Download Control of Cryptosporidiosis by Probiotic Bacteria

International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
Control of Cryptosporidiosis by Probiotic
Bacteria
Magda M. Sanad, Jamila S. Al-Malki, and Areej G. Al-Ghabban

of the intestinal mucosa surface has been shown to play an
important role in initiation of the mucosal immune response
(5,6). Epithelial cells, once infected, have increased expression
of inflammatory chemokines and cytokines and demonstrate
antimicrobial killing mechanisms, including production of NO
and antimicrobial peptides (7). Besides the definite role of
immunity, there is a clear evidence that resistance to C.
parvum infection can be mediated by non-specific
mechanisms associated with the presence of intestinal flora. In
other words, colonization of the intestine by Cryptosporidium
depends on the intestinal microflora because newborn or adult
germ-free mice are far more susceptible to this parasite than
flora-bearing adult mice (8).
To date, there is no totally effective and approved therapy
for cryptosporidiosis and a healthy intact immune system
seems to be the only solution. Probiotics, defined as live
microorganisms that when administered in adequate amount
confer a health benefit on the host (9), have been shown to
stimulate and regulate natural and acquired immune responses
and to enhance the mucosal barrier (10-13). The use of
probiotics in the treatment of specific diseases has evolved
into an extremely valuable option yet to be optimally used in
clinical medicine. Although modulation of Cryptosporidium
parvum infection by various probiotic bacteria species had
been controversially reported in immunodeficient (14) and
immunocompetent (15) animal models and in cell cultures
(16), but probiotics had been successfully used for treatment
of a girl with cryptosporidiosis (17). Promising health benefits
and efficacy of probiotics for preventing and treating a variety
of diseases attract increasing attention to use probiotics and
probiotic-derived factors for novel therapies. So, the aim of
the present work is to study the therapeutic efficacy of
consumption of a mixture of L.
acidophilus, L. helveticus and Bifidobacterium bifidum on
experimental cryptosporidiosis using immunosuppressed
mouse model.
Abstract—Cryptosporidiosis, an opportunistic parasitic disease,
has become a topic of great interest being life-threatening among
immunocompromised patients worldwide. To date, there is no totally
effective therapy other than a healthy intact immune system.
Probiotics, widely reported to stimulate both innate and acquired
immunity at mucosal and systemic levels, were investigated in the
present study for the therapeutic effects of daily administration of a
mixture of L. acidophilus, L. helveticus and Bifidobacterium bifidum
against Cryptosporidium parvum (genotype 2) infection in an
immunosuppressed mouse model. Although complete eradication of
the parasite was not achieved, but the used probiotics mixture
induced significant reduction in parasite burden, ultrastructural
changes in relation to parasite attachment, and internalization to
epithelial cells, partial reparation of the parasite-induced intestinal
mucosal damage and increase in the serum level of IFN-γ. These
results reveal beneficial effects of probiotics upon cryptosporidiosis
and indicate that they could help in reducing the risks of severe
disease in immunosuppressed patients. Hopping for a more beneficial
role against cryptosporidiosis, further studies are recommended to
exploit the role of specific strains in therapeutic intervention, and
possibility of their combination with chemotherapeutic agents.
Keywords—Cryptosporidium
parvum
(genotype
2),
immunosuppressed mouse model, probiotic bacteria, parasite burden,
intestinal mucosa, electron microscopy, IFN-γ.
I. INTRODUCTION
RYPTOSPORIDIUM has gained recognition as a
prominent enteropathogen of humans and animals. It is
an obligate intracellular protozoan parasite of worldwide
distribution in the environment, and proven to have potential
to
cause
watery
diarrhea
among
children,
immuneocompromised adults and newly born animals
particularly calves. The severity of clinical signs depends on
the immune status of the patient. In immunocompetent
individuals, the infection is self-limiting and patients can clear
parasites spontaneously within a few weeks (1). In
immunocompromised and immunosuppressed hosts such as
HIV patients, the infection is more severe; leading to
prolonged and persistent diarrhea with extensive alterations of
the intestinal epithelium and can be life threatening (2).
