bacteriophages for the control of vibriosis in...
TRANSCRIPT
Dr.C.R.Subhashini
Bacteriophages for the control of Vibriosis in Aquaculture
PHAGE FORMULATION AGAINST VIBRIOS
MANAGING DIRECTOR,
ARISTOGENE BIOSCIENCES PVT LTD
BANGALORE,
INDIA
Aristogene
ARISTOGENE BIOSCIENCES
DSIR (Government of India) recognized R&D centre
DBT project Grant Won “The BIRAC INNOVATOR AWARD” from Department of BIOTECHNOLOGY, Govt of India.
Awarded 2 projects in aquaculture by Department of Biotechnology, Government of India
Experienced team of scientists each with over 25 years of Industry experience
Bacteriophage (Phage)
• Definition - Obligate intracellular parasites
that multiply inside bacteria by making use of
some or all of the host biosynthetic machinery
• Definición - Obliga los parásitos
intracelulares que se multiplican dentro de las
bacterias haciendo uso de parte o la totalidad
de la maquinaria biosintética del huésped.
Bacteriophage (Phage)
Phages typically carry only the genetic
information needed for replication of their
nucleic acid and synthesis of their protein coats.
They require precursors, energy generation and
ribosomes supplied by their bacterial host cell.
Los fagos típicamente llevan solo la información
genética necesaria para la replicación de su ácido
nucleico y la síntesis de sus capas proteicas.
Requieren precursores, generación de energía y
ribosomas suministrados por su célula huésped
bacteriana.
Bacteriophage (Phage)
• Bacteriophages are among the most common and diverse entities in the
biosphere.--Los bacteriófagos se encuentran entre las entidades más comunes
y diversas de la biosfera.
• Phages are widely distributed in locations populated by bacterial hosts--
Los fagos se distribuyen ampliamente en lugares poblados por huéspedes
bacterianos.
• It has been estimated that there are more than 100 different phage
species and at least 10 phages for each bacterium.--Se ha estimado que
hay más de 100 especies de fagos diferentes y al menos 10 fagos para cada
bacteria.
Where we can find phages
• In human and animal intestines ( En intestinos humanos y animales)
• In running water (En agua corriente)
• Effluents (Efluentes)
• In the soil (En la tierra)
• Sewage (Aguas residuals)
¿Dónde podemos encontrar fagos?
What is Phage Therapy?
• Treatment of infections using specific natural agents (bacteriophages) that attack only bacteria
• Tratamiento de infecciones utilizando agentesnaturales específicos (bacteriófagos) que atacan solo a las bacterias.The first evidence for a viral-like agent with antibacterialproperties was reported by M. E. Hankin in 1896.
Found in the Ganges river in India, it was temperature sensitive,capable of passing through a porcelain filter, and could reducetitres of the bacterium Vibrio cholerae in laboratory culture.
Hankin suggested that it might help to decrease the incidence ofcholera in people using water from the Ganges.
¿Qué es la terapia de fagos?
LYSIS OF BACTERIA BY PHAGES
My enemy’s enemy is my friend
WHY PHAGE THERAPY IN AQUACULTURE?
¿POR QUÉ TERAPIA DE FAGA EN ACUICULTURA?
Disadvantages of antibiotics -Desventajas de los antibióticos.
• Emergence of multiple drug resistant strains which in turn enters the
human system - Aparición de múltiples cepas resistentes a los
medicamentos que a su vez ingresan al sistema humano.
• Problem of residues and export rejection -Problema de residuos y
rechazo a la exportación.
• Limited number of antibiotic in the pipeline
• Therefore, antibiotics are no longer the preferred treatment against
vibriosis in shrimp culture and alternative methods are being sought
-Por lo tanto, los antibióticos ya no son el tratamiento preferido
contra la vibriosis en el cultivo de camarones y se buscan métodos
alternativos.
Are phage our answer to antibiotic
resistance?
¿Son los fagos nuestra respuesta a la
resistencia a los antibióticos?
Need new approaches.. In aquaculture, impossible to vaccinate each and every
animal.
Need New Approaches to Combat Infections - Necesita
nuevos enfoques para combatir las infecciones
Bacteriophages appear to be natural plausible and
appropriate candidate to overcome the above problems
-Los bacteriófagos parecen ser plausibles y candidatos
apropiados para superar los problemas anteriores.
Already ubiquitously present in unprocessed foods
Advantages of Phage Therapy
• Self-replicating therapeutic Terapéuticoautorreplicante
• Self-limiting -Phages thrive in the presence of bacteria, and die out in their absence. Autolimitantes: los fagos prosperan en presencia de bacterias y mueren en su ausencia.
