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European Journal of Molecular & Clinical Medicine
ISSN 2515-8260 Volume 7, Issue 09, 2020 143
143
A PHARMACOLOGICAL COMPARISON
BETWEEN the PROBIOTIC PREPARED by
the LACTOBACILLUS ACIDOPHILUS
BACTERIA and NITAZOXANID in
ANIMALS INFECTED WITH
CRYPTOSPORIDIOSIS
Dr. Maysoon Mustapha Jasim#1Rsha shamil Hussin2& Mohammed Ahmed Mustafa 3,3
#1,2College of Education for Pure Science, University of Tikrit, Iraq.
3Department of pathological Analysis , College of Applied Sciences University of Samarra
3Department of Medical Laboratory Techniques , College of Technology , University of
Imam Ja far Al-Sadiq _ Dujail
#1mays.mus@gmail.com
2mohammed.alsad3@gmail.com
Abstract:
The study was conducted for the period from April 2016 to March 2017. The study of
lactobacilli, which was prescribed by acidophillus Lactobacilli (Probiotics), was
included in the treatment of the parasite Cryptosporidium parvam compared to
nitazoxanid, based on several criteria including therapeutic efficacy and some blood
criteria. The number of egg cyst in the first day of the treatment reached 6640 syst /
eggs with a therapeutic efficiency of 22.9%. In the first and second concentrations, the
ratio reached 7180, 6460 and 5560 with a therapeutic efficiency of 16.7,25.0 and 35. The
number of egg cyst continued to decline to the fifth day. The number of eggs cyst was 40
cysts / eggs and 99.4% The number of red blood cells in total infection compared to
control was 5.782 × 109 / L, while there was no significant difference in control. The
results showed no significant differences in hemoglobin ratios and the volume of red
blood cells between total negative control, total probiotic treatment group, and groups
treated with drugs in concentrations II and III, and in the number of white blood cells,
granulocyte and monocyte were no differences Between the probiotic group and the
negative control group, while all the remaining groups decreased.
Key words: Cryptosporidium parvam, bioenergy. Probiotic.
INTRODUCTION
Cryptosporidiosis is the most common human pathogen(Checkley et al ,2015)This disease is
the second most common cause of rotavirus in global causes of death among children under 2
years of age, causing mild to severe diarrhea (Shirley et al,2012) and causing 5-10 million
Case of death annually (Nemes,2009). Although the spawning parasite was discovered in
1907 by the Tyzer, it was discovered only as a cause of human infection in 1976 and in 2004
it was added to the World Health Organization's "Neglected Diseases Initiative", which
includes diseases that affect To people mainly in developing countries (Speich et al,2016)
(AL-Samarraie, et al,2019) .
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144
The parasite passes through a complex life cycle and the human or animal can put millions of
eggs in the stool. Although egg cysts are unable to reproduce outside the host, they can
survive in the environment for several months and resist disinfection, including chlorine.
(Ehsan et al,2015) also The transmission and spread of infection is particularly associated
with people who have frequent contact with animals and the use of manure and human stool
as fertilizers (Ya Yang et al,2017). The egg cyst contains infective oocyst. Four sporozoites
are introduced with feces to spread the infection and when eaten by the host, the egg sac
releases 4 of the spores that bind to the superficial epithelial cells of the stomach, small
intestine and colon. (Bowman et al,2003)
It has been scientifically proven that the continued and excessive use of antibiotics leads to
the appearance of bacterial resistant. Therefore, alternative methods of antibiotics, which are
used as therapeutic, prophylactic, or growth stimuli, have been directed to patients who want
to use natural and safe methods of treatment (Maria et al,2015) . In recent years, there has
been a wide trend towards the use of types of lactic acid bacteria isolated from human and
animal sources as probiotics, and the emergence of a new generation of these
biopharmaceuticals, either in known pharmaceutical forms or in the form of therapeutic food
products in many countries in Europe, North America and the Far East. Its use in solving
many problems related to food and consumer health. (Singhi & Kumar,2016) Hence the
objectives of this study, which include:
Isolation and diagnosis of parasite Cryptosporidum Parvum of calves with diarrhea.
