Sinbiotic Effect of Giving a Combination of Lactobacillus acidophilus and

Spirulina platensis on the Relative Amount of CD4 Cells and

Ileal Histopathology white rats induced
Salmonella enteritidis
Eka Wulandari
Email : ekawulan12042gmail.com

ABSTRACT
Salmonellosis is a disease that occurs in the gastrointestinal tract caused by gram
negative bacteria, one of which is Salmonella enteritidis. Salmonellosis control usually uses
chemical antibiotics that can potentially cause antibiotic residues. The combination of
probiotics from Lactobacillus acidophilus and prebiotics from Spirulina platensis as
synbiotics can work synergistically to prevent the attachment of pathogenic bacteria in the
small intestine so that it can reduce the population of Salmonella enteritidis bacteria. This
study aimed to determine the effect of synbiotic combination of Lactobacillus acidophilus
and Spirulina platensis on the relative number of CD4 cells and ileal histopathology in white
rats induced by Salmonella enteritidis. This study was an experimental study using a
completely randomized design method (CRD) with 5 treatments and 4 replications. Giving
synbiotics in mice was carried out for 3 weeks in groups P1, P2, and P3. The synbiotic
concentration given is 0.2%, 0.4%, and 0.6% which is mixed in rat feed. Salmonella
enteritidis was given in the treatment group of 0.5 ml orally with a concentration of 1.5x108
CFU / ml. The parameters measured were the relative number of CD4 cells and ileal
histopathology. The relative amount of CD4 was calculated by the flowcytometry method
which was then analyzed using One Way ANOVA and followed by the Tukey test (α = 5%),
while the ileal histopathology was analyzed descriptively. The results of the statistical
analysis showed that the relative number of CD4 in the administration of synbiotics with a
concentration of 0.6% gave a very significant effect (p <0.01) in decreasing the relative
amount of CD4 and minimizing damage to the ileal epithelium in white rats induced
Salmonella enteritidis.

Key word : Sinbiotic, salmonellosis, Rattus novergicus, ileal histopathology, CD4.

PENDAHULUAN
Salmonellosis is caused by salmonella sp. (OIE, 2008). Salmonella sp. Bacteria. has a
variety of species that have specific hosts, for example S. enteritidis. Salmonella enteritidis
bacteria are one of the serotypes of the subspecies of S. enteritica and belong to the family
member Enterobacteriaceae (OIE, 2000).
Economically, Salmonellosis is very important because it is related to the case of
foodborne disease (Portillo, 2000). In Indonesia, S. enteritidis was first discovered in 1991
from chickens obtained from Chicken Slaughterhouses in Jakarta. Cases due to S. enteritidis
were initially found from Day Old Chick (DOC) originating from the nursery of grandparent
nurseries and the parent stock (Poernomo, 2000). According to Poeloengan (2005), newly
hatched chicks can contract the mother and occur within weeks 2-3 with a high mortality rate
of 85%
Salmonella enteritidis is an intracellular facultative bacterium that can live well in
macrophages, has a lipopolysaccharide virulence factor (LPS) which can stimulate the
immune response in hosts through phagocytosis by macrophages activated by IFN-γ
cytokines produced by T cells. Then IFN-γ will stimulate macrophages to kill through the
respiratory burst, with oxidative processes that produce free radicals, nitric oxide and
activation of CD4 T cells that will lyse infected cells (Abbas, 2003).
One method used in the prevention of Salmonellosis is the use of antibiotics that can
leave a residue on the tissue (Haryati and Supriyati, 2010). The residues caused by antibiotics
in food products from animals will enter the human body that consumes these animal
products, and ultimately can result in the emergence of resistant microbes. In recent years
many alternative antibiotic substitutes have been developed which can reduce the potential
for salmonellosis. One of them is synbiotics, which are safer without leaving a residue
(Sinurat et al., 2017)
Sinbiotik is a combination of probiotics and prebiotics (Haryati and Supriyati, 2010).
Probiotics are feed supplements in the form of non-pathogenic living microorganisms which
if given in sufficient quantities can provide health benefits to the host through improving the
balance of microbes in the intestine. While Prebiotics are carbohydrate fragments that are
included in experimental animals through mixing in feed to support the growth of
microorganisms both in the digestive tract (Safingi et al., 2013).
Probiotics used are derived from the Lactobacilus acidophilus bacteria which is a lactic
acid bacteria that is resistant to acids and bile and can also produce antimicrobial substances
(Sunaryanto, 2014). While the prebiotics used are Spirulina platensis algae which contain
oligosaccharides in the form of mannose and rhamnosa which can stimulate the growth of
non-pathogenic microbes (Gupta et al., 2017). From the background above the authors are
interested in conducting research on the effect of synbiotic combination of Lactobacillus
acidophilus and Spirulina platensis on the relative number of CD4 cells and ileal
histopathology of white rat (Rattus novergicus) induced by Salmonella enteritidis bacteria.