• One phage/bacterium is sufficient
• “Green Natural Alternative” "Alternativa verdenatural"
• Highly host-specific✓No damage to normal (beneficial ) flora
• Sin daño a la flora normal (beneficiosa)
Ventajas de la terapia con Phages
Phages: safety. Theoretical considerations 2
Bacteriophages are numerous and ubiquitous:Los bacteriófagos son numerosos y ubicuos:
NumerousEstimate of total number of tailed phage particles on Earth:
4-6 x 1031 = 10-fold of number of prokaryotes. Bergh. 1989. Nature 340: 467-468Whitamn et al. 1998. PNAS 95: 6578-6583
UbiquitousUp to 109 phages per ml of surface watersIn animal sera, in vaccines, in foodE. coli phages in 11% of faeces of healthy personsB. fragilis phages in 68% of faeces of healthy persons
"We live in a sea of phages" "Vivimos en un mar de fagos"
Phage therapy: efficacy, animal studies 1
• Phage Therapy as an Approach to Prevent Vibrio anguillarum
Infections in Fish Larvae Production. Yolanda J. Silva et al , 2014
• Biswas et al. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect Immun 70: 204-210.
• Wagenaar et al. 2005. Phage therapy reduces Campylobacter jejuni colonization in broiler chickens. Vet. Microbiol. 19: 275-283.
Institute for Animal Disease Research in Houghton, Cambridgeshire, UK
Diarrhoea causing E. coli in mice, calves, lambs and piglets,
Treatment with bacteriophages
reduces the number of bacteria from 107 to 102 in 2 hours, and
stops the associated fluid loss.
--> survival of all treated animals, compared to the placebo group
• Smith WH and Huggins MB. 1983. Effectiveness of phages in treating experimental
Escherichia coli diarrhoea in calves, piglets and lambs.
J. Gen. Microbiol. 129: 2659-2675.
• Smith, et al. 1987. The control of experimental Escherichia coli diarrhoea in calves
by means of bacteriophages. J. Gen. Microbiol. 133: 1111-1126.
Phage therapy: efficacy, animal studies 2
Phage therapy efficacy: applications in humans 1
"Vaccination" study in Tbilisi, Georgia (1965)
30,769 children aging 6 months to 7 years old
17,044 children ingested bacteriophages against Shigella dysenteriae13,725 children, living at the opposite side of the streets, served as a control group.
[Dysentery incidence in control group is 2.6 fold higher than phage treated group]
Babalova et al. 1968. Preventive value of dried dysentery bacteriophage. Zh. Mikrobiol. Epidemiol. Immunobiol. 2: 143-145.
To sum up
Phages are everywhere: We live in a sea of phages.
Phages are safe
In-adequacies of previous Phage therapy era are well understood
More information & better tools are available now
Well controlled animal studies done in recent years have confirmed efficacy & safety of phage therapy
Polish studies have shown that Phage-therapy can tackle drug resistant pathogens
The world is re-considering Phage-therapy
It could be a stand alone therapy or Valuable adjunct to probiotics
Ultimate clean, green, eco-friendly disease control system
Vibriosis
• All the diseases caused by vibriospecies is called as vibriosis
• Constitutes natural flora of shrimp
• Opportunistic pathogen-causes disease when shrimp are stressed / weak.
VIBRIOSIS
• Septic hepatopancreatic necrosis (SHPN),
• Luminescent vibriosis,
• Swollen hindgut,
• Shell disease, appendage necrosis or rot, splinters
• EMS/ AHPND (Acute hepatopancreatic necrosis disease)
• White feces
• RMS
EPIZOOTIOLOGY
• All life stages affected (E, L, PL, J, A).
• Enteric, systemic or external infections.
• Antibiotic resistance easily developed.
• Many vibrio infections are opportunistic or secondary.
• Worldwide occurrence.
• Some strains/species may be highly pathogenic - AHPND
LUMINISCENCE BACTERIA (BACTERIAS DE LUMINISCENCIA)
White feces
• White fecal matter floating on the water surface• Reduced feeding• Size variation• Poor growth• Loose shell• Slow mortality
RMS-Signs and symptoms
• Antennae –red and necrotic- stage 1
• Red tail and red legs- stage 2
• Yellow cephalothorax- stage 3
• Reddish yellow cephalothorax- stage 4
• Anorexia
• Mortality
VIBRIOSIS IN HATCHERIES
• Hatcheries seed production often suffer setbacks due
to vibrio and suffer enormous economic losses.
• Vibriosis continue to cause chronic mortalities of up
to 30% under stressful conditions.
Why vibrio loads are high hatchery environment?
¿Por qué las cargas de vibrio son un entorno de alta incubadora?