Isolate and diagnose Lactobacillus acidophilus from milk and then prepare the lactic acid
from this bacteria as a bio-booster (Probiotics).
Study of the therapeutic effect of L.acidophilus on C.parvam parasite compared to
nitazoxanid and some blood parameters.
THE METHODS
Preparation of solutions, delays and dyes.
Carbol dye Fuxin alkaloid prepration according to (Baxby,1984), green malachite dye,
saturated sucrose solution, according to (John & Petri ,2006) and PBS solution as describe by
(Myers,1995), McFarland solution according to (Baron,1994) Central sugar fermentation as
(Harrigan & McCance,1979) center of MRS-CaCO3 (Henriksen & Pohlenz, 1981)
The parasite sample
Cryptosporidium parvam samples were collected from the dung of calves infected with
diarrhea (Samarra village and AL-Alam city) on 2016/4/1. The parasite was diagnosed
according to the method (Anderson , 1981), as well as by the flotation method of the diabetic
solution. (Arrowood & sterling , 1987) the isolation of parasite and Eggs collaction don as
(Savage,1984) The injection dose was determined by calculating the number of sacs and
determining the injection dose by calculating the number of sacs in the amount of 0.5 ml and
the dose was determined by 40000 sacs for each rabbit to be injected orally. The parasite bags
were investigated in infected rabbit stools daily after the mixture to ensure the occurrence of
parasitic infection. The preparation of several swabs of infected rabbit stools on a glass slide
and examined under a microscope and watch cysts and calculated using the method of
flotation with the diabetic solution.
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Sample of bacteria
Lactobacillus acidophilus bacteria were isolated and implanted into the MRS medium.
According to method of (Savage,1984). The morphological characteristics of the colonies
were studied, the cultural and the biochemical tests of bacteria were carried out to ascertain
the traits that would enable them to be used as a probiotics.
Cultural characteristics
The striking method was used to vaccinate bacterial models on selective media and incubated
dishes at 37 ° C for 18-24 hours.
Catalytic examination
The isolates were grown in the center of the fluid MRS media and then transferred to 1 ml of
sterile test tube and 1 ml of H2O2hydrogen peroxide was added (10% H / H) and the
emergence of bubbles was observed.
Examination of ammonia composition of arginine
0.3% of Argenine-monohydrochloride was added to the MRS liquid medium and sterilized at
121 ° C for 20 minutes. Then healed the isolates and incubated for 7 days at 37 ° C. then took
1 mL of the fertilized medium and placed in a test tube and added 1 ml of the NSLER
detector.
Carbohydrate fermentation test
Sugars fermentation test solated and developed after the replacement of the carbon source
from the sugar fermentation medium in one of the sugars: Fructose, Glucose, Galactose
Raffinose, Ribose, Lactose, Maltose and Saccharose by 1% The addition of sugars after the
sterilization of the primary mean of the balance as the sterilization of sugars using sheets
Millipor filtration size 0.22 mm and incubated isolates cultivated for 24-48 hours.
Determination of positive fermentation through color change to yellow, after isolating the
isolates, was indicated for identification during use in subsequent tests.
Testability of adhesion to Lactobacillus spores
This test was conducted according to (Fuller, 1975) as follows:
Part of the intestines were taken. In the current study, laboratory rabbits were used under
sterile conditions. The intestines were opened and washed twice by 100 mL of (PBS) solution,
pH 7.2. The intestinal lining was cut by a clean, sterile glass slide and suspended with 10 mL
of phosphate solution .
Preparation of 10 ml of Lactobacillu sacidophilus bacteria on the liquid MRS medium. The
germ cells were then mediated by the centrifuge device at 2500 cycles / min for 10 minutes.
The liquid was separated from the germ cell deposit and completed with the same phosphate
solution (PBS).
