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Cytogenetic Study for the Effect of Exotoxin a in Mice Caused by Pseudomonas Aeruginosa Isolated from Mastitic Cow
Assistant professor, Faculty of veterinary medicine, Zagazig university, Egypt
Submission: September 20, 2018; Published: October 15, 2018
*Corresponding author: Amira SHH, Assistant professor, Faculty of veterinary medicine, Zagazig university, Egypt
How to cite this article: Amira S. Cytogenetic Study for the Effect of Exotoxin a in Mice Caused by Pseudomonas Aeruginosa Isolated from Mastitic Cow. Dairy and Vet Sci J. 2018; 8(1): 555726. DOI:10.19080/JDVS.2018.08.555726
Environmental Mastitis in cow commonly caused by P. aeruginosa which found everywhere in farms, water and medical equipment’s so it is zoonotic transmission. Forty-five pathogenic bacterial isolates were identified as P. aeruginosa which isolated from Eighty-two cows suffering from mastitis, five cases were diagnosed as gangrenous mastitis. twenty-five isolates were found producing exotoxin A as detected by specific ELISA kit. Isolate PA15 was found able to produce 32.08ng/ml of active exotoxin A (ETA) with a protein of about 0.063 mg/ml. Molecular weight of the toxin was determined to be 65,000 Dalton. three dose of ETA (125, 250 and 500) ng/ml were given orally for seven days to experimental animals (mice) to measure the cytogenetic effects of this toxin by employing the following parameters (mitotic index (MI), micronucleus (MN), chromosomal aberrations (CAs) and sperm abnormalities) in compression with negative control PBS (phosphate buffer saline).
Pseudomonas aeruginosa is an opportunistic pathogen that causes extensive morbidity and mortality in animals and human who are immunocompromised or have underlying infection such as urinary tract, respiratory tract and skin infections and primarily causes of nosocomial infections, and it is frequently resistant to commonly used antibiotics and disinfectants [1,2]. P. aeruginosa was found only produce exotoxin A which released normally outside the cells . Pseudomonas aeruginosa produces large numbers of extracellular toxins, which include phytotoxic factor, pigments, hydrocyanic acid, proteolytic enzymes, phospholipase, enterotoxin, exotoxin, and slime . The most important factor in the pathogenicity of P. aeruginosa is the elaboration of a group of exotoxins (protein in nature). These exotoxins could cause leukopenia, acidosis, circulatory collapse, necrosis of liver, pulmonary edema, hemorrhage, and tubular necrosis of kidneys. Passive administration of antitoxic sera against these exotoxins can protect against lethal infections with P. aeruginosa in the absence of antibody against the cellular antigens .
Bacterial isolates Samples were collected according to the method suggested by Collee . All samples were immediately streaking on Mac Conky’s agar, blood agar and cetramide agar, incubated for overnight at 37°C under aerobic conditions for 24hrs followed by determination for protein concentration according to Bradford .
The ability of exotoxin A production for the isolates was tested using ELISA kit. This kit standardized to detect even trace amount of exotoxin A in cultures. This kit also found to be specific only for the exotoxin A produced by Pseudomonas aeruginosa.
Precipitation of Protein by Ammonium Sulphate, Segel : The supernatant (crude extract) was fractionated with ammonium sulphate at (0, 10, 20, 30, 40, 50, 60, 70, 80, 90) % saturation. The precipitated product of 90% saturation was used to obtain complete precipitation of the toxin, and then the precipitant was separated by centrifugation at 10,000rpm for 30 min. The precipitant was resuspended in 10ml normal saline.
