Identification of Vibrio sp.

as cause of white feces

diseases in white shrimp Penaeus vannamei and
handling with herbal ingredients in East Lampung
Regency, Indonesia
Supono, Wardiyanto, Esti Harpeni, Annisa H. Khotimah, Astri Ningtyas

Department of Aquaculture, Faculty of Agriculture, Lampung University, Lampung

Bandar Lampung, Indonesia. Corresponding author: Supono,
supono_unila@yahoo.com

Abstract. White feces disease (WFD) is one of the diseases that attack white shrimp (Penaeus
vannamei). This disease causes cultivation failure and huge loss for shrimp farmers. The cause of this
disease is thought to be due to the abundance of the Vibrio bacteria population in cultivation media. This
study was aimed to determine the abundance of Vibrio sp. and the presence of Vibrio sp. as a trigger for
WFD in P. vannamei ponds and to study the use of several herbal ingredients in suppressing the growth
of Vibrio sp. Samples were taken from secondary canals, tertiary canals, primary canals, shrimp pond
waters that were not infected with WFD, shrimp pond water infected with WFD, and shrimp infected by
WFD. Samples taken from those locations were then inoculated; the total population of the bacteria was
calculated, identified and tested for anti-bacterial activity using several herbal products. According to the
results of the study, Vibrio abundance was obtained as follows: water sample was positive of WFD by
3.5±0.9×105 CFU mL-1, shrimp intestine was positive of WFD by 4.4±0.1×105 CFU mL-1, primary canal of
3.9±2.×104 CFU mL-1, secondary canal of 1.0±0.1×105 CFU mL-1, tertiary canal 3.2±1.1×105 CFU mL-1,
shrimp pond 1 of 2.2±0.3×105 CFU mL-1, shrimp pond 2 of 1.3±0.3×105 CFU mL-1, shrimp pond 3 of
5.2±1.0×104 CFU mL-1, and healthy shrimp intestine of ≤2.5±0,5×104 CFU mL-1. The type of Vibrio
identified and suspected of triggering WFD disease were V. vulnificus, V. parahaemolyticus, and V.
alginolyticus. Antibacterial test showed that mangrove leaf extract (Rhizophora apiculata) had the best
inhibitory effect on V. parahaemolyticus (zone of inhibition of 5.61 mm), followed by ketapang leaf
extract (4.9 mm zone of inhibition) and papaya leaf extract (zone of inhibition of 4.5 mm). The best
concentration of mangrove leaf extract in suppressing the growth of Vibrio parahaemolyticus was 700 mg
L-1.
Key Words: Vibrio abundance, WFD triggers, antibacterial activity, Vibrio parahaemolyticus, Rhizophora
apiculata.

Introduction. White shrimp Penaeus vannamei is a type of shrimp that is widely

cultivated in Indonesia. This is because these shrimp have promising prospects and
profits (Babu et al 2014). However, disease attacks often occur in P. vannamei
cultivation. Diseases are commonly caused by bacteria, parasites, viruses, and fungi.
This disease attack occurs when environmental conditions, pathogens, and cultivan are
not balanced (Engering et al 2013). Pathogens that often attack P. vannamei are Vibrio
(Khamesipour et al 2014; Widowati et al 2018).
One of the diseases that appear in shrimp farming is white feces disease (WFD).
This disease is characterized by the appearance of white feces on the surface of the
culture medium. WFD is allegedly caused by an abundance of Vibrio sp. on cultivation
media (Jayadi et al 2016). The increase in the number of Vibrio sp. is due to adverse
environmental conditions, especially high organic matter content. According to Taslihan
et al (2015), increasment of the shrimp production can be maintain by monitoring the
quality of water, providing the proper feed, and appropriate administration of probiotic or
antibiotic doses. However, the use of antibiotics is not recommended in shrimp farming
because it leaves a residue that endangers consumers. Safe ingredients that have
antibacterial potential can be obtained from some plants. Herbal ingredients or medicinal