Take 0.4 ml of the epithelial cell suspension and add 0.1 mL of bacterial suspension and then
put in a water bath for 30 min. At 35 ° C and at 20 ° C. After incubation, take drops and place
on a glass slide and dye using a Gram and Gemsa stain. To determine adhesion, germ cells
take violet while the epithelial cells appear red.
After the tests were carried out, the bacteria were counted by using the McFarland tube
method as indicated in (Baron,1994). The bacteria isolated in the center (MRS) were frozen
as indicated in (Contreras,1997) and then the lactic milk was prepared from the isolated
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146
bacteria. (Jandal,2007) Lactate by means of a glaucoma in the mouth directly by a special
syringe prepared for this purpose by two milliliters twice a day.
Laboratory animals
In this study, adult male New Zealand white rabbits obtained from the Erbil Research Center
were aged between 10-18 months and weighed between 1000-18,00 kg. The animals were
placed in iron cages and the ground was sprayed with sawdust, Taking into account the daily
cleanliness of cages and the exchange of sawdust, as well as sterilization of cages disinfectant
every two days, as well as the animals fed ready-made in addition to providing water
throughout the study.
Experience Design:
Forty-four laboratory animals were used for the treatment trials, randomly divided into 6
groups, 7 animals per group, the first group left untreated, the remaining five groups were
injected with cryptosporidium parvum parasites 4 × 10 3cells / ml and monitored for 7 days
until the state of diarrhea, and the division was as follows:
Control group (C-ve), This group was left untreated throughout the experiment.
Infection group (C + ve), Infection group This group was used to compare with the other
groups in terms of infection development and was injected orally from the parasite
Cryptosporidium prvum, 0.5 mL and 4 × 103cells / mL as a dose of experimental infection.
After the infection was confirmed, the infected rabbits were treated with nitazoxanid (3, 4 and
5 groups ) with 0.5, 1, 1.5 mg / 100 g respectively of animal weight. They were administered
every 24 hours for five consecutive days.
Group 6: Animals were treated with a probiotic. The animal was given 2 ml orally from the
dose prepared in paragraph (3-11). Each ml contains 2 × 105bacteria, every 12 hours for a
week, taking into consideration the stool daily Calculate the number of sacs and calculate the
therapeutic efficiency. And then monitor and analyze the stool for a period of 5 days to
follow up the occurrence of the disease or not. The animals were monitored and monitored
during the treatment period by examining or previewing the changes in the general state of
the animal, the nature of the appetite, the nature of the stool, the temperature, and comparing
it with the normal rates referred to by (Mohammed, 2000).and calculate the number of eggs
cysts offered every 24 hours.
Number of eggs cysts (gm) number of eggs in tow roomsthe weight of the stool sample ×200
Since the weight of the model is 0.2 g, the number of egg bags x 100, the method of
(Ghazal ,1974). After the experiment was over, the animals were anesthetized by chloroform,
and blood samples were withdrawn from the heart directly in a cardiac catheterization
procedure.
MATERIALS AND METHODS
The animals were monitored during the treatment period by examining or previewing the
changes in the general state of the animal, the nature of the appetite the nature of the stool,
the temperature, and comparing it with the normal rates referred to in (Mohammed, 2000).
The rabbits' stools were examined daily in a manner that was used to determine whether or
not the injury occurred.
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The therapeutic efficiency was calculated according to (Xiao et al,1996) according to the
following equation:
Therapeutic efficiency=
The average number of egg bags in control group -The average number of egg bagsthe
everagr of the eggs cyst number in the control group
After the experiment was over, the animals were anesthetized by chloroform, and blood
samples were withdrawn directly from the heart in a cardiac punch. Approximately 8-10 ml
of blood was withdrawn and 1 ml of blood was placed in a container containing anticoagulant
for blood testing (Complete blood film) by a self-blood analysis device.