Preparation of ion exchange column (DEAE-cellulose) gel: The DEAE-Cellulose was prepared according to the method suggested by Whitaker & Bernhard . 20gram from ion exchange resin were suspended in 1 liter distilled water, left in graduated
cylinder to stagnate, after that the supernatant was removed,
this step was repeated many times, until the supernatant become
clear, the ion exchange resin was filtered by using Bukhner’s funnel
under vacuum (without drying the ion exchange resin), then
the resin was activated in 250ml from buffer which contain 0.25M
sodium hydroxide and 0.25M sodium chloride for 30 minutes,
the resin was re filtered and washed under vacuum using distilled
water, then the resin was suspended in 250ml hydrochloride
acid 0.25M with agitation for 30 minutes, after that, the resin
was washed with distilled water under vacuum, the resin was
suspended in Tris-HCl buffer (1μM, pH=8.0) and the ion exchange
resin was degassed by using vacuum, the resin was packaged gently
in glass column (2.5×16cm), the equilibration was achieved by
the same Tris-HCl buffer.
Separation through ion exchange resin (DEAE-Cellulose):
Ten ml solution was loaded on ion exchange column, the separated
fractions were collected at flow rate 20ml/hour (approximately,
3ml for each fraction), the wash was obtained of by using Tris-
HCl buffer (the same buffer used in equilibration), the elution was
achieved by the same buffer with gradual increase in concentration
of sodium chloride, the flow rate was 20ml/ hour too, the protein
concentration of the fractions was measured at wavelength
280nm to the washed and eluted fractions, protein concentration
then was calculated.
Determination of the void volume of the column: Sepharose-
4B column (56×1.5 cm) was prepared and packed according
to the instructions of the manufacturing company (Pharmacia
Sweden). The column was equilibrated overnight with 0.02M Tris-
HCl buffer pH 8.0 with a flow rate of 50ml/hour. A 2ml blue dextran
2000 solution was passed through the column, and 225ml of
Tris-HCl buffer pH 8.0 was added to the column. Fractions of 5ml
were collected. The absorbency at 600nm for each fraction was
measured. The column void volume (Vo) was determined, by estimation
of total volume of fractions as characterized with start
point movement of the blue dextran to that of climax of absorbency
of the blue dextran.
Determination of ETA Elution volume (Ve): Sepharose-4B
column (56 × 1.5 cm) was prepared, packed and equilibrated for
a second time. A 3 ml of purified exotoxin A sample was passed
through the column carefully and equilibrated with 0.02 M Tris
HCl buffer pH 8.0, with a flow rate of 50ml/hour. Fractions of 5ml
were collected. The elution volume (Ve) was estimated for the separated
fractions of purified exotoxin, by following the absorbency
Measurement of Standard Protein Elution Vole (Ve): Different
standard proteins were applied through sepharose-4B column,
and then eluted with 0.02 M Tris-HCl buffer pH 8.0, with a
flow rate of 50 ml/hour, as shown in (Table 1). The elution volume
was estimated for each standard protein by following the absorbency
for the separated fractions at wave length 280 nm. The (ve/
vo) ratio was calculated for each standard protein and for the separated
fractions of purified ETA, then standardization was done,
by plotting the elution volume (Ve) of each standard protein to the
void volume (Vo) of the blue dextran 2000 (Ve/Vo) versus the log
value of molecular weight [11,12]. The ETA molecular weight was
Albino Swiss (20Females&20 males), which were obtained
from the veterinary laboratory Center / Stanford University, were
used. For cytogenetic study of the effect of exotoxin A. Their ages
were ranged between (8-12) weeks and weighting (25-30) gm.
They were divided into subgroups, and each group was put in a
separate plastic cage. The cages were kept in a room temperature
(23-25)°C. The animals were fed with a suitable quantity of water
and complete diet.
Animals in this experiment were treated with a cumulative
dose of ETA for seven. The main aim of this experiment was to
evaluate the acute treatment effect of ETA in normal mice. The
LD50 of P. aeruginosa exotoxin A is 2.3 μg . Four groups of mice
were used in this experiment (half of them used to determine MI,
CAs and the other half for MN and sperm abnormalities) treated
a. Group I: Negative control (10 mice), treated with (0.1ml) of PBS.
b. Group II: ETA treatment (10 mice), treated with (0.1 ml)
of ETA (125ng/ml).
c. Group III: ETA treatment (10 mice), treated with (0.1ml)
of ETA (250ng/ml).
d. Group IV: ETA treatment (10 mice), treated with (0.1ml)
of ETA (500ng/ml).