Protection against this parasite has been largely
associated with cell-mediated immune response and
production of interferon gamma (IFN-γ) (3-4). Epithelial cells
C
II. MATERIALS AND METHOD
A. The parasite
C. parvum (genotype 2) oocysts ( originally isolated from
fecal samples of infected calves) were maintained in suckling
Swiss albino mice at Zoology Department, Faculty of Science,
Al Taif University. Oocysts from feces of infected mice were
collected and concentrated by the sugar centrifugal floatation
method after being stored in 2.5% potassium dichromate
solution at 4°C to be used within one month.
Jamila S. Al-Malki, Department of Biology, College of Science, Al-Taif
University, Saudi Arabia
Areej G. Al-Ghabban, Department of Biology, Girls College of Education,
Tabuk University, Saudi Arabia
Corresponding author: Magda M. Sanad, Professor
of
Medical
Parasitology, Faculty of Medicine, Zagazig University, Zagazig, Egypt,
e.mail: [email protected].
http://dx.doi.org/10.15242/IICBE.C0415017
B. Experimental animals
A total of 60 Laboratory-bred Swiss albino mice, 1-2
49
International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
subjected for
separation of sera. Concentrations of IFN-γ
were determined by commercially available ELISA Kit
(Genzyme, Thermo Fisher Scientific, USA). Detection limit =
100 pg/ml. IFN-γ assay in duplicate.
months old and free from intestinal parasitic infections were
immunosuppressed (I.S.) by hydrocortisone acetate (25
mg/ml.) in a daily intramuscular dose of 0.1 ml/mouse for 5
weeks.
3. Histopathological study:
C. Experimental infection
Oocysts were washed with phosphate buffered saline (pH
7.4) by centrifugation at 670 g for 7 minutes just before
inoculation. Infection was done on the 14 th day of
immunosuppressive therapy by oral inoculation of 10 5
oocysts/mouse in 0.1 ml PBS.
At the end of the probiotics treatment period, hematoxylin
and eosin stained ileum sections from mice of all groups
(3/group)
were
examined
microscopically
for
histopathological changes.
4. Transmission electron microscopy ( TEM ) study:
At the end of the probiotics treatment period, small tissue
samples from the distal ileum of sacrificed mice (3/group)
were collected and immediately fixed in cold buffered
glutaraldehyde (2.5 %) in o.1 M phosphate buffer (pH 7.4) for
1-4 days. Post-fixation was done in 1% OSO4 in the same
buffer at 4°C for 2-3 hours, transferred to 1% uranyl acetate
and dehydrated in ascending grades of ethyl alcohol to be
embedded in Epon resin. Ultra-thin sections were stained with
uranyl acetate and
lead citrate and examined for
morphological changes in invasive parasitic stages using a
Zeiss EM 906 transmission electron microscope..
D. Administration of the probiotics
A commercial mega Acidophilus, obtained from GNC
Inc., Saudi Arabia, was provided as capsules each containing
1.5 x 109 CFU of a mixture of three probiotic bacteria species:
Lactobacillus acidophilus; Lactobacillus helveticus and
Bifidobacterium bifidum. The orally inoculated daily dose /
mouse was freshly prepared and adjusted to a concentration
of 20 x106 CFU in 0.1 ml PBS.
E. Animal groups
A total of 60 immunosuppressed mice were maintained in
the laboratory as 4 groups, 15 mice each: Gr. I (infection
control): only infected, Gr. II: (treatment control): only
treated, once daily for 3 weeks from the 14 th day of
immunosuppression therapy until sacrifice, Gr.III (early
treatment): infected and treated once daily from the 1 st to the
21th post-infection (P.I), Gr. IV (late treatment): infected and
treated once daily from the 7th to the 21th post-infection (P.I)
day. All procedures including euthanasia procedure were
conducted in accordance with the National Institute of Health
Guide for the Care and Use of Laboratory Animals, Institute
for Laboratory Animal Research (NIH Publications No. 80-23;
1996) and the Ethical Guidelines of the Experimental Animal
Care Center ( Al Taif University, Saudi Arabia)
IV. STATISTICAL ANALYSIS
Data were computerized and statistically analyzed using the
arithmetic mean and standard deviation, Chi square test and
one way ANOVA.