RESULTS AND DISCUSSION
Diagnosis of parasite
The parasite was diagnosed with the Zell-Nelsen mutant, where the spherical egg sacs
appeared red and shiny distinct from the green background of granular cytoplasm and
contained 4-6 spores.
Diagnosis of bacteria
Lactobacilli colonies are circular convex in green because of the presence of Bromo Cresol
Green stain in the contents of the center axis and regular edges(Tietz, 2005). Lactobacillus is
one of the most important microbiology used in Probiotic, effectively contributing to the ideal
balance of microorganisms that form intestinal flora, which binds to the epithelial layer lining
the digestive tract, and thus colonizes the gastrointestinal tract(Al-saadi,2010) L.acidophillus
was a negative result of this test and is consistent with the study (Miles & Bootwala,2006).
Lactobacillus is negative for this test due to its inability to produce Peroxidase, which
analyzes the H2O2peroxide of H2O and O2, which appears in the form of bubbles giving a
positive result.
The test of ammonia production of arginine was not given a positive result when
Lactobacillus was exposed. On the analysis of arginine our results were identical to the
results of(Nizamuddin & ahaa,2002). The carbohydrate test gave all the sugars used as a
positive result except for the of the raffinoz and the ribose sugar gave a negative result.
Isolation showed high susceptibility to adhesion through a large increase in the number of
bacteria attached to epithelial cells. The compound responsible for the adhesion of L.
acidophilus bacteria is the S S-layer protein interferon.
The wall of the germ cell, which is shared with the adhesion of the receptors located on the
walls of the epithelial cell, which consists mainly of manganese sugar ) Nawaf, 2005)
The presence of this protein layer and its containment of glycoproteins, which consist of 0.9 -
1.4% carbohydrates and 3% water - resistant amino acids, play a key role in adhesion
(Bernet,2009) ( Fadhil et al .,2019).
Experimental Study
Clinical signs
The experimental infection in the animal groups (infection group) was shown to begin with
the appearance of the clinical signs observed through changes in the animal's appetite,
general condition and the nature of the stool. The results of the clinical signs were very clear
when the infection group was given a dose of carbotosporidium parasite. Her movement did
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148
not show a distinctive clinical marker in the precursors of the bio-booster as well as the
animals in the control groups.
The use of biopharmaceuticals in feed processing increased the appetite of Prohibited animals.
(Abas et al,2007) An increase in rabbit appetite was also observed in acid-resistant
lactobacillus bacteria.
The results of the examination the nature of the face's with the naked eye of the infection
group in the first days showed that it was changed from the solid to the softer, and then when
the infection developed, the stool appeared to be a liquid evidence of severe diarrhea,
indicating the severity of the parasite. In one animal noted that her health had deteriorated,
and die.
The
measuring of the temperature of experimental animals, a significant increase was observed
since the third day of the experiment in the infection group at 39.4 ° C compared with the
first day in which all groups with temperatures between 36 - 37.5 ° C, and continued rise in
the animals of the infection group. And slow movement of animals in the infection group and
this is a satisfactory sign (Muhammad,2008).
Results of the study of the effect of nitazaxonide with different concentrations and the
probiotic on the rate of deposition of cysts of parasite eggs in the rabbits.
This study shows that the probiotic had the greatest effect on the therapeutic efficiency rate.
The rate of cysts began to decrease gradually from the first day of treatment, reaching 6640
cysts of eggs and decreased in the second day to 1740 ,and continued to decrease to the fifth
day of treatment, reaching 40 cysts of eggs with a therapeutic efficacy rate of 99.4% as
shown in Table (1). The efficiency of the probiotic in treatment is attributed to the prevention
of adhesion of parasite cyst to bacterial susceptibility to high density adhesion on the surfaces
of epithelial cells. In addition, they produce a substance as a type of Biosurfacton called
Sarlactin, which is a product of microorganisms that separates two bodies. To prevent the
adhesion of most pathogens to host cells (Velra,1998). It was noted that the efficacy of L.
