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Vibrio in shrimp aquaculture

Causes, symptoms and prevention measures

Members of the microorganism genus Vibrio have become a major constraint on production and trade in shrimp industry. They are responsible for several diseases and mortalities of up to 100 percent, causing global losses of around US$ 3 billion. Vibrio-related infections frequently occur in hatcheries, but epizootics also commonly occur in grow-out pond. 

Vibriosis is a bacterial disease caused Vibrio which is Gram-negative, motile, facultative anaerobe bacteria of the family Vibrionaceae. It is ubiquitous throughout the world and all marine crustaceans, including shrimp. 

Vibrio spp. are natural micro-flora of wild and cultured shrimps (Sinderman, 1990), and become opportunistic pathogens when natural defence mechanisms are suppressed (Brock and Lightner, 1990). In intensive systems, shellfish species are often exposed to stressful conditions due to the high stocking density, leading to secondary vibriosis.  

Pathogenic strains

Vibrio usually associated with multiple etiological agents. However some Vibrio species have been identified as primary pathogen, several species of Vibrio including V. parahaemolyticus and V. harveyi have been described as the main pathogenic species in shrimp.  

V. harveyi is one of the most important etiologic agents of mass mortalities of penaeid larval rearing systems. Epizootics occur in all life stages but are more common in hatcheries.  

V. parahaemolyticus is a halophilic bacterium distributed in temperate and tropical coastal waters throughout the world. Some strains can cause acute gastroenteritis in humans, often after the consumption of contaminated seafood (Matsumoto et al., 2000).  

There are some other pathogenic strain Vibrio that have been reported in association with shrimp disease and caused massive epidemics such as V. vulnificus, V. anguillarum, V. campbelli, and V. splendidus (Chen 1992; Nash et al., 1992). 

Isolation and Identification  

Thiosulfate-citrate-bile salts-sucrose agar (TCBS agar), is a type of selective agar culture plate that is used in microbiologylaboratories to isolate Vibrio species. TCBS agar is highly selective for the isolation of Vibrio species.  

Vibrio isolates may be identified by a number of methods, including: Gram stain, motility, oxidase test, mode of glucose utilisation, growth in the presence of NaCl, nitrate reduction and luminescence. Vibrio species may be identified rapidly in the field using the PCR (polymerase chain reaction). 

Shrimp diseases associated to Vibrio 

A bacteria’s capacity to cause disease, or virulence, is a complex process affected by many variables, including host, Vibrio strain, developmental stages, physiological conditions, environmental stress, and infection method.  

Luminous disease is caused by V. harveyi. In hatchery both white shrimp and black tiger shrimp, V. harveyi is the major pathogen. Luminous bacteria associated with exoskeleton, gills and gut were isolated and quantified 

Early mortality syndrome or acute Hepatopancreatic necrosis (EMS/AHPND) is a disease caused by the bacterium V. parahaemolyticus. AHPND typically affects shrimp that have not reached marketable size (40 days or younger). It causes large scale deaths among cultivated shrimp and infected shrimp pond can be entirely wiped out. 

White feces disease (WFD) is associated to several pathogen including Vibrio. Somboon et al. (2012) reported seven species of Vibrio were isolated from WFD shrimp (L. vannamei) in the study. These species were 
V. vulnificus, V. fluvialis,
V. parahaemolyticus, V. alginolyticus,
V damselae (Photobacterium damselae), 
V. mimicus
 and V. cholera (non01).  

 

Vibrio species

% of disease infected shrimp with each species of Vibrio

Haemolymph

Intestine

V. vulnificus

80

80

V. fluvialis

44

44

V. parahaemolyticus

26

26

V. alginolyticus

20

20

V. damselae (Photobacterium damselae)

18

18

V. mimicus

8

8

V. cholera (non01)

6

6

Jayasree et al. (2006) reported occurrence of five types of diseases found in P. monodon in culture pond in Andhra Pradesh, India: tail necrosis, shell disease, red disease, loose shell syndrome (LSS) and white gut disease (WGD). Among these, LSS, WGD, and red disease caused mass mortalities in shrimp culture ponds. There are six Vibrio species are associated to these disease such as V. harveyi, V. parahaemolyticus, V. alginolyticus, V. anguillarum, V. vulnificus and V. splendidus