V. RESULTS
Oocysts count ( Tables 1 & 2):
In the present study, the infecting dose of
10 5
oocysts/mouse achieved 100% take up of infection. The early
treated mice (Gr.III) showed significant (P<0.05) decrease in
the number of oocysts shed on 6, 11, 16 and 21 P. I. days in
comparison with the non-treated (Gr. I) and the late-treated
(Gr.IV) groups. In addition, the early treated group showed
significantly (P<0.05) higher percent reduction in oocysts
shedding in comparison to the late treated one.
III. ASSESSMENT OF THERAPEUTIC EFFICACY OF THE
PROBIOTICS
1. Determination of intensity of infection by:
TABLE I
CRYPTOSPORIDIUM OOCYSTS COUNT AMONG IMMUNOSUPPRESSED (I. S.) MICE
a. Determination of Cryptosporidium oocysts count:
Three mice from each of the infected groups were subjected
to collection of fresh fecal samples of each individual mouse
on days 6, 11, 16, 21 P.I. Counting of shed oocysts in 10
microscopic fields (x400) of a modified Zeil Neelsen stained
smear, calculation of the mean oocyst count and the percent
reduction in each group was determined.
TREATED WITH PROBIOTICS VERSUS UNTREATED CONTROL AT DIFFERENT
POST-INFECTION DAYS
b. Determination of parasite count in stained ileal sections:
Three mice from each of the infected groups were dissected
on days 6, 11, 16, 21 P.I. and 1-cm fragments from the distal
ileum were collected, fixed in 10% buffered formalin,
processed to paraffin sections and hematoxylin and eosin
staining. Parasite stages were counted in 10 microscopic fields
(x 400).
No of examined mice at each time point / group = 3 (a, b, c): means
bearing different letter within the same day are significantly different
2. Quantification of mouse IFN- γ in the serum:
At the end of the probiotics treatment period,
exsanguinations of mice from all groups (3/group) were
http://dx.doi.org/10.15242/IICBE.C0415017
50
International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
TABLE II
PERCENT REDUCTION IN CRYPTOSPORIDIUM OOCYSTS SHEDDING AMONG I.
S. TREATED GROUPS AT DIFFERENT POST-INFECTION DAYS
Fig 1 (a) and (b)
Fig.1(a & b): Sections in the ileum of an I. S. C. parvum infected
non-treated mouse (on the 21th P. I. day. (a) shows edema, exudative
inflammation and sloughing of intestinal epithelial cells with evident
vacuolation and complete cell necrosis (H&E, X 400). (1b) shows
excess parasite stages (arrows) along the surface of epithelial cells,
evident changes in the nuclei and nucleoli indicating cell necrosis
(H&E, X 1000)
(a, b): means bearing different letter within the same day are significantly
different
Number of parasitic stages in ileum sections (Table 3):
On 6, 11, 16 and 21 P. I. days, significant (P<0.05) decrease
in the number of endogenous parasite stages were noticed in
ileal sections along the intestinal mucosal surface of the early
treated mice (Gr.III) in comparison with the non-treated )Gr.I)
and late treated groups (IV). On days 11, 16 and 21 P. I. days,
the number of endogenous parasite stages in the late treated
group was significantly ((P<0.05) less than that in the nontreated but significantly (P<0.05) more than in the early
treated group.
Fig 2 (a) and (b)
Fig.2 (a & b): Sections in the ileum of an I. S. early-treated mouse
(Gr. III) on the 21th P. I. day. (a) shows partial regression of
edematous and necrotic changes in intestinal epithelial cells and
improvement in villous architecture. (H&E, X 250). (2b) shows less
endogenous parasite stages (arrows) with partial reparation of the
epithelial cell surface. (H&E, X 1200)
TABLE III
NUMBER OF PARASITIC STAGES IN ILEUM SECTIONS OF
IMMUNOSUPPRESSED (I. S.) GROUPS TREATED WITH PROBIOTICS VERSUS
UNTREATED CONTROL AT DIFFERENT POST-INFECTION DAYS
No of examined mice at each time point / group = 3 (a, b, c): means
bearing different letter within the same day are significantly different
Fig 3 (a) and (b)
Figs. 3(a & b): Sections in the ileum of an I. S. late-treated mouse
(Gr. IV) on the 21th P. I. day. (a) Shows edematous changes and
sloughing of epithelial cells. Shortening of villi and increased crypt
depth were noticed ((H&E, X 250). (b) Shows less parasite stages
along the epithelial cell surface. Nuclear changes were evident
indicating cell necrosis (H&E, X 1200)
Histopathological changes (Table 4 and Figs 1-3):
The non-treated infected control (Gr. I) showed severe
mucosal and submucosal inflammatory changes represented
by shortening, broadening and fusion of villi; hydropic
degeneration, edematous and exudative inflammation,
desquamation and sloughing of intestinal epithelial cells and
evident vacuolated epithelial cells due to complete cell
necrosis. In both early and late treated groups, partial
regression of edematous and necrotic changes in intestinal
epithelial cells and improvement in villous architecture were
noticed at the end of the probiotics administration period. In
the non-infected treated control (Gr.II), no pathological
changes were noticed.