• Vibrio is halophilic- sea water is the suitable medium - halófilo
• High temperature during LRT (31-32°C) favorable for vibrio multiplication-
• Following inputs supply sufficient nutrients for the vibrio to multiply
Inputs which are responsible for vibrio loads
Artemia biomass (Biomasa de artemia) Unfertilized eggs (Huevos no fertilizados)
Dead eggs (Huevos Muertos) Egg shells (Cáscaras de huevo)
Inputs which are responsible for vibrio loadsDead nauplii and exuvia - Nauplios muertos y exuvia
• 5 stages• 5 moults• For example 2 milloin nauplii in holding tank, it has 10 million exuvia• Exuvia= chitin• Vibrio is chitinoclastic
Inputs which are responsible for vibrio loadsDead algae Vibrio isolated from outdoor algae
Inputs which are responsible for vibrio loadsFecal matter Dead larvae
• Suppose 2 million naupliistocked
• 40% final survival
• 1.2 million dead larvae
Inputs which are responsible for vibrio loadsTank bottom sediments
Dead artemianauplii
Artemiashells
Symptoms of vibriosis
Necrosis in mysis Necrosis in PL
ATROPHY OF HEPATOPANCREAS
Electron microscopic view of eggs with vibrio
Vibriosis in shrimp grow-out cultureThe shrimp pond is like a bank! –
A lot is deposited; very little is withdrawn -
¡El estanque de camarones es como un banco!
Se deposita mucho; Se retira muy poco.
Rearing pond
Vitamins, additives, binders, uneaten
pellets
+ Vibrios
Thousands of postlarvae with
associated bacteria+ Feces, metabolites,
old exoskeleton, mortalities + Vibrios
S h r i m p Algae and zooplankton grow and die Organic matter builds-up
Water quality changes Microorganisms grow Biofilms
form
+Vibrios
Shrim
p
Advantages of using Bacteriophage therapy in Aquaculture
Outline of workdone
Outline of workdoneHost range determination: All the isolated phages were checked on the various isolates and standard strains by spot test. Few of the phages showed a very good host range
Testing of phages on Vibrio isolate from Ecuador
Assay showing the bacterial lysis
0
0.5
1
1.5
2
2.5
3
1 2 3 4 5 6 7 8 9
Control V15
φ1a 1MOI
ΦB1 0.1 MOI
φVH17 0.1MOI
Φvh20 0.1MOI
φVH21 0.1MOI
TimeControl φ1a ΦB1 φVH17
Φvh20 φVH21
(in hours) V15 1MOI 0.1 MOI 0.1MOI
0.1MOI 0.1MOI
0 0.021 0.024 0.019 0.0260.021 0.029
1 0.109 0.115 0.123 0.1410.1
2 0.125
2 0.198 0.171 0.168 0.1890.153 0.15
3 1.051 0.651 0.762 1.0090.632 0.798
4 1.233 0.315 0.456 1.2510.421 0.286
5 1.489 0.329 0.436 1.310.319 0.201
6 1.532 0.289 0.357 1.5640.278 0.192
24 1.971 0.082 0.098 1.9230.085 0.096
48 2.531 0.079 0.042 1.9870.069 0.067
Time in hours
Ab
sorb
ance
at
60
0n
m
Whole Genome sequencing:
• Phage 1a purified by PEG method from the lysate• Phage DNA isolated and purified by silica column after
removal of host DNA using DNAse.• Whole genome sequenced and annotated• This had 90% similarity with the genome of Vibrio
vulnificus phage SSP002 which is a lytic phage.
SDS-PAGE analysis of phage proteins
External Lab trials
• External lab trials were conducted at National centre for aquatic animal health, Cochin
• Two trials of challenge studies were carried out
• Controlled challenge studies demonstrated complete protection against Vibrio challenge in VIBRIOSHIELD treated post larvae as against 55% mortality in untreated group. Challenge was done using Vibrio harveyi at a dose of 106 cfu/ml of water.
