DOI: https://doi.org/10.52973/rcfcv-e32148
Received: 11/05/2022 Accepted: 15/06/2022 Published: 31/07/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32148, 1 - 9
ABSTRACT
This work aims to evaluate looks at the antibacterial ecacy of BACTI-
NIL®AQUA when added to the feed of Penaeus vannamei in experimental
infection with Vibrio parahaemolyticus (M0904AHPND+strain).
Results show that adition of BACTI-NIL®AQUA at 3,000 and 5,000
part per million (ppm), causes inhibition growth zones of 15.00 ± 0.50
milimeters (mm) and 17.00 ± 0.30 mm, respectively. The challenge with
V. parahaemolyticus resulted in 60% survival for organisms fed doses
3,000 ppm of BACTI-NIL®AQUA and 60% for those fed 5,000 ppm of
BACTI-NIL®AQUA, resulting in twice the amount of survival as opposed
to 13.33% (4 organism) in the positive control at 24 hours post-infection.
Histopathological alterations in the hepatopancreas with hemocytic
inltration within the intertubular connective tissue were observed.
Also, tubules with severe cell detachment and tubular atrophy were
detected in the positive control organisms, and organisms treated
with of BACTI-NIL®AQUA only had vermiform structures in the tubular
lumen, cell detachment and inltration of hemolymph in intertubular
connective tissue. According to the analysis of the studied variables,
it can be concluded that of BACTI-NIL®AQUA is a promising alternative
for V. parahaemolyticus control in shrimp culture.
Key words: BACTI-NIL®AQUA; Vibrio parahaemolyticus; Penaeus
vannamei; acute hepatopancreatic necrosis disease;
early mortality syndrome
RESUMEN
El objetivo de esta investigación fue evaluar la ecacia de dos dosis de
BACTI-NIL®AQUA adicionada al alimento en una infección experimental
de 24 horas (h) en juveniles de Penaeus vannamei. Los resultados de
este estudio mostraron que BACTI-NIL®AQUA, a concentraciones
de 3.000 y 5.000 partes por millón (ppm) inhibe el crecimiento
de Vibrio parahaemolyticus (cepa M0904AHPND+), causante de la
enfermedad de necrosis hepatopancreática aguda en camarones.
En estas concentraciones, se observaron zonas de crecimiento de
15,00 ± 0,50 milímetros (mm) para 3.000 ppm y 17,00 ± 0,30 mm para
5.000 ppm, respectivamente. Se obtuvo una sobrevivencia del 60%
para ambas dosis, el doble de sobrevivencia que el control positivo
13,33% (4 organismos) a las 24 h post-infección. En los organismos
control positivo se observaron alteraciones histopatológicas en los
túbulos del hepatopáncreas con desprendimiento celular severo e
inltración hemocítica dentro del tejido conectivo intertubular. En
los organismos tratados con BACTI-NIL®AQUA solo se observaron
estructuras vermiformes en el lúmen de los túbulos del hepatopáncreas.
Con los resultados del presente estudio se puede concluir que BACTI-
NIL®AQUA adicionado al alimento es una alternativa prometedora para
el control de V. parahaemolyticus en cultivo de camarón.
Palabras clave: BACTI-NIL®AQUA; Vibrio parahaemolyticus;
Penaeus vannamei; enfermedad de la necrosis
hepatopancreática aguda; síndrome de la mortalidad
temprana
Ecacy of BACTI-NIL®AQUA in Experimental infection with Vibrio
parahaemolyticus in juvenile of Litopenaeus vannamei
Ecacia de BACTI-NIL®AQUA en infección experimental con Vibrio parahaemolyticus
en juveniles de Litopenaeus vannamei
María Soledad Morales-Covarrubias
1
* , María del Carmen Bolan-Mejía
1
, Noemí García-Aguilar
1
, María-Mercè Isern-Subich
2
,
Gilberto Hernández-González
2
and Waldo Gabriel Nuez-Ortín
2
1
Center for Research in Food and Development A.C., Mazatlán Unit in Aquaculture and Environmental Management. Sinaloa, México.
2
ADISSEO. Antony, France.