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plants are all types of plants that can be used as medicinal herbs, both singly and in a
mixture that is considered and believed to cure a disease or can have an influence on
health. Herbal ingredients are used because they are safe and do not produce residues in
cultivated organisms so that they are safe for consumption and friendly to the
environment (Yuhana et al 2008; Aminzare et al 2015; Ventola 2015).
Some herbal ingredients that can be used include papaya (Carica papaya),
ketapang (Terminalia catappa), and mangrove (Rhizophora apiculata) leaf extracts. C.
papaya leaf extract contains flavonoids, tannins, alkaloids, vitamin C, karpain,
cyanogenic, glucosides, and papain enzymes so that it has the potential as an
antioxidant and antiseptic (Eleazu et al 2012). While T. catappa leaf extract has an
effective antibacterial chemical content (bactericidal).
One area of shrimp farming in Indonesia that faces an epidemic of WFD is Pasir
Sakti District, East Lampung Regency, Lampung Province. This region is one of the
shrimp producing regions in Lampung Province. The condition of the shrimp ponds in
that location has been attacked by White feces disease (WFD) which causes a huge loss
for shrimp farmers. The study was aimed to determine the cause of WFD and its
treatment needs to be conducted to reduce the incidence of WFD and prevent failure of
shrimp farming using herbal ingredients.

Material and Method

Study site. The research was conducted in East Lampung Regency, Indonesia. Samples
were taken from secondary canals, tertiary canals, primary canals, shrimp pond waters
that were not infected with WFD, shrimp pond water infected with WFD, and shrimp
infected by WFD. Research location can be seen in Figure 1.

Figure 1. Research location.

Sample collection. Sampling was carried out by taking 100 mL water samples using a
sample bottles. Samples were taken from 6 locations with 3 replicates for each location.
Taken Samples were directly inoculated on TCBS media. Samples also were collected
from healty and WFD infected P. vannamei.

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Total bacterial count. After incubation for 24 hours, a growing bacterial colony was
calculated by placing a petri dish on top of the Colony Counter device and the bacterial
colony was then calculated and counted using the formula:

N=
Where:
N = Number of colonies (CFU mL-1)
= Number of calculated of bacterial colonies on the petri dishes
= Dilution Factor
= Sample Volume

Bacterial identification. Identification of Vibrio was carried out by a quadrant stroke

method with several stages to obtain 1 pure isolate. The isolate were then identified.
Identification of Vibrio sp. was carried out using MICROBACTTM 24 E Gram Negative
Identification System (OXOID) and read using software microrobact 2000. Observation
of cell morphology includes gram staining, cell shape, and motility test. Physiological
properties include catalase test, indole test, MR-VP test, Citrate Simmons test, and TSIA
test.

Bacterial medium. A total of 3.2 g of Nutrient Agar (NA) media was weighed and then
placed into erlenmeyer and diluted with 160 mL of sea water. Erlenmeyer tube
containing nutrient agar was heated using a hot plate until the solution was
homogeneous, then autoclaved for 15 minutes with a temperature of 121°C under a
pressure of 1 atm. A total of 20 mL of sterile nutrient agar was poured into a petri dish
and leaved until it solidified. The pouring was done in the laminar air flow to prevent
contamination.

Bacterial cultivation. Nutrient Broth (NB) media was weighed as much as 0.09 g then
transferred into erlenmeyer and 90 mL of sea water was added. The erlenmeyer tube
containing media NB was heated using a hot plate until the solution was homogeneous
and heated by autoclaving for 15 minutes at a temperature of 121°C under a pressure of
1 atm. Pure culture of Vibrio parahaemolyticus was inoculated aseptically into 2 test
tubes containing 15 mL sterile Nutrient Broth (NB) media. Test tube was incubated in
shaking incubator for 4 to 5 hours (Tampemawa et al 2016).

Antibacterial activity test of herbal ingrediennts. A total of 20 µL of liquid isolate V.

parahaemolyticus with a density of 10 7 CFU mL-1 were dropped on Nutrient Agar (NA)
media and was leveled with spreader. Disc paper was placed on the media containing a
spread of bacteria with a little pressure. A total of 40 µL of C. papaya leaf extract, T.
catappa leaf extract, and R. apiculata leaf extract with a concentration of 500 mg L-1 in
each extract were dripped on a paper disc with a diameter of 6 mm. The petri dish was
then incubated for 24 hours. Furthermore, measurement of the zone of inhibition of the
extracts against bacteria was carried out. The ability to inhibit bacteria is characterized
by the formation of a clear zone around the tested disc papers.