acidophilus in the treatment of diarrhea was due to the prevention of adhesion of diarrhea-
causing intestinal pathogens. (Vandenplas,1999) L. reuteri was used as a probiotic to control
C. parvum infection because of its efficiency in rapidly clearing intestinal cells from the
parasite out of the epithelial layer of the rat intestines infiction with parasite and it weakens
the immune system (Alak,1997) Previous studies have also indicated that Lactobacillus
species can bind to intestinal epithelial cells until they are planted on tissue farms and thus
compete with the intestinal mucosa (Dorevitch et al,1015)
The efficiency of probiotic treatment follow the third concentration of nitazaxonid which
decrease at the first day of treatment to 5550 as a efficiency treatment 35% and continuous
decreasing to 2360,1080, 420, 60 cyst/eggs mg at the next day, third, fourth and, fifth day of
treatment, respectively. The Treatment efficiency was 73.3%, 77.5%, 94.0% and 99.1%,
respectively. In the same Table, the efficiency of the second concentration of nitazacxonide
was found to reach 80.9%. On the fifth day, the number of egg cysts reached 80 cysts / eggs.
This indicates the efficiency of the dose given to rabbits in reducing the number of parasite
cysts raised with stool and this indicate full recovery. This is consistent with (lqbal,2015).
Nitazoxanide has been shown to be highly effective in the treatment of cryptosporidiosis
patients as well as patients with immunosuppressive agents. Studies have shown a significant
reduction in the incidence of egg sacs in nitazoxanide-treated mice for seven days and a 94%
cure rate of 50-200 mg / kg bw(painter,2015). The same drug with a dose of 100-200 mg /
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90% reduction in the incidence of cysts and recovery in small goats for five days
(Valeur,2004), and recovery rate was 85% in calves infected with Cryptosporidiosis and
treatment with nitazoxanide for four days. The interpretation of these contradictions is due to
the composition of drugs by different manufacturers, Animal species used in research, as well
as the difference in doses and the duration (schlagenhauf et al,2015)
Nitazoxanide's mechanism of action is to inhibit PFOR (pyruvate ferredoxin oxido reductase),
the primary enzyme in the anaerobic metabolism of the parasite, and after the human takes
the drug, its 2: 3 ratio is cast with feces (pensabene et al,2015).
Table (1) Effect of nitazaxonide with different concentrations and probiotic on the rate of egg cysts in rabbits infection with
Cryptosporidium parasites
Eggs
cysts
number
after First
day of
treatment
efficiency
Eggs cysts
number
after First
second day
of treatment
efficiency
Eggs
cysts
number
after First
third day
of
treatment
efficiency
Eggs
cysts
number
after First
fourth
day of
treatment
efficiency
Eggs
cysts
number
after First
fifth day
of
treatment
efficiency
Nitazoxanie
con.1
7180
b
16.7%
4200
b
52.5%
2260
B
53.1%
3180
b
54.7%
700
B
90.5%
Nitazoxanie
con.2
6460
c
25.0%
2140
cd
75.8%
1180
C
75.5%
540
c
92.3%
80
c
98.9%
Nitazoxanie
con.3
5560
d
35.4%
2360
c
73.3%
1080
Cd
77.5%
420
c
94.0%
60
c
99.1%
Probiotic
con.3
6640
c
22.9%
1740
d
80.3%
920
D
80.9%
180
d
97.4%
40
c
99.4%
Positive
control
8620
a
8860
a
4820
A
7020
a
7400
a
Different characters in one column mean significant differences at probability level 0.05
While the first concentration of nitazaxonide has decreased the number of parasite eggs on
the first, second and third day, as follows: 7180, 4200, 2260 egg cysts / mg, but on the fourth
day of treatment, the number of bags increased to 3180 cysts as efficiency rate / 53.7% and
decreased on the fifth day to 700 cysts of eggs / mg and at an efficiency rate of 90.5%, the
increase in the number of bags of eggs during the period of treatment is called relapse after
treatment and this is supported by al- Rifai (Citation et al,2015) because resistance shown by
parasites when the inefficiency of the dose used when activated others have a subsequent
injury and the phenomenon of self-healing is not observed, which has been recorded by
several researchers and may be attributed to the immunity of the host as well as auto infiction,
which plays an important role in the continuity of infection and increase the number of eggs
cysts raised.