*Email: marisol@ciad.mx
BACTIL-NIL®AQUA in infection with Vibrio parahaemolyticus in Litopenaus vannamei / Morales-Covarrubias et al. __________________________
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INTRODUCTION
Marine shrimp farming has increased dramatically, from around
100 tons in the 1980s to about 4.5 million tons in 2018; the top ve
producers being China, Thailand, Vietnam, Indonesia, and Ecuador.
The Pacific white shrimp (Litopenaeus vannamei) is the main
cultivated aquatic species in the world, with a value of USD 18.46
billion in 2018 [10].
Despite this success, shrimp farming continues to suffer important
economic losses due to the impact of mainly viral diseases. More
recently, more concerns arised when bacterial diseases such as
Acute hepatopancreatic necrosis disease (AHPND), formerly known as
early mortality syndrome (EMS) started to showed. This is a bacterial
shrimp disease due to the action of Pir A and B toxin secreted by
Vibrio parahaemolyticus [31] capable of destroying the cells (E, R, F,
and B) of the hepatopancreas, causing detachment of the tubular
epithelial cells, hemocytic inltration, and very marked necrosis of
the hepatopancreas [36]. In the terminal phase, in addition to the
shedding of epithelial cells, a massive secondary bacterial infection
occurs [26, 36]. Vibrio parahaemolyticus, V. campbellii, V. owensii, and
V. punensis have been proved to cause AHPND.
However, the mechanisms underlying the burgeoning number
of Vibrio species that cause AHPND is not complete known. All of
AHPND-causing Vibrio bacteria (V
AHPND
) harbor a highly homologous
plasmid (designated as pVA1-type) carrying pirAB
vp
toxin genes [12].
AHPND is characterized by sudden and massive mortalities (100%)
in post larvae or juveniles with 30 to 35 days (d) of culture [12, 25].
Antibiotics are used in aquaculture to control the development of
bacteria during the production process. However, misuse has led
to antibiotic resistance in both humans and animals. At present,
the use of these compounds is being restricted by the resistance
they can induce in different groups of microorganisms, either by
mismanagement of the effective doses or by new mechanisms that
allow them to generate resistance [1, 7]. The shrimp industry requires
other alternatives to inhibit microbial ora in production systems.
Among the sustainable strategies applied to modulate the intestinal
microora of shrimp, wide varieties of natural compounds are used,
such as organic acids (OA).
Specic OA alone or in combination are one of the alternatives for
nutritionally sustainable and environmentally friendly production. OA
are oxygenated compounds derived from hydrocarbons, and have
been widely used in formulations for animal nutrition. OA included
in balanced food function as preservatives, lowering the pH and
reducing microbial growth; although the main application is as an
antimicrobial with action in the digestive tract [1, 27].
The main effect of OA in shrimp is due to the fact that undissociated
molecules penetrate the cell walls of Gram-negative pathogenic
bacteria and acidies their cytoplasmic pH, for which the bacterial
cell needs to neutralize its pH through the proton pump H
+
-ATPase,
causing excessive energy expenditure. As undissociated OA molecules
continue to penetrate the bacterial cell walls, they rapidly deplete
their energy reserves, ultimately leading to cell death [27]. However,
there are very few studies on the antibacterial ecacy of OA in vivo
and in vitro, in shrimp with experimental infections. The primary
antimicrobial action of BACTI-NIL®AQUA (a synergistic blend of OA
mixture) is by altering the cell cytoplasm pH of bacteria and those that
are sensitive to such changes are inhibited, thus reducing harmful
bacteria within the gastrointestinal tract of the host animal.
The aim of the study was to determine the effect of dietary
supplementation of BACTI-NIL®AQUA (a synergistic blend of OA
mixture) on survival to V. parahaemolyticus (M0904AHPND+strain)
infection in L. vannamei and alterations in hepatopancreas using
wet analysis and histopathological analysis.