Chromosomal preparation from somatic cells of the mouse
bone marrow: This experiment was done according to  as
a. Each animal was injected with 0.25ml of colchicine with
a concentration of (1mg/ml) intra peritoneal (I.P) 2hr before
sacrificing the animal.
b. The animal was sacrificed by cervical dislocation.
c. Then the animal was fixed on its ventral side on the
anatomy plate and the abdominal side of the animal and its
thigh region were swabbed with 70% ethanol.
d. The femur bone was taken and cleaned from the other
tissues and muscles, then gabbed from the middle with
forceps in a vertical position over the edge of the test tube,
and by sterile syringe 5ml of PBS were injected to wash and
drop the bone marrow in the test tube.
e. The test tube was taken and centrifuged at speed of
2000rpm for 10min.
f. The supernatant was removed and 5ml of potassium
chloride (0.085) M was added as a hypotonic solution, then
the test tubes were left for 30min in the water bath at 37°C
and shacked from time to time.
g. The tubes were centrifuged at 2000rpm for 10min.
h. The supernatant was removed, and the fixative solution
was added (as drops) on the inside wall of the test tube with
the continuous shaking, the volume was fixed to 5ml and the
content shacked well. I. The tube was kept at 4°C for 30min to
fix the cells.
i. The tubes were centrifuged at 2000rpm for 10min.
The process was repeated three times and the cells were
suspended in 2ml of the fixative solution. By a pasture pipette,
few drops from the tube were dropped vertically on the chilled
slides from a height of 3 feet at a rate of (4-5) drops to give the
chance for the chromosomes to spread well. Later the slides
were kept drying at room temperature.
j. The slides were stained with Giemsa stain and left for
15min, then washed with distilled water.
k. Two slides per each animal were prepared for cytogenetic
Micronucleus test in mouse bone marrow cells: This assay
was adapted from that described by .
a. The femur bone was cleaned from tissue and muscles,
then gapped from the middle with forceps in a vertical
position over the edge of a test tube, and by a sterile syringe
1ml of human serum was injected to wash and drop the bone
marrow in the test tube.
b. The test tube was centrifuged at 1000rpm for 5min.
c. The supernatant was removed, and a drop from the
pellet was taken to make a smear on clean slides. The slides
were kept at room temperature for 24hrs.
d. The slides were fixed with absolute methanol for 5min,
then stained with Giemsa stain for 15min, then washed with
distilled water and left to dry.
e. Two slides for each animal were prepared for
Mitotic Index (MI) Assay: The slides were examined under
the high dry power (40X) of the compound light microscope and
(1000) of divided and non-divided cells were counted and the
percentage rate was calculated for only the divided ones according
to the following equation : MI= number of divided cells/total
number of cells (1000) X 100.
Chromosomal Aberrations (CAs) Assay: The prepared slides
were examined under the oil immersion lens for 100 divided cells
per each animal, and the cells should be at the metaphase stage of
the mitotic division, where the chromosomal aberrations are clear,
and the percentage of these aberrations was estimated .
Micronucleus (MN) Test: The number of MN in (1000) cells
of polychromatic erythrocytes (PCE) in mice was scored under
the oil immersion lens, and the percentage of MN was calculated
Sperm Abnormality: This assay was adapted from that
described by Wyrobek and Bruce. Animals were sacrificed after
37 days after injection. The epididymides extracted and the sperm
were sampled. Both epididmides from each mouse were minced
with small scissors in 4 ml PBS (Phosphate Buffer Saline), and
then left at least 2 min for the spermatozoa to diffuse into the
saline. Pipetted and then filter in test tube to exclude large tissue.
More than one slide was prepared for each animal by placing a
drop of suspension on cleaned microscopic slide and smearing
with a clean cover slip. Then the slides were air dried and the fixed
in absolute alcohol for 1 min before staining in Eosin Y for 15 min.