RBC count
Table (2) shows the effect of nitazoxanide and probiotic in the treatment of diarrheal cases in
male rabbits on the blood picture after infection.
The results showed that there were no significant differences in the number of RBC between
the animal groups treatment with nitazoxanide (I and III) and the probiotic dose compared
with the control group which were 4.837 × L / 109. The above groups were 4.825, 4.712 × L /
109 for the first and third concentrations of nitazoxanide. The second concentration was
significantly different from the 4.597 x L / 109. The RBC population in the treated rabbits
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group was 4.727 × L / 109, which was not different from the negative control group, but
differed from the infection group formed the highest increase in the RBC count.
concentration of hemoglobin (Hb).
As shown in Table (2), the level of hemoglobin Hb did not differ significantly in all the
groups of rabbits used in the study, except for the infection group has increased blood
hemoglobin significantly, where the level of hemoglobin Hb in the control group was 12.273
g /dl. The level of Hb increased to 13.442 g / dL in infection group, and when the animals
were similar to recovery, it was as in the negative control group. The levels in the three
concentrations of the drug and the probiotic were 12.890, 12,251, 12.385 and 12,281,
respectively.
Table (2) Changes in total red blood cell count, hemoclobin ratio and volume of compressed red blood cells in rabbits treated with different
concentrations of nitazoxanide and biochemist
Total RBC count × 109
Hb g/dl
PCV %
control
mean
±
SD
mean
±
SD
mean
±
SD
Parasite group
4.837
b
±
0.507
12.273
C
±
0.320
38.409
c
±
0.487
Nitazoxanie con.1
5.782
a
±
0.439
13.442
A
±
0.521
40.976
a
±
0.920
Nitazoxanie con.2
4.825
b
±
0.467
12.890
B
±
0.720
38.776
b
±
0.832
Nitazoxanie con.3
4.597
c
±
0.539
12.251
C
±
0.475
38.371
c
±
0.345
probiotic
4.712
bc
±
0.524
12.385
C
±
0.466
38.505
bc
±
0.977
4.727
bc
±
0.422
12.281
C
±
0.486
38.496
c
±
0.816
Different letters in one column mean significant differences at probability level 0.05.
Packed cell volume P.C.V
Table (2) also shows the PCV for control and infection groups and all treatment groups. PCV
in the control group was 38,409%, and the infection rate increased to 40.976%. PCV
decreased at the end of the treatment period in the three concentrations of nitazoxanide with
38.776% in the first concentration. The PCV in the other two concentrations of the drug was
38.371% and 38.505% and did not differ significantly from the negative control group, but
differed from the infection group, and finally A probiotic group was (38.496%), which is
statistically similar to the first and second concentration groups of nitazoxanide.
The results of the present study were agreed with a study (Al-rifai,2009) which stated that
there were no significant differences in hemoglobin Hb, RBC and PCV between the extract
and the treatment of spiramycin between the concentrations used in the experiment. There
was also no significant difference between Control and treatment groups. The values
remained at the normal level compared with the negative control group. These results
were not consistent with the study (salem,2015). For both PCV and Hb in Microsporidiosis
patients, the rates were normal and not consistent with (Abdulsadah et al,2013) Children who
suffer of the infection with hidden spores and that they suffer from anemia. The differences
between the results may be due to the duration of infection. The parasite may be chronic for
months. Side effects such as loss of fluids from the body due to diarrhea are associated with
anorexia leading to malnutrition. This leads to a decrease in the amount of Hb and PCV, it
was observed in Experimental rabbits that there was no significant different between
treatment groups and control, it is also shown that this parasite does not affect physiologically
on both PCV, Hb, and RBC.