Serum level of mouse IFN- γ (Table 5):
Sera of treated mice (Gr.II, Gr.III, Gr.IV) showed
insignificant (P > 0.05) increase in the level of IFN- γ in
comparison to the recorded level in sera of the infected nontreated control ( Gr. I).
TABLE V
IFN-γ LEVEL IN SERA OF DIFFERENT I.S. STUDIED GROUPS
TABLE IV
NUMBER OF PARASITIC STAGES IN ILEUM SECTIONS OF
IMMUNOSUPPRESSED (I. S.) GROUPS TREATED WITH PROBIOTICS VERSUS
UNTREATED CONTROL AT DIFFERENT POST-INFECTION DAYS
Mouse groups
IFN- γ level (pg/ml) ± SD
Infection control (Gr.I)
a 234 ± 6.4
Treatment control (Gr.II)
a 437 ± 15.1
Infected & treated early (Gr.III)
a 416± 18.2
Infected & treated late (Gr.IV)
a 388 ± 35
Statistical analysis
P > 0.05
(a): means bearing the same letter within the same column are
insignificantly different
‫ـ‬absent; + mild to absent; + mild; ++ moderate; +++ severe
http://dx.doi.org/10.15242/IICBE.C0415017
51
International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
Fusion of parasite and host-cell membranes was seen at the parasitecell contact forming a dense band (DB). An eccentric rounded to
ovoid nucleus, nucleolus and rough endoplasmic reticulum were
seen. Bar = 2μm
Fig 4-d Transmission electron micrograph showing section of C.
parvum zoite from the non-treated I. S. group (Gr.I), attached to the
enterocytes membrane. A vacuole was detected in the cytoplasm at
the base of the zoite representing feeder organelle (FO) attached to
the host cell membrane. Bar = 1μm
Electron microscopy results (Figs 4-5):
In the non-treated control group zoites were mostly located
among the microvilli, attached to the enterocytes surface and
surrounded by a four-membrane complex (MC), the most
inner one appeared more electron-dense at its apical end. In
attached forms, the parasite pellicle was mostly separated from
the host cell membrane at the base of the parasite, membrane
fusion was seen at the parasite-cell contact (with some
serrations) and the pellicle and host cell membrane became
unclear. The margin of attachment area showed high electron
density (DB). A vacuole was detected in the cytoplasm at the
base of the zoite "representing feeder organelle" (FO) attached
to the host cell membrane. Each parasite was existed in a
parasitophorous vacuole (PV) and completely covered with a
double-layered membrane derived from numerous microvilli
(MV) that elongated besides and around the parasite.
In treated groups a lot of extracellular stages were noticed
and evident ultrastructural changes were seen in the invasive
non- attached zoites. These were represented as lacking
normal integrity with degeneration of their contents, a fuzzy
uneven outer surface with more electron dense serrations in
some zones, ill-defined nucleus, ill-defined parasite
membrane, less electron dense area surrounding the cytoplasm
and incomplete covering by markedly redundant microvillous
membranes.
Fig 4 (e) and (f)
Fig 4-e Transmission electron micrograph showing section of C.
parvum zoite from the non-treated I. S. group (Gr.I), attached to the
enterocyte surface. A three-membrane complex (MC) is tightly
adhered to certain zones of the parasite, with a fourth more electrodense membrane appeared at apical end. A dense band (DB) and a
feeder organelle (FO) were clearly visible Bar = 0.5μm
Fig 4-f Transmission electron micrograph showing section of C.
parvum zoite from the non-treated I. S. group (Gr.I), located within a
parasitophorous vacuole and attached to the enterocytes membrane.