MATERIALS AND METHODS
Bacterial suspension preparation (inoculum)
A sample of V. parahaemolyticus used in this experiment was isolated
from shrimp farms affected by AHPND in north-western Mexico and
cryopreserved (Panasonic-U53VA-PA. USA) at –80°C [33]. The strain
was recovered from cryovials, inoculated in 10 mililiters (mL) of tryptic
soy broth (TSB) + 2.0% NaCl (TSB+ Bioxon), and incubated in a rotary
shaker (nb-205L N-BIOTEK. México) at 30°C for 24 hours (h). Bacterial
cells were washed by centrifugation (Refrigerated centrifuge FELISA-
TE-CR12-México) (2330 x Gears (G) for 20 minutes (min) at 20°C) and
the optical density (OD600 nanomer (-nm-) was adjusted to 1.0 [21].
One hundred microliters (µL) aliquot were inoculated in 40 mL of
TSB in triplicate; these samples were incubated in a rotary shaker
(VWR-Scientic-1516, USA) at 30 ± 1°C for 24 h, bacterial growth was
estimated by total viable count (TVC) on TCBS agar plates (BD Difco).
Minimum inhibitory concentration (MIC) of BACTI-NIL®AQUA
(a synergistic blend of organic acids) against V. parahaemolyticus.
The MIC was determined in triplicate at concentrations of 500;
1,000; 1,500; 2,000; 3,000; 4,000; 5,000 (provided by the supplier),
8,000; and 9,000 parts per million (ppm) with pH 7.0, 7.5, and 8.0,
adding 100 μL of the bacterial inoculum 1x10
8
Colonies Former Units
(CFU)·mL
-1
and incubated (Shaking incubator-NB205L-Biotek-USA) at
30 ± 1°C for 24 h with constant stirring at a 125 revolution per minutes
(rpm). The samples were visually evaluated and those that did not
show turbidity were established as MIC [18, 20].
Minimum bactericidal concentration (MBC) of BACTI-NIL®AQUA
(a synergistic blend of organic acids) against V. parahaemolyticus.
MBC was determined in triplicate with the concentrations that did
not produce turbidity (MIC), by adding 100 μL of the bacterial inoculum
1x10
8
CFU·mL
-1
in glass tube with tryptic soy broth (Bioxon
©
TSB, Mexico)
and 2.0% NaCl, for 24 h, at 30 ± 1°C. The lowest concentration, in which
colonies former units (CFU) did not occur, was considered MBC [20].
A positive (bacterial inoculum 1x10
8
CFU·mL
-1
) and a negative control
(only tryptic soy broth) were used to ensure adequate bacterial growth
during the incubation period and sterility of the mediums [15, 39].
Sensitivity of BACTI-NIL®AQUA against V. parahaemolyticus
The bactericidal capacity of the BACTI-NIL®AQUA was determined
by Bauer et al. [3] with each experiment done in triplicate. A colony
was selected and placed in a sterile saline solution until its turbidity
matched a Mac Farland standard 0.5 solution [21]. The test was
performed as follows:100 μL of bacterial suspension was added at
a concentration of 1×10
8
CFU·mL
-1
and swabbed on the surface of
Mueller-Hinton agar (MHA) plates, supplemented with 2.5% NaCl,
pH 8.4. Sterile OXOID discs (OXOID antimicrobial susceptibility test
discs) measuring 8 milimeters (mm) diameter were impregnated with
three concentrations of with the BACTI-NIL®AQUA (10, 20 and 50 μL)
in triplicate including a negative control (only sterile OXOID discs)
and incubated at 30 ± 1°C for 24 h. According to the test for bacterial
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sensitivity to antibiotics, effectiveness was classied according to
the inhibition diameter proposed by Celikel and Kavas [8], and it
was achieved.
Food preparation
Commercial feed (Purina 35) was pulverized using a domestic mill.
Subsequently, BACTI-NIL®AQUA was added to make a pre-mix and,
then, distilled water was added at 40°C (Fisherbrand Termo Fisher
Scien – 37200t-China). Pellets were restored using a domestic meat
grinder (3 mm in diameter), dried in an oven (LBI, 30CH220V, MEXICO)
at 40°C for 12 h, and stored (Samsung, RT45VNSW5, MEXICO) at 4°C,
according to Morales-Covarrubias et al. [22].