MATERIALS AND METHODS

Place and time of research
This research was conducted in May 2018 - July 2018 at the Laboratory of
Epidemiology at the Faculty of Animal Husbandry, Brawijaya University, Animal Husbandry
Nutrition and Food Laboratory, Universitas Brawijaya, Microbiology Laboratory, Faculty of
Veterinary Medicine, Pathology Laboratory BBVET Wates Yogyakarta, and Molecular
Biology Laboratory University of Brawijaya.

Tools and materials

The tools used in this study include: Freeze dryer, mouse cage, feedlot, drinking water,
digital scales, autoclave, incubator, glove, mask, syringe, Biosafety Cabinet (BSC), ose,
surgical scissors, mortar, setrifugator, glass measuring, petri dish, vortex, bunsen, test tube,
erlenmayer tube, glass object, glass cover, Falcon tube, eppendorf tube, micropipette, yellow
tip, blue tip, white tip, ice box, flow meter, tube rack, microtom.
The materials used in this study were male white rats, disinfectants, sawdust, rat feed,
water, Salmonella enteritidis, Lactobacillus acidophilus, standard Mc. Farland 0.5, Spirulina
platensis 100%, NB media, MRSA media, MRSB media, PBS, SSA media, NaCL 0.9%,
CD4 extracellular antibodies, Alcohol, Xylol.

Research design
This study uses the Experimental method using a completely randomized design
(CRD). This study used male wistar strain white rats (Rattus novergicus). The rat's body
weight is around 100 grams and is 4-6 weeks old. The number of experimental animals used
in this study were 20 which were grouped into 5 treatments with each treatment having 4
replications.
Implementation of Research
1. Sinbiotic Preparation
Sinbiotik used is a combination of probiotics from standard Lactobacillus
acidophilus bacteria Mc. Farland 0.5 (1.5 x 108 CFU / ml) and prebiotics from Spirullina
platensis algae 100%. Sinbiotic production is based on Permatasari (2013) research. The first
step is to increase the dosage of Lactobacillus acidophilus in the MRSB media. Then mixing
probiotics and prebiotics with a ratio of 1: 2 is as much as 28 ml of standard Lactobacillus
acidophilus bacteria Mc. Farland 0.5 (1.5 x 108 CFU / ml) and as much as 56 grams of
spirulina platensis algae 100%. The synbiotic mixture is then stirred using a glass stirrer until
the mixture is homogeneous. Then the synbiotic mixture was put into 194 ml of distilled
water containing maltodextrin 58 grams. The mixture is homogenized by stirring using a
glass stirrer. Homogeneous mixtures are dried using a freeze dryer for 4-5 days until
microcapsules are formed.
2. Preparation of Try Animals
The experimental animals used in this study were 20 rats (Rattus Novergicus) aged 4-6
weeks weighing 100 grams obtained from the Laboratory of Animal Physiology, State
Islamic University, Malang. Mice are adapted for 7 days first and placed in a plastic box
measuring 50 x 30 cm. Mice are placed in a clean place, free of smoke, pollutants, noise, and
with good ventilation. Feed and drinking water are given by adlibitum. Feed is given as much
as 10% of the body weight of the mouse which is as much as 10 grams in the form of BR-2
pellets
3. Preparation of Salmonella enteritidis bacteria
Salmonella enteritidis bacteria were obtained from the Microbiology Laboratory,
Faculty of Medicine, Universitas Brawijaya. Salmonella bacteria are then propagated in
Nutrient Broth (NB) liquid media and re-incubated at 37 ° C for 24 hours. Furthermore,
Salmonella enteritidis bacteria were made by following turbidity on the Mc Farland standard
0.5 (1.5 x 108 CFU / ml) (Larasati, 2016).
4. Treatment of Try Animals
After 7 days of adaptation, rats were given a synbiotic combination of Lactobacillus
acidophilus and Spirulina platensis by mixing them with feed according to the concentration
of 0.2% / kgPK, 0.4% / kgPK, 0.6% / kgPK. Another study regarding additive feed has been
carried out by Galik et al., (2015), that giving an additive feed of as much as 0.5% and 0.75%
mixed in rat control feed as an animal model gave a significant positive effect. After
synbiotic administration for 2 weeks, then 0.5 ml per head of Salmonella enteritidis bacteria
was induced. Then re-maintenance is carried out by giving feed and synbiotics for 1 week.
According to Naughton et al., (1997), in rat animals the dose of S. enteritidis 103 did not
cause infection and could cause infection at higher doses of 108 CFU / ml or gram.
5. Euthanation and Necropsy
Euthanation in rats was done by servical dislocation, carried out on the 28th day of
maintenance, before euthanation of rats was weighed to determine the addition of body
weight. After euthanation, surgery is performed with an incision in the abdominal line, then
the spleen is taken and a flowcytometry is performed to measure CD4 levels.
6. Flowcytometry
Examination of Flowcytometry aims to calculate the relative number of CD4 cells by
the method of flowcytometry. This examination begins with the preparation of the spleen
organ. The spleen organ is taken from the decropped mouse then rinsed using 0.9% NaCl,
then placed in a petri dish containing 5 ml of NaCl, crushed using the base of the syringe,
taken using a syringe and then inserted into the eppendorf tube. Then centrifuged at 2500 rpm
for 5 minutes at 4 ° C, then the liquid supernatant was removed and the pellets were taken.
After the pellets were obtained, 50µl of cellular extract antibody was added using CD4
staining using FITC, then inserted into flowcytometry cuvettes and then running (Puspitasari,
2014).
7. Making Preparat Histopathological
The process of making histopathological preparations according to Jusuf (2009), is as
follows. The ileal organs taken 1 cm long from the decropped rats were then put into a
sample pot containing 10% formalin. The ileal organs that have been fixed in formalin 10%
then go through several stages including dehydration, clearing, paraffin infiltration,
embedding, sectioning, mounting. Furthermore, the preparations were colored with stages
including deparafinization, rehydration, Hematoxylin coloring, defferentiation, eosin staining,
dehydration, clearing, and Mounting.