At the parasite-host cell contact, membrane fusion with serration and
formation of a dense band was seen. Bar = 0.5μm
Fig 4 (a) and (b)
Fig 4-a: Transmission electron micrograph showing cross section
of an invasive non-attached C. parvum zoite from the untreated I. S.
group. The parasite was surrounded by 2 membranes, parasite
pellicle was dense at some areas, micronemes (M) were seen (arrow).
Bar = 1μm
Fig 4-b Transmission electron micrograph showing longitudinal
section of invasive non-attached C. parvum zoite from the untreated
I. S. group. The parasite membranes are not distinct, serrations were
noticed all around the parasite and less electron dense area was seen
surrounding the cytoplasm. Bar = 0.5μm
Fig 5 (a) and (b)
Fig 5-a Transmission electron micrograph showing cross section
of an invasive C. parvum zoite from the early-treated group (Gr.III).
The parasite was located among the microvilli and had abnormal
fuzzy uneven ill-defined membranes with some areas of electron
density. The pellicle is seen separated from the host cell membrane.
Bar = 0.5μm
Fig 5-b Transmission electron micrograph showing cross section
of an invasive C. parvum zoite from the early-treated group (Gr.III)
incompletely surrounded by redundant membranes derived from the
microvilli. The pellicle was not clearly observed at the base of the
parasite and nucleus was ill-defined. Bar = 0.5μm
VI. DISCUSSION
To date, the infection of cryptosporidiosis in the
immunocompromised patients is largely out of treatment.
Immunocompetent individuals can get rid of the infection
because parasite eradication relies on innate and
acquired immunity. So, in the preset study, mouse model was
maintained immunosuppressed throughout the experiment and
probiotic treatment was used to promote human health due to
Fig 4 (c) and (d)
Fig 4-c Transmission electron micrograph showing C.
parvum zoite from the non-treated I. S. group (Gr.I), located within a
parasitophorous vacuole and attached to the enterocytes membrane.
http://dx.doi.org/10.15242/IICBE.C0415017
52
International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
parasite has been largely associated with production of IFN- γ
; a major player not only in cell-mediated immunity, but in
early innate immune responses as well. In vitro studies have
demonstrated that IFN- γ directly prevents the parasite from
invading host cells [32]. Additionally, a case of persistent
cryptosporidiosis has been described in a child with primary
IFN- γ deficiency [33], suggesting that this cytokine is also
important in human infections. The important protective roles
for IFN-y may also include apoptosis induction in infected
intestinal epithelial cells, and modulation of mucosa epithelial
integrity [6].
In the present work, the ultrastructural changes seen in the
invasive non- attached zoites may suggest interference with
gliding motility of the sporozoites and affection of the
membrane-associated proteins located in apical organelles
of C. parvum sporozoites. Chen et al (34) and Sibley (35)
provided evidence that blockage of apical discharge by the
inhibition of parasite actin and tubulin polymerization and the
chelation of intracellular calcium in C. parvum sporozoites
significantly decreased the gliding motility of the parasite.
In conclusion, this study provides evidence that a mixture
of the probiotics; L. acidophilus, L. helveticus and
Bifidobacterium bifidum; is a good choice to improve the
mucosal immune system and may have implication in the
therapy of cryptosporidiosis during immunosuppressive states.
However, further investigations are needed to move forward in
this direction, probiotics have to be precisely characterized at
a strain level and mechanisms through which they work have
to be elucidated
their effects on luminal microbial ecology and immune
modulation, particularly through balance control of proinflammatory and anti-inflammatory cytokines (18). Also,
probiotics are important to stimulate the proliferation of
mucosal epithelial cells which are considered as the first line
of defense against intestinal pathogens (19). In the present
study, a mixture of Lactobacillus acidophilus; Lactobacillus
helveticus and Bifidobacterium bifidum was assessed because
these species are most widely used to prevent and treat
intestinal disorders (20, 21). The significant reduction in the
number of oocysts and in percent reduction in oocysts
shedding detected in fecal samples collected from the early
treated group on days 6- 21 P. I. in comparison to the late
treated mice, together with the significant decrease in the
number of endogenous parasite stages in ileum sections along
the intestinal mucosal surface of the early treated mice in
comparison with the non-treated, all these results confirm the
effective therapeutic role of probiotc bacteria used in the
present study against Cryptosporidium and are in agreement
with Mc Donald,et al (7) and Alak et al ( 14). Histologically,
morphological and cellular alteration of microvillus membrane
integrity revealed that probiotic administration ameliorated the
mucosal damage in both early and late probiotic- treated I S
mice, compared with the severe microvillus damage,
edematous and vacuolated epithelial cells in non-treated mice.