For scoring, in each animal about 1000 sperm were examined
for morphological abnormality. Mammary gland and nibbles of
female mice: preparation smear same as sperm in vital cells all
cells became inflamed, bucket filled with granulation tissue and
most cell dead with gangrenous
A one-way analysis of variance was performed to test whether
group variance was significant or not. Data were expressed as
mean ± standard deviation and statistical significances were
calculated using ANOVA test .
ELISA kit was used for detection of exotoxin A produced by
isolates of Pseudomonas aeruginosa. Average the duplicated
reading for each standard, control, and sample and subtract the
average zero standard optical density. Create a standard curve by
reducing the data using computer software capable of generating
a four-parameter logistic (4-PL) curve-fit. As an alternative,
construct a standard curve by plotting the mean absorbance for
each standard on the x-axis against the concentration on the y-axis
draw a best fit curve through the points on the graph. The data
may be linearized by plotting the log of the PEA concentrations versus the log of the O.D. and the best fit line can be determined
by regression analysis. This procedure will produce an adequate
but less precise fit of the data. If samples have been diluted, the
concentration read from the standard curve must be multiplied by
the dilution factor. Forty-five isolates only twenty-five were found
to produce exotoxin A. The kit was also used for concentration
measurement as the results indicated in (Table 2). Screening of
these isolates indicates that, most of the isolates show positive
results. However, three isolates were selected according to their
highest productivity as well as their distribution in different sites
if infections, three isolates namely PA 10, PA 11 and PA 15 which
produce (23.31) (19.01) (29.73) ng/ml and distributed in burn,
UTI infection and wound respectively.
These results were nearly agreed with Warren who found
that registered concentration between 0.3 and 0.6 ng/ml at least
concentration, and Michael  found the maximum concentration
detected by using ELISA approximately 0.75 μg/ml.
From previous results, it was shown that, the isolate PA15
capable of producing exotoxin A at concentration of 29.37ng/
ml which considered as basal synthesis. To improve synthesis,
TSBD medium was amended with nitrilotriacetic acid (NTA) and
then fallow productivity. Results shown in (Table 3) indicate that
protein concentration was found to be higher in cultures contain
NTA than that of free cultures, this may be due to the ability of NTA
for inhibiting protease activity and hence stop protein turnover as
reported by several research articles Matthew .
Number of purification steps was followed to obtain pure
exotoxin A. These steps were reduced to minimum if the quantity
of toxin is low and could be lost during purification. These steps
Precipitation of proteins by ammonium sulphate: To concentrate
the crude extract of toxin and remove as much as possible
water, ammonium sulphate was used at (10, 20, 30, 40, 50,
60, 70, 80, 90) % saturation, the saturation ratio of 90% was chosen
to precipitate exotoxin A. This step allows the salting out of
molecules from water. Since ammonium sulphate can neutralize
charges at the surface of the protein and to disrupt water layer
surrounding the protein, it will eventually cause a decrease in the
solubility of protein which, in turn lead to the precipitation of the
protein by the effect of salt [18,19]. Ammonium sulphate is widely
used because of availability of ammonium sulphate, high solubility,
low cost and it stabilizes the protein .
Partial purified of exotoxin A by ion-exchange chromatography:
Purification of exotoxin A was done by ion-exchange chromatography
using (DEAE-cellulose). (Figure 1) showed the wash
and elution of DEAE-cellulose column for three isolates. No exotoxin
A was detected in the wash steps, while the eluted fractions
revealed. Results indicate the presence of three peaks. However,
only one peak for each elution of three isolates shows activity as
detected by ELISA kit. The amounts of partial purified proteins
shown in (Table 4), indicate as much as 0.063, 0.044 and 0.051
mg/ml of protein produced by isolates PA15, PA11 and PA10 respectively.