Sensitivity of BACTI-NIL®AQUA in food
The test was performed as follows: 100 μL of bacterial suspension
was added at a concentration of 1×10
8
CFU·mL
-1
and swabbed on the
surface of Mueller-Hinton agar (MHA) plates, supplemented with
2.5% NaCl, pH 8.4. The feed pellets with BACTI–NIL®AQUA including a
negative control (feed pellets without BACTI–NIL®AQUA) and incubated
at 30 ± 1°C for 24 h.
Food consumption and palatability were assessed using the method
proposed by Morales-Covarrubias et al. [22]. Acrylic aquariums
(25x30x28-CIAD-México) with ve liters (L) of water each were used
in the present bioassay. One organism was placed in each aquarium,
using ve repetitions with each concentration of 3,000 and 5,000
ppm of BACTI-NIL®AQUA, and control (food without OA).
The organisms were fed the diets, providing 30% of their body
weight for ve consecutive d. After a period of 4 h, the uneaten
food (without feces) was recovered from each aquarium, rinsed with
distilled water to remove the salt, and, subsequently, dried in the
oven (40°C). They were cooled (LG GR 282SVF-USA) and weighed to
estimate food consumption with the following formula:
Weight of food consumed = A – B
Where A is the total weight of the initial food, and B the weight of
recovered food.
Experimental animals
A total of 200 juvenile L. vannamei were purchased from local
commercial hatchery with a certificate specifying that were not
detected of
white spot syndrome vírus (WSSV), infectious hypodermal
and hematopoietic necrosis vírus (HHNV) and V. parahamenolyticus.
The organisms were acclimated in Center for Research in Food and
Development (CIAD) for 1 week in 600 L tanks with ltered (10 micro
mol –μM-) seawater (33 salinity) disinfected by ultraviolet (UV) radiation.
Each tank had individual aeration, constant temperature (30 ± 1°C), and
a photoperiod of 12 h light: 12 h dark. Shrimp were fed Camaronina™
daily, which contains 35% protein and 9% lipids at 3% total biomass.
Shrimp were fed 3 times at d.
Before the assay, 25 shrimp (10% prevalence [16]) were removed
from the batch (200 juvenile) to determine their health status by
bacteriological analysis, wet mount analysis [16, 24]. Polymerase
Chain Reaction (PCR) using commercial kits (IQ2000
TM
GeneReach
Biotechnology Corp., Taiwan) and histological analysis for acute
hepatopancreatic necrosis Disease (AHPND), white spot syndrome
vírus (WSSV), infectious hypodermal and hematopoietic necrosis vírus
(IHHNV) and Necrotizing hepatopancreatitis bacterium (NHPB) [16, 36].
Bioassay (for effectiveness and survival record)
A bioassay was conducted for 24 h in 10 L glass tanks with 10
shrimps (3-4 grammes (gr)), not detected pathogens and intermolt
stage) with three replicates per treatment and constant aeration. In
total, 2 treatments of the organic acids mix were used: 3,000 ppm;
4,000 ppm and two controls (positive and negative). Before infection,
an acclimatizing period of 24 h was allowed. The established control
conditions during the test were: 30 ± 1°C, seawater 30% salinity, pH
7.5 – 8.0, ammonium < 0.1 miligrams (mg) L
-1
and oxygen 6 – 8 mg L
-1
.
Infection (bacterial inoculum)
Fifty mL of bacterial inoculum concentration of 1×10
8
UFC·mL
-1
was added directly to an experimental aquarium nal concentration
water 1×10
6
UFC·mL
-1
containing 10 shrimps, for all treatments and
controls. For the negative controls the same concentration of
autoclave heat inactivated bacteria was added (120°C for 15 min by
autoclave (FE-405U-México)).
First feeding was administered after 15 min of inoculation and then
every 4 h until the end of the experiment (24 h) [22].
To evaluate survival rate, 3 replicates from each treatment were
used with 10 shrimps replicate, for a total of 30 shrimps in each
treatment and 120 shrimps overall. The survival rate was calculated
as the survival probability at any particular time (S
t
) [11]. A total of
30 shrimps were used to evaluate the AHPND disease by wet mount
and histological analysis. The surviving shrimps were also xed in
Davidson´s solutions at the end of the experiment.