TANK NO TIME
INTERVAL
Total Bacterial count in cfu/ml
Luminescent Bacterial count in cfu/ml
Total vibrio harveyi count in cfu/ml
Larval survival in
%
1. Control tank (No bacteria
and no phage)
0hours 4×103 _ _ 100
24 hours 3×104 _ _ 94
48 hours 9.6×104 _ _ 90
72 hours 90
2. Only
bacteria
0hours 2×106 2.2×105 4.2×105 100
24 hours 2.8×106 4×104 6.8×105 73
48 hours 4.9×105 _ 3×104 61
72 hours 55
3. Bacteria +
lyo. Φ cocktail 1
( Φ1a, ΦVH3, ΦVH20)
0hours 2×106 2.2×105 4.2×105 100
24 hours 2.6×104 _ 1.6×102 96
48 hours 1.1×104 _ 2×101 91
72 hours 90
Bacteriophage Therapy under laboratory conditions Conducted at
National Centre for Aquatic Animal Health (NCAAH), Cochin
TANK NO TIME
INTERVAL
Total Bacterial count in cfu/ml
Luminescent Bacterial count in cfu/ml
Total vibrio harveyi count in cfu/ml
Larval survival in
%
4. Bacteria +
lyo. Φ cocktail 2
( Φ1a, ΦVH3, ΦVH19)
0hours 2×106 2.2×105 4.2×105 100
24 hours 2.3×104 _ 9.4×103 91
48 hours 2.3×104 _ 1×101 87
72 hours 87
5. Bacteria + antibiotics
0hours 2×106 2.2×105 4.2×105 100
24 hours 3.28×104 _ _ 94
48 hours 2.1×104 _ _ 90
72 hours 89
6. Bacteria + Φcocktail
lysate ( Φ1a, ΦVH3,
ΦVH20)
0hours 2×106 2.2×105 4.2×105 100
24 hours 1.6×104 _ 4.8×103 93
48 hours 1.9×104 _ 1.3×102 88
72 hours 87
Bacteriophage Therapy under
laboratory conditions
Bacteriophage Therapy- NCAAH, Cochin
0
10
20
30
40
50
60
70
80
90
100
Mean Larval survival (+/-SD)
Field Trial 1– Blue star marines
BACTERIOPHAGE 1MOI
Field Trial 2 - Kannur- India
• Here phage cocktail was applied to a 10000 l tank containing 6.4 lakhs of nauplii for 10 days.
Field Trial 2
Field Trial 2
BacteriophageCONTROL
Decrease of Vibrio count on tcbs agar after bacteriophage treatment (FIELD TRIAL )
24 hours0 day
72 hours 96 hours
48 hours
Total vibrio count in Probiotics VsVibrioshield tanks - Hatchery 1 kakinada
No of days Probiotics Vibrioshield
Day 14 x 10 6 x 10
Day 2 4.4 x 102 1.6 x 102
Day 3 5.2 x 103 1.7 x 103
Day 4 4.3 x 103 1.6 x 103
Day 5 2.6 x 104 2.3 x 103
Day 6 4.9 x 104 2.7 x 103
Day 7 2.3 x 104 4.5 x 103
Day 8 3.0 x 104 1.9 x 103
Day 9 3.7 x 104 2.9 x 102
Day 10 1.7 x 105 2.1 x 102
Day 11 1.2 x 105 2.1 x 102
0
1
2
3
4
5
6
0 2 4 6 8 10 12
Log
VIB
RIO
CO
UN
T C
FU/m
l
DAYS
Total vibrio count in Probiotics Vs Vibrioshield tanks
Probiotics Vibrioshield
Survivability- Hatchery 1 kakinada
Average Highest
Probiotics 30 60
Bacteriophage 40 70
Tank capacity: 12 tons: Average water filling : 8 tons
Stocking density: 150 naupli/litre
Comparison of probiotics with Bacteriophage
• Place: Kakinada
• 13 th Oct 2017
• Temperature -31.50c, Salinity -30ppt, alkalinity -105 PPM, pH -7.5, Nitrite -0, Nitrate -0, Ammonia -0
• 300 l of water with 40000 naupli each
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
MIC-F Bactocell EPICIN -D ECOTECH-H Vibrioshield
Surv
ivab
ility
%
Probiotic A PB-B PB-C PB-D Bacteriophage
Results of Bacteriophage performance-Hatchery 2 Marakkanam
0
10
20
30
40
50
60
Surv
ivab
ility
%Survivability %
Control VibrioshieldBacteriophage
Tank Capacity: 12 tons
Stocking quantity: 1.5 million
Date: 8/11/17 to 25/11/17
Place: Marakkanam, Tamil Nadu
Survivability
Bacteriophage 57 %
Probiotics 45%
Feedback of farm
• Drastic reduction of total vibrio counts
• Reduction of running mortality syndrome
• Reduction of White faecal disease where there is no EHP involvement
Bacteriophage therapy
• Several hatcheries have reported that the larvalsurvivability increased markedly with the bacteriophagetherapy.
• There is a drastic reduction in vibrio counts in the LRTs
• Bacteriophage therapy can be stand alone therapy or canbe used adjunct with probiotics
•ROAD SHOWS
Road Shows
BIRAC Innovator award from DBT
Team
DR.C.R.SUBHASHINI
MANAGING DIRECTOR,
ARISTOGENE BIOSCIENCES PVT LTD
BANGALORE, INDIA
EMAIL: [email protected]