401
2
3
4
5
6
94
Fig-61-Various staph vaccines influence on plate
lets counts in albino rats.
In this study, various staph
vaccines were tested for their efficacy. The
maximum amount of WBC total counts,
Haemoglobin, packed cell volume and total
RBC counts were observed in killed vaccine.
The second maximum total WBC counts,
Haemoglobin, packed cell volume and total
RBC counts were observed in mixer vaccine
0
0.5
1
1.5
21
2
3
4
5
6
95
treatment. Remaining treatment has low immune
responses compared to these two treatments.
The maximum platelets counts were
observed in Heat stress protein vaccine; Plasmid
DNA vaccine and killed vaccine have same
range of platelets counts. Mixer vaccine has
third range of platelets count. In the packed cell
volume, more or less same values observed in
Toxoid vaccine and Heat stress protein vaccine.
Our previous Studies reported that various
vaccines efficacy of Aeromonas hydrophila
pathogen. They observed killed vaccine produce
maximum immune responses second maximum
immune response was observed in protein
vaccine. But they recommended plasmid DNA
vaccine, because the killed vaccine and protein
vaccines slowly lose their efficiency during long
96
duration. The DNA vaccine merged to host
DNA produce long term immunity.
In the present attempt, similar thing was
observed, but the mixer vaccine produce second
highest immune responses. The efficacy of the
mixer vaccine is better because the killed
vaccine part and protein vaccine part induce
immediate highest immune responses. The
toxoid protein vaccine and Heat stress protein
vaccines are also act as good bio-advents and
vaccine.
The plasmid DNA vaccine gives
long term immunity. So during long duration,
these mixer vaccine shows good efficacy
compared to other vaccines So the mixer vaccine
is recommended for New generation staph
vaccine development program for human use.
97
Conclusion and Recommendation
Normally killed vaccines stimulate
immune responses. But the mutant strain
killed vaccine stimulates slightly higher
immune responses.
Naked plasmid DNA act as vaccines,
similarly mutant strain naked plasmid
DNA produce slightly higher immune
responses.
The Engineered plasmid DNA also acts as
good vaccines.
Heat stress proteins are act as good
boiadjuvents and vaccines.
Especially 055 C temperature exposed
pathogens (at 10 minutes time) produced
98
Heat stress- protein gives good immune
responses.
The mixer of plasmid DNA with various
proteins such as Heat stress protein,
Toxoid protein and killed vaccines i.e.)
mixer vaccines are also act as best vaccine
against Staphylococcus aureus infections.
Because the killed vaccines and protein
vaccines stimulate immediate cellular
immune responses, the DNA vaccine has
long term viability.
So the combination of all these things is
act as good vaccine. This is also
recommended for new generation vaccine
preparations.
99
Bibliography
Moran.G.J. ,Krishanadasan.A, Gorwitz.R.J.,
Fosteim.G.E., Mc. Dougal L.K., Carey.R.B. (2006)
Methicillin- resistant S. aureus infections among
patients in the emergency department.J of medicine.
val. 355 pp. 666-74.
Kerttuls.A, Lyytikainen .O , S.Vgopio – varkilla.J.
(2004) changing epidemiology of Methicillin –
resistant Staphylococcus aureus in Finland.J . horp.
Inf. 58: 109 – 114.
Lawrence.C. , Cosseron.M., Durand.P, Costa.Y.,
Leclerig.R. (1996), consecutive isolation of
homologous strains of Methicillin-resistant and
Methicillin-susceptible Staphylococcus aureus form a
hospitalized child. J. Horp. Inf. 33 :49-53.
Liu.Y, Wang.H, Dlu.N, Sten.E, Chen.H, Niu.J (2009).
Molecular evidence for spread of two major
Methicillin-resistant staphylococcus aureus clores
with a unique geographic distribution in chinese
hospitals, Antimicrob. Agents. Chemother. 53: 512-
518.
Michael.S., Truchiya.H, Miyazaki.J, fujiwara.S,
Yamaguchi. R, Kureshiro.. (1996) Antibacterial
activity of hydroxy chaicore against Methicillin-
resistant Staphylococcus aureus. Int. J.Antimicro.b.
Agents. 6: 227-231.
100
Scribel.L.V., Silva-carvalho.M.C., Souza.R.R.,
Superti.S.V., Kvitko.C.H.C., Figuerieredo.A.M.S.
(2009). clinical and molecular epidemiology of
Methicillin-resistant staphylococcus aureus carrying
sec mec IV in a university hospital in portoalegre.
brazil. Diag. Microbiol. Inf. Dis. 65:457-461.
Razieh Amini, Abdulamir.A.S., Fatemeh Jahanshiri,
Lee Chye Shan, Alihematian, Yasaman Amini,
Zamberi Sekawi and Farid Azizi Jalilian. (2012).