Wet mount analysis
Immediately after the survival challenge test, diagnosis through
wet mount analysis was done to asses if the surviving or moribund
shrimps had organ and tissue alterations. Their organs and tissue were
removed, dissected and squash mounted with sterile seawater then
examined under the light microscope Olympus BX 60-USA and photo-
documented using an Olympus Innity 2 camera-USA [16, 23, 34].
Histopathological analysis
Bioassay organisms displaying behaviors such as positionment in
aquarium bottom, decubitus and movement of the scaphognathite
(moribund) were extracted and fixed with Davidson solution for
conventional histological processes [4, 24, 36]. Specimens were
paran-embedded, cut into 4 μM sections, stained with hematoxylin
and eosin, and reviewed under the light microscope to detect AHPND
and alterations in hepatopancreas [4, 14, 33, 36].
Lesion severity was graded accordingly to the G-grading system
[16] with G0 being negative and G4 as the highest severity of AHPND.
Briey, tissues graded as G0 are without lesions associated with
AHPND; G1 are mild focal lesions; G2 and G3 are moderate, locally
extensive to multifocal lesions; and G4 are severe, multifocal to diffuse
lesions. Slides were observed under Olympus-USA (BX60) microscopy
and photo-documented using an Olympus (Innity 2) camera.
Statistical analysis
Statistical analysis was conducted with R 3.3.1 (R Windows®)
software. The experimental infection was analyzed by two-way ANOVA
(2x2) (α<0.05). Factor A levels were (P) organic acid group, and (C)
positive control group. Factor B levels were (V) tested with V. harveyi,
MIC test showed that the BACTI-NIL
®
AQUA had
the activity of inhibiting the growth of V. parahaemolyticus
(M0904AHPND+strain) bacteria with a minimum concentration of
3,000 ppm (OA3000) appear clear, while those that appear cloudy
indicate bacterial growth is found in positive control (POSITIVE)
BACTI-NIL
®
AQUA at the doses of 5,000 and 3,000 ppm, with inhibition
Agar diffusion method with food MHA with BACTIL-
NIL®AQUA at the doses of 5,000 (blue arrow) and 3,000 ppm (red
BACTIL-NIL®AQUA in infection with Vibrio parahaemolyticus in Litopenaus vannamei / Morales-Covarrubias et al. __________________________
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and (S) negative control with the same amount, though the bacteria
were inactivated by heat. When the analysis of variance indicated
the difference between the factors, the Holm-Sidak test was used
with a signicance level of 0.05 [19, 40].
RESULTS AND DISCUSSION
Determination of the MIC and CMB
The results of observations of the MIC test showed that the
BACTI-NIL®AQUA had the activity of inhibiting the growth of V.
parahaemolyticus (M0904AHPND+strain) bacteria with a minimum
concentration of 3,000 ppm (FIG. I).
Results of observations of the MIC test visually in tubes to 3,000
ppm (appear clear), while those that appear cloudy indicate bacterial
growth is found in positive controls and tubes with concentrations of
500 ppm to 1,500 ppm. The observations showed control (-) there was
no growth of of V. parahaemolyticus bacterial colonies and no growth
of other bacterial colonies, meaning that there was no contamination
during the dilution of BACTI-NIL®AQUA. The observations showed
that control (+) there was a growth of bacterial colonies, meaning
that the suspension of V. parahaemolyticus 1.0 x 10
8
CFU·mL
-1
that
was used for the living conditions was not contaminated by other
bacteria, as evidenced by the formation of bacterial colonies by
bacterial colonies of V. parahaemolyticus for which the MIC values
also corresponded to MBC.