Determination of the best concentration. A total of 20 µL of liquid isolate Vibrio

parahaemolyticus with a density of 107 CFU mL-1 were dropped on Nutrient Agar (NA)
media and was leveled with spreader. Disc paper was placed on the media containing a
spread of isolate with a little pressure. A total of 40 µL of R. apiculata leaf extract was
dropped on paper disks measuring 6 mm with concentrations of 300, 400, 500, 600, and
700 mg L-1. Positive control was represented by giving paper discs containing
chloramphenicol antibiotics while negative control was neutral disc paper (water
disinfectant only). All treatments were incubated for 24 hours. After the incubation
period, the diameter of the zone of inhibition formed around the disc paper was observed
and measured.

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Statistical analysis. The data of Vibrio sp. abundance in the study field were presented
in table form and analyzed descriptively according to the research objectives. Data of
antibacterial activities were analyzed using Anova followed by least significant difference
(significance of differences at p<0.05).

Results. In the primary canal or the main canal, abundance of Vibrio sp. was
3.9±2.1×104 CFU mL-1 so that this canal is still at safety level for shrimp culture
(Taslihan et al 2015). The total abundance of Vibrio sp. on secondary canals was
1.0±0.1×105 CFU mL-1 so that it exceeds the safe limit in the waters. While in the
tertiary canal which is the inlet and outlet canal, the total abundance of Vibrio sp. was
3.2±1.1×105 CFU mL-1. Moreover, the total abundance of Vibrio sp. in shrimp pond 1
was 2.2±0.3×105 CFU mL-1 and 1.3±0.3×105 CFU mL-1 in shrimp pond 2. While total
abundance of Vibrio sp. in shrimp pond 2 was 5.2±1.0×104 CFU mL-1. The abundance of
Vibrio sp. in all study sites is presented in Table 1.

Table 1
The abundance of Vibrio sp. in all study sites

Sample Abundance of Vibrio (CFU mL-1)

Water (+) 3.5±0.9×105
Primary canal 3.9±2.1×104
Secondary canal 1.0±0.1×105
Tertiary canal 3.2±1.1×105
Shrimp pond 1 (+) 2.2±0.3×105
Shrimp pond 2 1.3±0.3×105
Shrimp pond 3 5.2±1.0×104
Shrimp intestine (+) 4.4±0.1×105
Healthy sShrimp intestine ≤2.5±0.5×104
+ = infected with WFD.

Color of bacterial colony in TCBSA media. TCBSA media is a selective medium that
can inhibit the growth of undesirable bacteria and can distinguish Vibrio into 2 groups,
namely a group that ferments sucrose characterized by yellow colonies and a group that
does not ferment sucrose characterized by green colonies.
The characteristics of the colonies of V. parahaemolyticus and V. vulnificus on the
TCBSA media included round, green or bluish green colonies in the middle of colonies
with a diameter of 2-3 mm and did not ferment sucrose. Colonies of V. parahaemolyticus
and V. vulnificus were found in secondary, tertiary, shrimp pond 1 and shrimp pond 2
samples. Primary canal and shrimp pond 3 samples showed morphological characteristics
of yellow colonies and the ability of the colony to ferment sucrose (Figure 2). This was
seen from colony changes from green to yellow on TCBSA media. The characteristics of
these colonies are typical V. alginolyticus, V. fluvialis, Listonella anguillara, and others.

A
B

Figure 2. Color of Vibrio sp. in the media. A - Yellow-colored Vibrio sp. colony, B - Green-
colored Vibrio sp. colony.

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Bacterial identification. Identification of Vibrio sp. at the study sites was carried out
by taking the most formed colonies on TVC (Total Vibrio Count). Identification was
carried out using the MICROBACTTM 24 E Gram Negative Identification System (OXOID)
and the results were read by the microbact 2000 software. The results of the
identification carried out are presented in Table 2.