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The reason for these high results in tretment rabbits is due to the drought of infected animals
due to a decrease in fluid volume in the body leading to an increase in the concentration of
Hb, PCV and RBC (Al-samarrai,1999).
Total and differential count of white blood cells:
WBC
The results of the present study showed a difference in the total number of white blood cells
(Table 3). The statistical analysis showed significant differences (P <0.05). There was a
decrease in the total number of white blood cells of the positive group compared to the
negative group. The number of white blood cells in the negative rabbit group was 8.174 ×
109/ L and the number decreased to 7.174 × 109/ L when rabbits were infected with the
parasite. There was no significant difference in the infection group in the nitazoxanide treated
rabbits for the three concentrations, which were 7.158, 7.345, and 7.337 × 109/ L,
respectively but the height number was in the group treatment with the probiotic where the
WBC number was 8.137 × 109/ L, which is not significantly different from WBC in the
negative control group.
The reduction of the parasite may be due to the fact that the white blood cells are rapidly
stimulated when the antigen enters, leading to large numbers of migration to the location of
the parasite by the adsorbents that are attracted to them. So the redaction of WBC number to
half can noticed , because it can kill and destroy the pathogenic organisms as the molecules
resulting from the break-up can stimulate T cells and thus stimulate the acquired immune
system. (Feldman et al,2002) As for the increase in the total number of white blood cells in
the group of rabbits treated with probitic, these results confirm the effect of lactic acid
bacteria in raising the immune response to the body compared to the two sets of antibody,
which decreased the number of lymphocytes, which indicate an increase in production of T &
B cells (Schnare et al,2001).
Lymphocyte
As shown in Table (3), we observe the significant differences in the lymphocyte count of all
the groups under the study. The most significant feature is the decrease in the number of
lymphocytes in the positive control group (infected) of 1.914 × 109/ L. Negative control was
3.393 × 109/ L. When rabbits treated with nitazoxanide at the binging, the numbers of
lymphocyte increased. The numbers in the second and third concentrations of the drug
reached 3.357 and 3.308 × 109/ L. They did not differ significantly from the number of
lymphocyte in the negative control group , And from the results in this table also is increase
the number of lymphocytes in the rabbit probiotic treatment group compared to all treatments
where the number reached to 4.334 × 109/ L. This result was agreed with (Valeur et al,2004)
which reached an increase in WBC and lymphocytes in probiotic rabbits.
The reason for the reduction in the numbers of lymphocytes in animals infected by parasite is
due to the phenomenon of vascular dilation of lymphocytes. And may cause the decline of
numbers of lymphocytes around the blood vessels, but these cells do not have the ability to
phagocytosis, but have a specific ability to the movement of amoebic and in most
circumstances these cells out of the blood vessel but near it, so the role of lymphocytes in
inflammation is to contribute to the immune response Leading to the secretion of toxic
substances and the formation of antibodies that have a direct and effect against the parasite
(Al-saadi,2010). The change in the differential count of white blood cells may indicate
antibody production (Roitt et al,1998)
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Table (3) Changes in total and differential CBC rates in rabbits treated with different concentrations of nitazoxanide and bioenergy
Total WBC
Lymphocyte ×109/L
granulocyte×109/L
Monocyte
control
mean
±
SD
mean
±
SD
mean
±
SD
mean
±
SD
Parasite group
8.174
a
±
0.531
3.393
b
±
0.370
4.059
a
±
0.402
1.344
b
±
0.115
Nitazoxanie con.1
7.174
b
±
0.556
1.914
d
±
0.205
2.935
b
±
0.509
0.795
b
±
0.317
Nitazoxanie con.2
7.158
b
±
0.577
2.732
c
±
0.526
3.291
b
±
0.501
0.888
b
±
0.545
Nitazoxanie con.3
7.345
b
±
0.488
3.357
b
±
0.695
2.916
b
±
0.635
1.067
b
±
0.586
probiotic
7.337
b
±
0.439
3.308
b
±
0.306
3.307
b
±
0.619
1.219
b
±
0.290
8.137
a
±
0.599
4.334
a
±
0.517
4.332
a
±
0.634
1.519
a
±
0.609
The current results have agreed with (Thongsong et al,2008) the rise of lymphocytes in
animals treated with bacterial probiotic and their filtration. The role of natural
microorganisms enhances the role of flora in the defense of the body by preventing the
proliferation of harmful bacteria, as well as stimulating the action of natural and acquired
immunity, and this is confirmed by (Rastall &Gibson,2002) in his study, he pointed out that
the use of microbial preparations increases the effectiveness of the immune system in terms
of Stimulating the humoral and cellular immunity together in the chicken used in its study.