RESULTS AND DISCUSSION

1. Relative CD4 level
In this study the relative number of CD4 cells was measured using the flowcytometry
method with samples in the form of spleen cells labeled using CD4 extracellular antibodies.
Flowcytometry Test results were analyzed by One Way Anova Test followed by Tuckey Test
(α = 5%) indicating that the synbiotic combination of Lactobacillus acidophilus and Spirulina
platensis can significantly reduce CD4 levels. Tables and graphs of relative CD4 levels can
be seen in Tables 1 and 2 and Figure 1.

Tabel 1 Average Table of Relative CD4 Levels (%)

Repeat
Treathment Total Average
1 2 3 4
K+ 28,98 33,04 35,03 37,02 134,07 33,52±3,43a

K- 29,73 29,00 28,79 27,87 115,39 28,848±0,766ab

P1 29,40 26,76 27,56 28,37 112,09 28,023±1,129ab

P2 21,14 31,18 25,29 24,91 102,52 25,63±4,15b

P3 26,38 23,42 17,48 27,01 94,29 23,57±4,35b

Description: different notations show significant differences (p <0.01)

Table 2 Relative Amount of CD4 ANOVA Table

S.V. df x SS MS xx F xxx Calc F5% F1%

Treatment 4 225,4 56,348 5,66 3,06 4,89

Error 15 149,4 9,960

Total 19 374,8

Description: Fcount> Ftable 1% (4.89), then there is a significant difference between

treatments (p <0.01) with α = 5%.
 Interval
33,52±3,43 a
Plot of k-; k+; ...
95% CI for the Mean
38

28,848
36 ±0,766ab
28,023±1,129ab
Average of
34

CD4 32 25,63±4,15b
30
23,57±4,35b
Data

28

26

24

22

20

k- k+ p1 p2 p3

The pooled standard deviation is used to calculate the intervals.

Treatment

Image 1. Relative CD4 Level Average Graph

In this study the negative control group (K-) had a different average of the positive
control group (K +) but was not significant. In the negative control group (K-) there is a
higher CD4 compared to the treatment group this is caused by the presence of un activated
CD4 T lymphocytes or commonly called naïve T cells in healthy white mice that will
proliferate when there are antigens through the Precenting Cell Antigen (APC ) (Akrom,
2013). The positive control group treated with control feed and Salmonella enteritidis
bacterial infection had an average increase compared to the average negative control group
which was only given control food, this is in accordance with the research conducted by
Mittrucker et al., (2000), that bacterial infections can induce a Th cell response 1. Most CD4
+ and CD8 + cells will be activated after infection. Positive control (K +) mice infected with
S. enteritidis experienced an increase in CD4 cell count compared to the negative control (K-)
given only control feed.
In this study the positive control group (K +) was not significantly different from P1,
but was significantly different with P2 and P3. Treatments 1, 2, and 3 are treatments with
synbiotic administration with a multilevel concentration of 0.2% / kgPK, 0.4% / kgPK,
0.6% / kgPK. Sinbiotik is a balanced combination between probiotics and prebiotics in
supporting the survival and growth of beneficial bacteria in the digestive tract of living things
(Yolanda, 2017). Giving synbiotics which is a combination of probiotics Lactobacillus
acidophilus and prebiotics Spirulina platensis has an antibacterial and anti-inflammatory
effect.
Decreased relative levels of CD4 in the treatment group given synbiotics were caused
because lactic acid bacteria contained in synbiotics had antimicrobial and anti-inflammatory
effects. The antimicrobial effects caused by Lactobacillus acidophilus bacteria produce
antimicrobial compounds in the form of bacteriocins so that they can suppress the growth of
enteric pathogenic bacteria, besides Lactobacillus acidphilus also produces lactic acid which
will disrupt the growth of pathogenic bacteria that are not low in pH (Andriani, 2008). The
anti-inflammatory effects caused can inhibit the appearance of proinflammatory cytokines.
BAL (Lactic Acid Bacteria) is a group of beneficial microorganisms that are not toxic to their
hosts and are capable of producing compounds that can kill pathogenic bacteria
(Klaenhammer, 2005).
The factors that influence the effectiveness of the use of sinbiotics is the dose of
probiotic bacteria used. Probiotics are living microorganisms which, when given to hosts,
both humans and animals, in sufficient quantities will provide health benefits (FAO / WHO,
2002). In this study the dose of probiotics contained in synbiotics was 6.25 x 107 CFU /
gram. The dosage is in accordance with FAO standards which say that probiotics that reach
the digestive tract up to 105 CFU / ml or grams will show the functional effects of probiotics
(FAO / WHO, 2002)