Table 2
Identification Results of Vibrio sp. at the study sites

Sample Kind of Vibrio

Feces (+) V. parahaemolyticus
Primary canal V. alginolyticus
Secondary canal V. vulnificus
Tertiary canal V. vulnificus
Shrimp pond 1 ( infected with WFD) V. parahaemolyticus
Shrimp pond 2 V. alginolyticus
Shrimp pond 3 V. alginolyticus
Shrimp intestine (+) V. parahaemolyticus

Antibacterial activity of herbal ingredients. The results of the measurement of

antibacterial activity from several extracts of herbal ingredients with a concentration of
500 mg L-1 incubated for 24 hours can be seen in Table 3.

Table 3
Observation of antibacterial activity of herbal ingredients

Inhibition zone diameter (mm)

Tested material Average (mm)
I II III
C. papaya leaf extract 3.57 4.68 5.25 4.5±0.85
T. catappa leaf extract 3.64 5.58 5.46 4.9±1.08
R. apiculata leaf extract 4.11 6.28 6.44 5.6±1.30

Concerning the antibacterial activity of herbal ingredients tested, based on statistical

analysis, there is no significant diffrenece among treatments.

The best concentration of mangrove leaf extract. The results of the measurement
of antibacterial activity were indicated by the presence of zone of inhibitions, namely
zone of inhibitions where bacteria did not grow around paper discs of growth of V.
parahaemolyticus after 24 hours incubation can be seen in Table 4.

Table 4
Observation of antibacterial activity of mangrove leaf extract

Concentration of R. Inhibition zone diameter (mm)

apiculata leaf Average (mm)
Sample I Sample II Sample III
extract (mg L-1)
300 4.87 4.98 5.44 5.09±0.30
400 5.26 5.14 5.79 5.39±0.35
500 6.42 6.50 7.57 6.83±0.64
600 7.11 7.19 7.36 7.22±0.13
700 8.25 8.11 8.16 8.17±0.07

In the antibacterial testing of herbal leaf extract in inhibiting the growth of V.

parahaemolyticus, positive controls were used in the form of chloramphenicol antibiotics
and negative controls in the form of distilled water. Antibacterial testing using
chloramphenicol showed that the diameter of the zone of inhibition produced was 10.98

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mm which was categorized as medium. Desinfected water do not form zone of inhibition
because it is a are pure water without the content of active compounds that have
antibacterial properties (Tampemawa et al 2016). Thus, each concentration shows a
different zone of inhibition. The greater the concentration of extract the greater the zone
of inhibition formed. The higher the concentration of R. apiculata leaf extract used, the
more antibacterial content will be, thus it can inhibit the growth of V. parahaemolyticus.
Statistical analysis showed that different concentration of R. apiculata leaf extract
affected on inhibition toward V. parahaemolyticus growth.