These results clearly show the anticryptosporidial effect of the
probiotics in vivo by modulating the gut cells to inhibit the
colonization and multiplication of Cryptosporidium , thus
reducing the severity of cryptosporidiosis.
Gupta and Grag (22) mentioned that probiotics induce
modulation of the intestinal environment by having the
capacity to control the proliferation of surrounding
microorganisms. Wollowski et al (23) and Madsen (24)
reported that probiotic bacteria influence the mechanisms of
innate immunity and the activation of B cells for mucosal
immunity important for protection against pathogens,
prevention of the penetration by foreign antigens, and
maintenance of mucosal homeostasis.
Close results were reported by Alak et al (25) since daily
ingestion of L. reuteri was efficient to prevent C.
parvum intestine colonization and tissue lesions in
immunodeficient mice. Waters et al. (26) suggested that
protection was due to secretion of as yet unidentified
antimicrobial products. Arvola et al (27) added that probiotics
have been shown to modulate release of cytokines (TNFα IFN-γ, IL-10, IL-12) which play a central role in maintaining
the delicate balance between necessary and excessive defense
mechanisms.
Gill (28) reported that the underlying mechanisms are
however not clear, involving stimulation of different subsets
of immune system cells to produce cytokines, which in turn
play a role in the induction and regulation of the immune
response, and to enhance intestinal IgA immune responses and
increase intestinal mucin production . Similarly, Borchers et al
(29) found that IL-10 and secretory IgA response, important
actors in an efficient anti-Cryptosporidium immune response,
have been shown to be induced by some probiotic strains.
In the present study, the increased serum level of IFN-y
ininfected treated groups is in agreement with Mead and You
(30) and Riggs (31) who found that protection against this
http://dx.doi.org/10.15242/IICBE.C0415017
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
53
DuPont, H.L, C. L. Chappell, C. R. Sterling, P. C. Okhuysen, J. B. Rose,
and W. Jakubowski. 1995. The infectivity of Cryptosporidium parvum
in healthy volunteers. N. Engl. J. Med. 332:855-859.
http://dx.doi.org/10.1056/NEJM199503303321304
Godwin, T.A.1991. Cryptosporidiosis in the acquired immunodeficiency
syndrome: a study of 15 autopsy cases. Hum. Pathol. 22:1215-1224.
http://dx.doi.org/10.1016/0046-8177(91)90103-V
Lean, I.S, Mc Donald, V, Pollok, R.C. 2002. The role of cytokines in the
pathogenesis of Cryptosporidium infection. Curr. Opin. Infect. Dis.
15(3):229-234.
http://dx.doi.org/10.1097/00001432-200206000-00003
Tessema, T.S, Schwamb, B, Lochner M, Förster I, Jakobi V, Petry F.
2009. Dynamics of gut mucosal and systemic Th1/Th2 cytokine
responses in interferon-gamma and interleukin-12p40 knock out mice
during primary and challenge Cryptosporidium parvum infection.
Immunobiol. 214 (6): 454-466.
http://dx.doi.org/10.1016/j.imbio.2008.11.015
Jung, H. C., L. Eckmann, S. K. Yang, A. Panja, J. Fierer, E. MorzyckaWroblewska, and M. F. Kagnoff. 1995. A distinct array of
proinflammatory cytokines is expressed in human colon epithelial cells
in response to bacterial invasion. J. Clin. Investig. 95:55-65.
http://dx.doi.org/10.1172/JCI117676
Lacroix-Lamandé S., R. Mancassola, M. Naciri and F. Laurent. 2002.