2. Ileal Histopathology

K- K+ P1
400x 400x 400x

P2 P3
400x 400x
Image 2. Ileal Histopathology

The results of the study of the effect of synbiotic combination of Lactobacillus

acidophilus and Spirulina platensis on experimental animals infected by Salmonella
enteritidis on examination of ileal histopathological images performed with Hematoxylin-
eosin (HE) staining were analyzed descriptively using a light microscope with 100x and 400x
magnification. The effect of synbiotic administration is indicated by reduced erosion in the
ileal epithelial layer and the number of goblet cells. The ileal histology in normal conditions
is shown in the negative control group. The ileal villi structure in the form of a bulge is used
to expand the surface. On the surface of the villous epithelium there are microvilli which can
increase absorption of nutrients. The mucosa of the ileum consists of the epithelial layer and
lamina propia. In the epithelial layer there are columnal epithelial cells lined with layers
attached to the basement membrane and goblet cells. Goblet cells are collections of glandular
and columnar epithelial cells. Goblet cells produce acid glycoproteins which function to
protect and lubricate the intestinal mucosa. In the lamina propia there are connective tissue,
blood capillaries, and secretory units (Junquira, 2007).
The ileal histopathology in the positive control group (K +) induced by Salmonella
enteritidis without synbiotics showed structural damage in the ileal tissue when compared
with negative controls. Columnar epithelial cells in the mucosa rupture and some cells
disappear. The histopathological description of the ileum in groups P1 and P2 treated with a
synbiotic administration of a combination of Lactobacillus Acidophilus and Spirulina
Platensis 0.2% and 0.4% showed structural damage to the ileal tissue when compared with
negative controls, but not as severe as positive controls. Histopathological histopathology in
P3 group treated with a synbiotic combination of Lactobacillus acidophilus and Spirulina
platensis 0.6% / kgPK showed improvements in the structure of ileal tissue when compared
with positive controls and other treatments. There is no erosion or rupture in the villous
epithelial layer.
On the histopathological observation of the ileum the treatment group with synbiotic
combination of Lactobacillus acidophilus and Spirulina platensis namely P1, P2, and P3
showed an improvement marked by reduced damage to the villous epithelium when
compared with the positive control group. According to Kara (2012), in addition to the
presence of inflammatory cell infiltration, ileal damage due to bacterial infection is also
characterized by damage to epithelial cells. This is also supported by research conducted by
Permatasari (2017), about the effects of Lactobacillus casei bacteria on ileum organs of white
mouse infected with Salmonella enteritidis which showed damage to the ileal epithelium.
Improvements due to synbiotic administration of the ileal epithelial layer in this study
are in accordance with the research conducted by Rahmadi (2013), that Lactobacillus bacteria
have antibacterial properties. Antibacterial activity is caused by medium chain fatty acids by
damaging the cell walls of S. enteritidis. In addition, lactic acid bacteria (LAB) are also
known to inhibit attachment and invasion of pathogenic bacteria in the ileal mucosa.

CONCLUSION
Based on the research that has been done, it can be concluded that the synbiotic
combination of Lactobacillus acidophilus and Spirulina platensis can prevent Salmonellosis
in experimental animals with Salmonella enteritidis-induced Rattus novergicus with an
effective dose of 0.6% / kgPK based on a significant decrease in CD4 levels in organs.
spleen.

SUGGESTION
Based on the research that has been done, it needs to be studied and developed further
about synbiotic doses that are more effective against Salmonella enteritidis infection and also
do research on synbiotic doses that can cause toxic.

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