Antibacterial sensitivity in Petri dish and food
TABLE I illustrates the results of antibacterial sensitivity (mm) with
respect to the concentration of the treatment, with an inhibition zone
greater than 20.0 ± 0.50 mm and 9,000 ppm of BACTI-NIL®AQUA,
i.e., ‘extremely sensitive’. The doses of 5,000 and 3,000 ppm, with
inhibition zone of 17.0 ± 0.03 mm and 15.0 ± 0.50 mm, were within the
‘very sensitive’ range (FIG. 2). The ‘sensitive’ doses were 1,500ppm, with
an inhibition zone of 11.0 ±0.10 mm, and 500 ppm, with a diameter of
10.0 ± 0.03 mm. In food, the dose of 5,000 ppm had a diameter of 14.0±
0.06 mm (very sensitive), and the dose of 3,000 ppm a diameter of 13 ±
0.1 mm (sensitive) (FIG. 3). Based on these results, it was decided to use
the concentrations of 3,000 and 5,000 ppm to perform the ecacy test.
TABLE I
Bacti-nil
®
aqua concentrations and measurements of
on oxid antimicrobial susceptibility test discs
BACTI-NIL®AQUA
Concentration (ppm) Halo (mm)
500 10.0 ± 0.03
1,500 11.0 ± 0.10
3,000 15.0 ± 0.50
5,000 17.0 ±0.03
9,000 20.0 ± 0.50
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Antibacterial capacity of BACTIL-NIL®AQUA assessed in vivo
The average consumption of food with or without acid was not
significantly different between the treatments and the controls
(P<0.05). All the shrimps of the positive control exhibited red antennae
and uropods ten min after inoculation (FIG.4A). After 30 min the
shrimps exhibited muscle opacity, cramping, and erratic swimming
with permanence at the bottom of the tank (FIG. 4B). After two h, the
shrimps had empty intestines with pale hepatopancreas (FIG. 4C), and
swam in the decubitus position with static lapses (FIG.4D).
Mortality occurred after 3 h of bacterial inoculation, ending after 19 h
with 86.66% (26 shrimps) of accumulated mortality. At the end of the test
period (24 h), the negative control showed normal swimming behavior,
without color change, and survival of 100% (30 organisms) (FIG. 5).
Organisms treated with BACTI-NIL®AQUA at a dose of 3,000 ppm
exhibited clinical signs 3 h after infection, with reddish coloration of
antennae and uropods, muscular opacity, cramping, discoloration of the
hepatopancreas, and swam in the decubitus position with permanence
at the bottom of the aquarium. Seven h after infection, mortality rates
were recorded (12 organisms). These rates decreased and there was a
survival rate of 60% at the end of the 24 h bioassay (FIG.5).
The shrimps treated with 5,000 ppm exhibited the same changes in
behavior and coloration as the organisms treated with the 3,000ppm.
Mortality (12 shrimp) occurred 6 h after infection, and there was a
survival rate of 60% at the end of the test (FIG.5).
TABLE II
Food consumption per day
Food consumption
Days
BACTIL-NIL®AQUA
3,000 ppm
(mg)
5,000 ppm
(mg)
1 30 25
2 31 27
3 32 23
4 29 26
5 30 24
Food consumption assessment
A favorable intake was observed thirty min after feeding, since
the organisms immediately captured the food. The intestines were
lled with continuous strand of feces when evacuated, for which it
was considered a positive acceptance for the consumption of the
food with the two concentrations of the mixtures OA. The shrimp
consumed approximately 30 miligrams (mg) of food per d with a dose
of 3,000 ppm, and 25 mg·d
-1
with a dose of 5,000 ppm (TABLE II).
Rates of survival at the end of the 24 h bioassay
Histological sections of tissues of Litopenaeus vannamei
BACTIL-NIL®AQUA in infection with Vibrio parahaemolyticus in Litopenaus vannamei / Morales-Covarrubias et al. __________________________
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Statistically, no significant differences were observed in the
organisms treated with 3,000 and 5,000 ppm, since the survival rate
was the same 24 h after infection (P>0.05). Signicant differences
were observed between controls and treatments when performing
the Holm-Sidak multiple comparison test (P<0.05).
Histological analysis
The organisms of the negative control did not exhibit changes in
hepatopancreas. They showed normal hepatopancreatic tubules,
with embryonic (E), brillar (F), reserve (R), and secretory or globular
cells (B) (FIG. 6A).