Role of Gamma Interferon in Chemokine Expression in the Ileum of
Mice and in a Murine Intestinal Epithelial Cell Line after
Cryptosporidium parvum Infection. Infect Immun. 70 (4):2090–2099.
http://dx.doi.org/10.1128/IAI.70.4.2090-2099.2002
McDonald V., D.S. Korbel, Barakat F.M., Choudhry N. and Petry F.
2013. Innate immune responses against Cryptosporidium parvum
infection. Parasite Immunol., 35 (2): 55–64.
http://dx.doi.org/10.1111/pim.12020
Harp J. A, W. Chen and A. G. Harmsen. 1992. Resistance of severe
combined immunodeficient mice to infection with Cryptosporidium
parvum: the importance of intestinal microflora. Infect Immun. 60(9):
3509-3512
International Conference on Agricultural, Ecological and Medical Sciences (AEMS-2015) April 7-8, 2015 Phuket (Thailand)
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Food and Agriculture Organization of the United Nations and World
Health Organization. 2001. Regulatory and clinical aspects of dairy
probiotics. Food and Agriculture Organization of the United Nations and
World Health Organization Expert Consultation Report. Online.
Shukla G., P. Devi and R. Sehgal. 2008. Effect of Lactobacillus casei as
a probiotic on modulation of giardiasis. Dig Dis Sci , 53 (10): 26712679.
http://dx.doi.org/10.1007/s10620-007-0197-3
Shukla G. P. and R. K. Sidhu. 2011. Lactobacillus casei as a probiotic
in malnourished Giardia lamblia infected mice: a biochemical and
histopathological study. Can J Microbiol., 57( 2): 127-135
http://dx.doi.org/10.1139/W10-110
Claude P., R. Champagne, P. Ross, M. Saarela, K. F. Hansen and D.
Charalampopoulos. 2011. Recommendations for the viability assessment
of probiotics as concentrated cultures and in food matrices. Int. J Food
Microbiol.,149(3) : 185-193
http://dx.doi.org/10.1016/j.ijfoodmicro.2011.07.005
Maldonado G. A., de Moreno de LeBlanc, G. Vinderola, M. E., Bibas B,
and G. Perdigón. 2007. Proposed Model: Mechanisms of
immunomodulation induced by probiotic bacteria. Clin Vacc Immunol. ,
14 (5): 485-492.
http://dx.doi.org/10.1128/CVI.00406-06
Alak J. I, B.W .Wolf, E.G Mdurvwa , G.E. Pimentel-Smith , S.
Kolavala, H. Abdelrahman, V. Suppiramaniam. 1999. Supplementation
with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding
of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.
Cell Mol Biol.45(6):855-863.
Guitard J, J. Menotti , A. Desveaux , P. Alimardani , R. Porcher , F.
Derouin , N. Kapel. 2006. Experimental study of the effects of probiotics
on Cryptosporidium parvum infection in neonatal rats. Parasitol Res.,
99(5): 522-527.
http://dx.doi.org/10.1007/s00436-006-0181-4
Glass M.D., P.D. Courteny, J.T. Lejeune , L. A. Ward. 2004. Effects of
Lactobacillus acidophilus and Lactobacillus reuteri cell-free
supernatants on Cryptosporidium viability and infectivity in vitro. Food
microbiol., 21: 423-429.
http://dx.doi.org/10.1016/j.fm.2003.11.001
Pickerd N. and D Tuthill. 2004. Resolution of cryptosporidiosis with
probiotic treatment. Postgrad Med J.; 80:112–113.
http://dx.doi.org/10.1136/pmj.2003.014175
Oelschlaeger T.A. 2010. Mechanisms of probiotic actions - a review. Int.
J. Med.Microbiol. 300(1):57–62.
http://dx.doi.org/10.1016/j.ijmm.2009.08.005
Isolauri E. , Y. Sütas, P. Kankaanpää, H. Arvilommi, S. Salminen. 2001.
Probiotics: effects on immunity. Am J Clin Nutr.73 (2):444S-450S.
Fang Y. and D. B. Polk. 2010. Probiotics: progress toward novel
therapies for intestinal diseases. Curr Opin Gastroenterol. 26(2): 95–101.
http://dx.doi.org/10.1097/MOG.0b013e328335239a
Shukla G., R. deep, K. Sidhu. 2011. Lactobacillus casei as a probiotic in
malnourished Giardia lamblia-infected mice: a biochemical and
histopathological study. Can J Microbiol, 57:(2) 127-135
http://dx.doi.org/10.1139/W10-110
Gupta V. and R. Garg 2009. Probiotics. Ind J Med Microbiol.