Discussion. Secondary and tertiary canals are generally closed canals that are not
crossed by heavy irrigation canals. In addition, both canals have a level of organic
matter that tends to be higher than of the primary canal due to waste from previous
crop. This causes a buildup of the amount of bacteria and total organic matter (TOM) in
the canal at low tide. Total organic matter will trigger the growth of Vibrio sp. (Eiler et al
2003). The high total organic matter in tertiary and secondary canals was due to waste
from shrimp pond which cannot be released totally into the sea. In this study area, inlet
and outlet of pond were in one canal. Pond it is filled when high tide occurs and emptyed
when at low.
The condition of shrimp pond 2 and shrimp pond 3 have not shown clinical
symptoms of WFD attack, however, total abundance of Vibrio ≥104 CFU mL-1 can trigger
WFD attacks. This can be seen from the results obtained in WFD positive water samples
and WFD positive shrimp intestines which have a total abundance of Vibrio sp. of
3.5±0.9×105 CFU mL-1 and 4.4±0.1×105 CFU mL-1 respectively. At the location of the
farm, previously were WFD and WSSV (White Spot Syndrome Virus) outbreaks, so that it
did not rule out the possibility of WFD disease attacks again. Healthy shrimp intestines
have Vibrio bacteria with a total abundance of ≤2.5±0.5×10 4 CFU mL-1, which indicates
that Vibrio presence in healthy shrimp does not become a pathogen because the nature
of the bacteria is opportunistic, but can be pathogenic in certain conditions. Research
conducted by Kharisma & Manan (2012) reported that, in vaname shrimp, Vibrio
abundance exceeding 104 CFU mL-1 was susceptible to attack by Vibriosis. According to
Taslihan et al (2015) the presence of Vibrio bacteria which exceeds 104 CFU mL-1 can
cause mass death in cultivated shrimp.
Low population of pathogenic bacteria in cultivation media will provide better
shrimp health conditions so that growth of shrimp is better (Nurbaya et al 2010).
According to Kharisma & Manan (2012), total bacteria that exceeds the threshold has
the potential to increase disease infection which in turn causes mass mortality of
cultured shrimp. Increased population of Vibrio sp. can be caused by high dissolved
organic matter from the rest of the feed and shrimp feces (Paena et al 2018).
Identification results showed several types of Vibrio, namely V. parahaemolyticus,
V. vulnificus, and V. alginolyticus. The group that was successfully isolated at the study
site showed a low variety of species. This indicates that the shrimp pond waters do not
contain many types of Vibrio.
Vibrio is the dominant flora in eggs, larvae and post-larvae of shrimp (Hameed et
al 2003). According to Otta & Karunasagar (2001), an increase in organic material
sourced is derived from feed and feces encourage the microflora to develop into
opportunistic pathogens. Limsuwan (2010) reported that V. harveyi, V. vulnificus, V.
parahaemolyticus, V. alginolyticus, and Vibrio sp. are pathogenic bacteria that are
always found in hatcheries and shrimp ponds. According to Taslihah et al (2015) and
Anjaini et al (2018), WFD is caused by microsporidia (from the Enterocytozoon group)
and gregarin (allegedly derived from the Nematopsis sp.) incorporated with Vibrio. Some
Vibrio are identified in WFD-infected shrimp, including V. vulnificus, V. fluvialis, V.
parahaemolyticus, V. alginolyticus, V. mimicus, V. cholerae, and Listonella damsela.
Ralalage et al (2017) found V. alginolyticus and V. fluvialis as the causes of white feces
diseases in P. monodon grow-out ponds in Sri Lanka.
The results of the measurement of the antibacterial activity of C. papaya, T.
catappa and R. apiculata leaf extracts showed the showed the antibacterial potential
againts Vibrio sp. They had different zone of inhibition values of which C. papaya leaf