Detection of exotoxin in fractions eluted from ion-exchange
chromatography was done using ELISA kit, the results shown in
(Table 5) indicate the presence of only one peak in the elution
steps of the three isolates giving positive result, as measured
spectrometric ally at 450 nm wavelength. The results indicate
that, isolate PA.15 is the highest producer for exotoxin A.
The sample passed through Sepharose-4B then fractionated on
the gel, fractions were collected up to 30 fractions. Exotoxin A was
present in fractions 21-23 depending on using acetic acid assay
and increase absorbency of fractions. The result of purification
showed the presence of one-peak as indicated in (Figure 2).
Molecular weight of exotoxin A was determined using
Sepharose4B column (56×1.5 cm). The void volume (Vo) of the
column was calculated by estimating the void volume of blue
dextran 2000 to the elution volume (Ve) for each one of standard
proteins and for the separated fractions of purified exotoxin. The
ratio of the elution volume of each standard protein as well as the
separated fractions of the purified exotoxin, to that of void volume
of the blue dextran 2000 was calculated. Results in (Table 6), show
that the (Ve/Vo) ratio of purified exotoxin was about (65000 D),
the ratio of (Ve/Vo) of each standard protein to the log molecular
weight of each standard protein was plotted. The ratio of (Ve/
Vo) of each standard protein to the log molecular weight of each
standard protein was plotted.
Results shown in (Figure 3) indicate that molecular weight
of purified exotoxin was estimated as 65000 Dalton, which near
molecular weight of bovine serum albumin as about 67000
Dalton. These results agree with the results described by Hamood
, who determine the molecular weight of exotoxin A as about
68000 Da. While Jilani determined molecular weight of exotoxin A
as about 66,583 Da.
Cytogenetic effect of ETA on mouse bone marrow cells:
188.8.131.52 ETA effect on mitotic index (MI). The treatment effect
of three doses of ETA of isolate (PA15) in addition to PBS (as a
negative control) on mitotic index of mouse bone marrow cells was
examined. Results shown in (Table 7) indicate that a significant
decrease in MI (P<0.005) for the all three doses of ETA in the order
(35.05%, 26.86% and 19.82%) after seven days of treatment at
doses of (125, 250 and 500 μl/kg) respectively as compared with
negative control (43.08%).
Attributed to MN minimization of MI occurred because of many
factors, mainly could be reduction in protein concentration that is
important for mitosis or death of bone marrow cells if ETA toxin
inhibit protein synthesis in cell. Or could be because on synthesis
of the mitotic spindles which is organized during cell division
 or during mitosis, these spindles composed of number of
microtubules which is responsible on capture and alignment
of chromosomes in metaphase and subsequent separation to
the two daughter cells at anaphase. Although the mechanism
of mitotic arrest by microtubule-targeted antimitotic agents is
poorly understood, it is believed to be a result of suppression of
microtubule dynamics, which prevents chromosome alignment at
the metaphase plate, resulting in a sustained block at or before
the metaphase-anaphase transition [16,22]. Many bacterial toxins
were shown to cause reduction in MI such as C. botulinum C2
toxin ADP-ribosylates that inhibit MI, Cells treated with C2 toxin
did not recover and did not start to proliferate again . At the
same time, MI was shown to be increase after treatment with
other kinds of agents, such as antioxidants (e.g., vitamin C which
is the active constituent of many plants), they act by inducing cell
division by acting as mitogens [24,25]. P. aeruginosa ETA was not
shown to have antioxidant activity, but in fact, it was shown to act
as many as bacterial toxin in decreasing MI, but the mechanism
Results shown in (Table 7) indicate the effect of three doses
of ETA (125, 250 and 500 μl/kg) on MN reduction. A percentage
of (1.74%, 1.92% and 2.46%) obtained for the three doses
respectively as compared with PBS as a negative control (0.54%),
and (Table 7) indicate the micronucleate cell in bone marrow
of mice per cell after treatment with three doses of ETA. These
indicate a significant increase in MN percentage was recorded
for the three doses. Micronuclei may be originated from acentric
chromosome fragments, either from the double stranded DNA
damage before cell division or after breakage of anaphase bridges
, as shown in (Figure 3).