27(3):202–209.
http://dx.doi.org/10.4103/0255-0857.53201
Wollowski, I., G. Rechkemmer, and B. L. Pool-Zobel. 2001. Protective
role of probiotics and prebiotics in colon cancer. Am. J. Clin. Nutr.
73:451S-455S
Madsen K. 2006. Probiotics and the immune response. J Clin
Gastroenterol. 40(3):232-234.
http://dx.doi.org/10.1097/00004836-200603000-00014
Alak J.I, B.W Wolf, E.G. Mdurvwa, G.E. Pimentel-Smith, O. Adeyemo.
1997. Effect of Lactobacillus reuteri on intestinal resistance to
Cryptosporidium parvum infection in a murine model of acquired
immunodeficiency syndrome. J. Infect. Dis. 175(1):218–221.
http://dx.doi.org/10.1093/infdis/175.1.218
Waters W. R, J. A. Harp, M. J. Wannemuehler, N.Y. Carbajal, I.A.
Casas. 1999. Effects of Lactobacillus reuteri on Cryptosporidium
parvum infection of gnotobiotic TCR-α-deficient mice. J. Eu. Microbiol.
46(5):60S–61S.
Arvola T, K. Laiho , S. Torkkeli, H. Mykkänen, S. Salminen, L.
Maunula, E. Isolauri. 1999. Prophylactic Lactobacillus G Greduces
antibiotic-associated diarrhea in children with respiratory infections:a
randomized Pediatrics. 104(5): e64.
http://dx.doi.org/10.1542/peds.104.5.e64
http://dx.doi.org/10.15242/IICBE.C0415017
[28] Gill H.S. 2003. Probiotics to enhance anti-infective defence in the
gastrointestinal tract. Bailliere’s Best Prac. Res. Clin. Gastroenterol.
17(5):755–773 .
http://dx.doi.org/10.1016/S1521-6918(03)00074-X
[29] Borchers A.T, C. Selmi, F.J.Meyers, C. L. Keen, M. E. Gershwin. 2009.
Probiotics and immunity. J. Gastroenterol. 44(1):26–46.
http://dx.doi.org/10.1007/s00535-008-2296-0
[30] Mead J. R. and X. You. 1998. Susceptibility differences to
Cryptosporidium parvum infection in two strains of gamma interferon
knockout mice. J. Parasitol. 84:1045-1048
http://dx.doi.org/10.2307/3284643
[31] Riggs M.W. 2002. Recent advances in cryptosporidiosis: the immune
response. Microbes Infect.4 (10):1067–1080.
http://dx.doi.org/10.1016/S1286-4579(02)01631-3
[32] Pollok R.C, M.J. Farthing, M. Bajaj-Elliott, I.R. Sanderson, V.
McDonald. 2001. Interferon γ induces enterocyte resistance against
infection by the intracellular pathogen Cryptosporidium parvum.
Gastroenterol. 120 (1):99–107.
http://dx.doi.org/10.1053/gast.2001.20907
[33] Gomez M.A, C. M. Ausiello, A. Guarino, F. Urbani, M. I. Spagnuolo, C.
Pignata, E. Pozio.1996. Severe, protracted intestinal cryptosporidiosis
associated with interferon γ deficiency: pediatric case report. Clin.
Infect. Dis., 22(5):848–850.
http://dx.doi.org/10.1093/clinids/22.5.848
[34] Chen, X, S. P. O'Hara, B.Q. Huang, J.B. Nelson, J. J. Lin, G. Zhu, H. D.
Ward, and N. F. La Russo. 2004. Apical organelle discharge by
Cryptosporidium parvum is temperature, cytoskeleton, and intracellular
calcium dependent and required for host cell invasion. Infect. Immun.,
72 (12): 6806-6816.
http://dx.doi.org/10.1128/IAI.72.12.6806-6816.2004
[35] Sibley, L. D. 2004. Intracellular parasite invasion strategies. Science
304:248-253.
http://dx.doi.org/10.1126/science.1094717
54