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extract was 4.5 mm, T. catappa leaf extract was 4.9 mm, and R. apiculata leaf extract
5.61 mm. R. apiculata leaf extract had the highest average zone of inhibition compared
to C. papaya leaf and T. catappa leaf extracts. The difference in the average zone of
inhibition of C. papaya, T. catappa, and mangrove leaf extract was due to the
antibacterial compounds contained in each of the herbal leaf extracts having different
amounts of active compounds.
Marshall et al (2015) reported that antibacterial compounds found in C. papaya
leaf extract are alkaloid of 0.05 g, flavonoids of 2.80 g, saponin of 0.07 g, and tannin of
1.05 g. Whereas T. catappa leaf extract contains antibacterial compounds such as
alkaloid of 1.20 g, flavonoid of 0.93 g, saponin of 2.67 g, tannin of 0.50 g. According to
Okpako et al (2017), the leaf of T. catappa contains saponin, tannin, phenol and
flavonoid as phytochemical compounds, which can surpress the growth of bacteria. A
study conducted by Ekwueme et al (2015) showed that R. apiculata leaf extract
contained active compounds in the form of alkaloid of 3.43 g, flavonoid of 2.67 g,
saponins of 1.97 g, tannin of 4.75 g, steroid of 0.86 g, and terpenoids of 0.87 g.
Antibacterial activity compounds found in herbal leaf extracts have different
mechanisms to suppress bacterial growth. According to Darsana et al (2012), the
mechanism of action of alkaloids as antibacterial is to disrupt the constituent
components of peptidoglycan in bacterial cells so that the cell wall layer is not formed
intact and causes cell death. Flavonoid compounds serve to inhibit cell membrane
function (Kumar et al 2012). While saponins serve to reduce surface tension so that
permeability will rise or cell leakage will occur so that intracellular compounds will come
out (Nuria et al 2009). Tanin works by inhibiting the synthesis of peptidoglycan so that
the bacteria are unable to divide and cells are lyses due to osmotic and physical pressure
which in turn bacterial cells die (Ngajow et al 2013).
The antibacterial activity test of R. apiculata leaf extract showed its ability as an
antibacterial. Antibacterial testing of R. apiculata leaf extract can be seen in Table 4
above which is characterized by the formation of a clear zone which shows that at
concentrations of 300 mg L-1, 400 mg L-1, 500 mg L-1, 600 mg L-1, and 700 mg L-1, R.
apiculata leaf extract was able to inhibit the growth of V. parahaemolyticus.
The measurement results of zone of inhibition diameter of R. apiculata leaf
extract at a concentration of 300 mg L-1 and 400 mg L-1 were categorized as weak of
which 5.09 mm and 5.39 mm, respectively. R. apiculata leaf extract at concentrations of
500 mg L-1, 600 mg L-1, and 700 mg L-1 were categorized as moderate of which 6.83
mm, 7.22 mm; and 8.17 mm, respectively. The value of antibacterial activity at a
concentration of 700 mg L-1 had the highest zone of inhibition with an average value of
the diameter of 8.17 mm which was categorized as moderate.

Conclusions. According to the research results, it can be concluded that:

1. The abundance of bacteria in the study site varied. The abundance of Vibrio sp. as a
trigger for WFD is around 105 CFU mL-1 or can be said exceeding the safe limit of 104
CFU mL-1.
2. Identification on positive control and positive shrimp intestine of WFD, type of Vibrio
sp. found in the study sites were V. parahaemolyticus, V. vulnificus, and V.
alginolyticus. The type of Vibrio sp. as a trigger for WFD is V. parahaemolyticus.
3. The best concentration of mangrove (R. apiculata) leaf extract in suppressing the
growth of V. parahaemolyticus was 700 mg L-1.

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Received: 22 February 2019. Accepted: 25 March 2019. Published online: 03 April 2019.
Authors:
Supono, Lampung University, Faculty of Agriculture, Department of Aquaculture, Indonesia, Lampung, 35145
Bandar Lampung, Jalan Sumantri Brojonegoro No. 1, e-mail: supono_unila@yahoo.com
Wardiyanto, Lampung University, Faculty of Agriculture, Department of Aquaculture, Indonesia, Lampung,
35145 Bandar Lampung, Jalan Sumantri Brojonegoro No. 1, e-mail: wardibdifp@gmail.com
Esti Harpeni, Lampung University, Faculty of Agriculture, Department of Aquaculture, Indonesia, Lampung,
35145 Bandar Lampung, Jalan Sumantri Brojonegoro No. 1, e-mail: edypeni@yahoo.com
Annisa Husnul Khotimah, Lampung University, Faculty of Agriculture, Department of Aquaculture, Indonesia,
Lampung, 35145 Bandar Lampung, Jalan Sumantri Brojonegoro No. 1, e-mail: annisa.husnulkh@yahoo.com
Astri Ningtyas, Lampung University, Faculty of Agriculture, Department of Aquaculture, Indonesia, Lampung,
35145 Bandar Lampung, Jalan Sumantri Brojonegoro No. 1, e-mail: astri.ningtias90@gmail.com
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Supono, Wardiyanto, Harpeni E., Khotimah A. H., Ningtyas A., 2019 Identification of Vibrio sp. as cause of
white feces diseases in white shrimp Penaeus vannamei and handling with herbal ingredients in East Lampung
Regency, Indonesia. AACL Bioflux 12(2):417-425.

AACL Bioflux, 2019, Volume 12, Issue 2. 425

http://www.bioflux.com.ro/aacl