Histopathological examinations of positive control organisms
revealed separation of hepatopancreas myoepithelial layer and
epithelium, inltration of hemocytes within the interstitial sinuses,
rupture and collapse of hepatopancreatic tubules, with cellular
detachment of the intestine proximal region (Grade III) (FIG. 6D).
The treated organisms in antennal gland exhibited inltration
of hemocytes and pyknotic nuclei (FIG. 6B), gill with inltration of
hemocytes apical zone, minor hyperplasia and rupture and collapse
of tubules (FIG. 6C) and hepatopancreas, with rupture and collapse
of hepatopancreatic tubules (Grade I) (FIG. 7).
Litopenaeus vannamei histological section of
hepatopancreas with separation of the epithelial membrane
hepatopancreatic cells (blue arrows) from proximal área to the
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The results of the present study indicated that BACTI-NIL®AQUA,
inhibites the growth of V. parahaemolyticus in vivo and in vitro, at
concentrations of 3,000 and 5,000 ppm. In vivo was suggested that
BACTI-NIL®AQUA was ingested and thus inhibited the growth of of V.
parahaemolyticus. However, at a higher pathogens, 5,000 ppm could
potentially show differences in comparison to 3,000 ppm. Adams and
Boopathy [1] found that formic acid, at a concentration of 5,000 ppm,
inhibited the growth of V. harveyi; whereas Ng et al. [27] observed that
lactic and citric acids, at a concentration of 10,000 ppm higher than
that reported in the present study, inhibited the growth of V. harveyi.
The survival percentage of the ecacy bioassay in the shrimps fed
with the addition of 3,000 and 5,000 ppm of BACTI-NIL®AQUA was
60%. In this regard, Ng et al. [27] reported that prawns fed with the
addition of 10,000 ppm of lactic acid had 65% survival when tested
by intramuscular injection with V. harveyi.
The histopathological analysis indicated that the organisms fed with
the doses of 3,000 and 5,000 ppm exhibited inltration of hemolymph
and hemocytes in the antennal gland, gills, intestinal epithelium, and
the lymphoid organ [37, 38]. In addition, the hepatopancreas exhibited
rupture and collapse of hepatopancreatic tubules (Grade I). Authors such
as Anuta et al. [2], Ng et al. [29] and Romano et al. [30] have documented
that by adding OA to shrimp (white and tiger) diets, tolerance to pathogens
increased. These authors performed experimental infections with V.
harveyi and observed minor changes such as rupture, cell detachment,
and collapse of the tubules in hepatopancreas.
It has been reported that synergistic activities between OA may
occur and provide a broader spectrum of antimicrobial protection
than the exclusive use of one type of OA [5, 6, 9, 28, 32, 35]. Probably,
this synergy favored tolerance to V. harveyi, since the present study
showed low mortality and minor changes in organs and tissues,
especially in hepatopancreas [13, 34], since only Grade I rupture and
collapse of hepatopancreatic tubules was observed in organisms fed
with 3,000 and 5,000 ppm of BACTI-NIL®AQUA during the ecacy test.
This result was relevant, because the hepatopancreas was
the largest organ of crustaceans and fullls different functions,
including secretion of digestive enzymes, digestion and absorption,
storage of mineral reserves and organic substances, metabolism of
carbohydrates and lipids, distribution of reserves stored during the
intermoult cycle, and catabolism of products of the ingested diets [17].
CONCLUSIONS
The results obtained in the present study allowed inferring that the
doses of 3,000 and 5,000 ppm of BACTI-NIL®AQUA added to food, in
experimental infection for ecacy, improved the biological response
regarding bacterial infections of shrimp L. vannamei in comparison
to those specimens who did not receive. This way, these results are
certainly encouraging, given that BACTI-NIL®AQUA added to food showed
benecial properties that can help reduce the dependence on antibiotics
in shrimp farming and, therefore, produce an antibiotic-free product.
ACKNOWLEDGMENT
This work was partially funded by CIAD grant 20657-MCMS.
CONFLICTS OF INTEREST
The authors of this paper deny any nancial or personal relationship
with other people or organizations that could inappropriately inuence
or bias the content of the paper.
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