Granulocyte cells of WBC (acidophil, basophil, neutrophil)
The results obtained in the present study, as shown in Table (3), showed a significant
decrease in the control group of granulocyte counts in the infedted and nitazoxanide groups
of the three concentrations, with a significant difference at a probability level of 0.05. The
infection group was 2.935 × 109/ L, In the first, second and third groups of nitazoxanide,
3.291, 2.916 and 3.307 × 109/ L, respectively, the number of granulocyte cells was
statistically similar to the negative control group at 4.332 × 109/ L. this Results
corresponding with a study of (Al-Saeedi,2004) decrease a white blood cells and
granulocytes and lymphocytes in infected animals with cryptosporidium parasite and the
absence of significant differences between infected animals and Nitazoxanide treatment
animals.
The cause of the reduction of the granulocytes in the blood after the entry of the parasite is to
the migration of these cells to the location of parasite, which leads to the decline and
eliminate it by the process of phagocytosis, so this cell is an important part of the phagocytic
system (Hamdy,2017).
The most important cells that contribute to the phagocytosis process are neutrophils, and the
characteristics of the infection are cell resurfacing (janssen,1997). acidophil have a
significant role in the immune response to parasitic infections by migrating to infection sites
due to the Eosinophil chemotactic factor, are highly interacting to remove granulocytes from
mast cells. These parasitic cells also attack by binding to immunoglobulin IgG and IgE,
which causes the loss of their granules and the release of their contents.
(janssen,1997).
The acidophil destroy the parasites through their enzymes, which affects the outer wall of the
parasite, which leads to its destruction. The acidophil that help in its function are mast cells.
IgE binds to the receptors on the mast cells when exposed to the parasite and attracts to
acidophil to the site of infection, and the cells of the current act to stimulate inflammation at
the site of the concentration of the antigen and work similar to the mast cells (Roitt et al,1998)
European Journal of Molecular & Clinical Medicine
ISSN 2515-8260 Volume 7, Issue 09, 2020 153
153
Monocyte cells (mast cell)
According to the results in the Table ( 3 ) The number of Monocyte in rabbits was decreased
in the group of infection. However, according to the statistical analysis, the decrease was not
significant for the control group at 0.795%, while the treated group of nitazoxanide in the
three concentrations was not significantly different from the Negative control group, and
reached 0.888, 1.067 and 1,219% in the three concentrations, respectively.
While the number of rabbits treated with biochemist was quite different. Monocyte cells
increased after completion of the treatment and the number reached 1.519%. This result is
significantly different from the negative control group at the probability level of 0.05. The
results of this study were consistent with the results of a decrease in the number of whole and
differential white blood cells in rabbits infected with Cryptosporidium parvam. The reduction
in the number of single cells in the parasite-infected animals may be due to the migration of
these cells from peripheral blood to infection sites because they are non- The important
quality against many of the infections, and their transformation into macrophage cells
(Madigan et al,2003) as these cells contribute to the phagocytosis to eliminate the parasite.
From the observation of the blood picture of white blood cells, we can point to the role of
lactic acid spores in stimulating the immune system of rabbits in the case of immunization
and treatment with these probiotic compared to the antibiotic(Levinson & jawetz,2000).
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