Although, there are agents that cause increased MN frequency,
at the same time several plant extracts were also found cause
decreased MN frequency. Several plants were shown to contain
active constituents such as rutin and quercetin which protect DNA
from damage by their antimutagenic and detoxification activities
[27,28]. Some chemicals could increase MN frequency such
fluoride due to its mutagenic effects and could give rise to DNA
The examination of chromosomal aberrations is important
for studying the effect of ETA in mouse. In this study, types of
chromosomal aberrations which have been observed were;
ring, gap, acentric, dicentric, chromosome break, chromatid
break and deletion. Three different doses of ETA were found
cause significant differences (P<0.05) in total and in all types of
CAs frequency in comparison to the negative control. Results of
(Table 5) revealed that treatment with different doses of ETA had
increased spontaneous CAs percentage (0.615, 0.926 and 1.318%)
at doses of (124, 250 and 500μg/Kg) respectively as compared
with negative control. As shown in (Figure 3).
Although Gopal et al. (1999), who suggested that endo/
exotoxin released by Mycobacterium tuberculosis act as chemical
mutagens in generating CA in affected individual. Other similar
infections caused by Mycobacterium Lepra  and number of
viruses, including A and B hepatitis  were demonstrated to
cause different CAs in human lymphocytes. These abnormalities
may involve the autosomes, sex chromosomes, or both. The
disruption of the DNA sequence could alter the genes carried on
the affected chromosomes results in a mutation. Such a change
may alter the protein coded by a gene. Often, however, a mutation
could also result in disruption of gene functionality, which result
in altered or missing proteins for metabolism which cause genetic
diseases. Only mutations occurring to the DNA in the gametes
will potentially pass on to the offspring. An elevated frequency
of structural chromosome aberrations could be directly caused
by an abnormally high incidence of DNA double-strand breaks.
Chromosomal breakage can result in several different structural
rearrangements, some of which give rise to abnormalities of
chromosomal segregation at mitosis. For example, terminal
deletions due to a break of a single chromatid will result in a
centric derivate chromosome plus an acentric fragment. Because
of its failure to bind the mitotic spindle, the fragment may be
permanently lost in the subsequent cell division and may be
seen body at metaphase or anaphase . However, although P.
aeruginosa ETA was shown to cause different CAs, but also the
mechanism still unknown.
It is known that the abnormality that occur in germ cells
transfer by the movement to subsequent generations and
thus constitute one of the most important genetic risk due to
carcinogenic effect while the distortions that occur in somatic
cells may cause a risk to the individual himself. The results in
(Table 3) indicating that, the abnormality in the sperm was
increase as the dose of ETA increase and this incensement is
significant when compared with negative control after seven days
of mice treatment. Sperm abnormality includes: amorphous head,
banana head, two head, head without hook, divided tail, coiled tail
and other abnormalities that shown in (Figure 3). It was noted
that the abnormality in divided tail more than other abnormality
because of toxin effect on protein concentration in tail which made
of protein in most contents. Another bacteria product decrease
sperm head abnormality like Lactobacillus acidophilus . Also,
some chemicals caused decreases in sperm head abnormality like
dimethoxyethyl phthalate, epichlorohydrin and formaldehyde
Pseudomonas aeruginosa was found able to produce
significant amount of exotoxin A in growth medium. Productivity
of exotoxin A was enhanced by using NTA in growth medium and
reduced in isolates producing proteases. Inhibitors were affecting
in protecting the toxin from degradation. The molecular weight
of purified ETA was found to be 65,000 Dalton. Numbers of
cytogenetic effects in mice caused by ETA were detected depending on changes such as (MI, MN, CAs and sperm abnormalities). My
Recommendations Further research is needed concerning the use
of this toxin for vaccination against P. aeruginosa. Genetic studies
include localization of structural and regulatory genes related to
exotoxin A synthesis. Genetic engineering used to more production
of exotoxin A of P. aeruginosa by cloning.