Isolation and Identification of Methicillin-resistant
Staphylococcus aureus from student’s coins
Aftrican.J. Biotech. Vol.II (50). pp. 11143-11149.
Climo.M.E., Patron.R.L. and Archer.G.L. (1999)
combinations of encomycin and beta- lactams are
synergistic against staphylococci with reduced
susceptibilities to vanomy(in. J. Che mot. vol. 43 pp.
Ligi, Liu Y.K., Mak.J.Y. Chen.L.L. and Lee.W.M.
(2005) Thermal responses of rat fibre sets stably
transfected with the human 70 K De heat shock
protein encoding gene. vol. 88. pp. 1681 – 1685.
Yues Le Loir, Florence Aron and Michal Gautier
(2003). Staphylococcus aureus and food poisoning
Geret mole Res. Vol.2 No.1 .pp. 63-76.
Cunta.B.A.(2006), New uses for older antibiotics
nitrofurantion, amikacin, colistin, polymyxin.B
doxyiyilire and mino cycline 90: 1089 – 1097.
101
Muruganandam. M. (2007) New DNA vaccine for
Aeromonas hydrophila infection, Aqua Tech, Aug:79.
Robert. G. whalen (1996) DNA vaccine for Emerging
infection’a Disease: what if? E,D., 2(3): 1-15 (web
reference).
Mohanta.K.N. and S.K. Majhi (2003) on Fish
vaccination, Fishing chimes, 23(7):39-41.
Muruganandam.M.( 2010). Engineered DNA vaccine
for Typhoid. J. Nat.Con.22(1):123 126
Muruganandam M. and Verrayee Kanna, (2010.)
Mutant strain vaccine for Staphylococcus aureus
J.Curr.Sci.15 (1):229-232.
Minnesota Department of Health Fact Sheet
(MDHFS), “causes and symptoms of Staphylococcus
aureus’, Revised Feb-2010. – Web reference.
Muruganandam.M and Verrayee Kanna. K.N. (2009).
Heat stress protein vaccine development for
staphylococcus aureus. J. Nest. Con. 21(2): 331-334.
Muruganandam.M (2010), plasmid DNA vaccine for
staphylococcus aureus J.Nat. Con. 22(1): 73 – 76.
Muruganandam.M (2011). Engineered plasmid DNA
vaccine for staphylococcus aureus. Int.J.bio. tech.
2(1): 7-10.
102
Muruganandam.M. (2012) Short sequence DNA
vaccine. –Book, Pub. by St. Engere university,
Republic of Zambia.. pp: 1-118.
Marina Sara Mathew, Thripty Mary Eopenm ,Dinesh
and Muruganandam, (2008). Evaluation of various
vaccines efficacy for Aeromonas hydrophila
infection National symposium on Emerging trends in
Medical Biotechnology, Medico ventures – 08 –
organized by Dept. of Biotechnology and
Biochemical engg., Sree Buddha college of
Engineering, Alappuzha, Kerala, Held on 6th & 7
th –
Mar-2008.
Muruganandam.M. (2010) Genomic DNA vaccine for
common food borne diseases. Int. Nat. J. Bio. Tech.
1(20): 107-109.
Muruganandam.M. (2010 ) DNA vaccine for bacterial
pathogen Escherichia coli. Int. Nat. J. Bio. Tech:
1(2): 110 – 112.
Muruganandam.M (2011) cocktail plasmid DNA
vaccine for common food borne diseases. Int. Nat. J.
Bio. Tech. 2(1): 1-3
Verrayee Kanna. K.N. (2008) Sam. S- HSP vaccine
development, B.Tech project, Anna University,
Chennai.
Kenneth Todar (2008 – 2012) Todar’s online
Textbook of Bacteriology – Staphylococcus – Chapter
Madison, Wisconsin – Web-reference.
103
Muruganandam.M.(2005) vitamin C enhance stress
tolerance limit to fishes, Aqua Tech-Oct pp:69-72.
Ittoop Varghese and Muruganandam.M (2008)
protein Vaccine for Aeromonas hydrophila infection,
Int.Nat.Con.of Biotech (INCOB-2008) VIT
University, India.
Beckmann.R.P., Mizzan .L.E., Welch.W.J.,
(1990)Interaction of Hsp 70 with newly Synthesized
protein : implication for protein folding and assembly
vol.248, pp.850-854.
104
About the Author
Dr.M.Muruganandam is
working in Einsteein Bio-Engineering
Research Foundation. He is an Editor
of African journal of Biotechnology and
International journal of Medicine and
Biomedical Research. He is also
Reviewer and Editorial board member
in Various National and International
journals.