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 Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32148, 1 - 9
ABSTRACT
This work aims to evaluate looks at the antibacterial ecacy 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
inltration 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 inltration 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 ecacia 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
inltración 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
Ecacy of BACTI-NIL®AQUA in Experimental infection with Vibrio
parahaemolyticus in juvenile of Litopenaeus vannamei
Ecacia 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 inltration, 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
microora of shrimp, wide varieties of natural compounds are used,
such as organic acids (OA).
Specic 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 acidies 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 ecacy 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-Scientic-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 classied 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 Innity 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
paran-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.
Briey, 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 (Innity 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,