Signs of Vibrio disease 

Vibrio infections are commonly known as black shell disease, tail rot, septic hepatopancreatic necrosis, brown gill disease, swollen hindgut syndrome and luminous bacterial disease, describing a number of clinical signs:  

  • Lethargy  
  • Loss of appetite 
  • Discoloured and necrotic hepatopancreas with the presence of “clumping“(aggregation of digestive cells)  
  • Red discolouration of the body  
  • Yellowing of the gill tissue  
  • White patches in the abdominal muscle  
  • Melanisation  
  • Granulomatous encapsulation, necrosis and inflammation of organs (lymphoid organ, gills, heart etc.) 
  • Luminescence 
Electron micrograph of a Vibrio parahaemolyticus cell
Electron micrograph of a Vibrio parahaemolyticus cell.
Scale bar = 1µm. Source: BIOMIN
L. vannamei with greenish fluorescence on tail
L. vannamei with greenish fluorescence on tail
(Courtesy Dariano Krummenauer)
Necrosis on the muscular fiber caused by colonies of V. parahaemolyticus
Necrosis on the muscular fiber caused by colonies of V. parahaemolyticus
(Courtesy Dariano Krummenauer)

Trigger and co-infection 

Diseases outbreaks caused by Vibrio may occur when environmental factors trigger the rapid multiplication of bacteria already tolerated at low levels within shrimp blood (Sizemore & Davis, 1985), or by bacterial penetration of host barriers.

As opportunistic bacteria and associated with multiple etiological agents, rapid multiplication also triggered by primary disease. Phuoc et al., (2008) reported on (Table 2) the rapid multiplication of V. campbellii in co-infection with white spot syndrome virus (WSSV). 

Table 2. Quantification of WSSV-infected cells and V. campbellii (mean±SD) in gills (G), stomach epithelium (SE), lymphoid organ (LO) and haematopoietic tissue (HP) of shrimp collected 6 h after V. campbellii injection (shrimp in dual treatment were moribund). 

Treatments VC (CFU ml-1 of haemolymph)

 

WSSV-infected cells in organs

 

G (cells mm-2)

SE (cells mm-2)

LO (cells mm-2)

HP (cells mm-2)

WSSV

189 ± 130a

14 ± 9a

59 ± 72a

210 ± 154a

-

VC

-

-

-

-

231 ± 445a

WSSV + VC

183 ± 51a

15 ± 6a

39 ± 18a

143 ± 86a

83430 ± 66871a

G=Gills; SE=Stomach epithelium; HP=Haematopoietic tissue; LO=Lymphoid organ; WSSV=White spot syndrome virus; VC=Vibrio campbellii; CFU=Colony forming unit. Numbers of infected cells in the same tissue or CFU ml−1 with different superscripts were significantly different between the two treatments (P<0.01).

The high V. harveyi density also observed in a field study at the end of the culture period and correlated with the outbreak of white spot disease Kannapiran et al. (2009). 

Management strategies for shrimp disease prevention and control 

Vibrio are difficult to eradicate because they adapt well to different environmental conditions and can adopt a dormant state when facing adverse conditions.  

In shrimp pond, Vibrio are living in shrimp gut and pond water. Managing pond water and gut health are important to control Vibrio. And frequent surveillance to know Vibrio population in shrimp gut and pond water to understand their level. 

Use of antibiotics to control these agents has led to problems of drug resistance and resulted in trade restrictions in export markets. Shrimp aquaculture continues to find more effective and environmentally friendly approaches of improving shrimp health and yields.  

There are several strategies to prevent and control Vibrio in shrimp aquaculture: 

  • Biosecurity 
  • Immunostimulants 
  • Probiotics 
  • Anti-microbials 
Biosecurity 

Filtration, disinfection and sanitation are such of biosecurity practices. Disinfectant may use to treat water source as biosecurity on hatchery and grow-out period. V. harveyi in the water column and can be inactivated by Chlorine. In Hatchery, luminescent vibriosis may be controlled in the hatchery by washing eggs with iodine or chlorine.  

Other approach is the prevention of infection by using specific pathogen free (SPF) shrimp. Such shrimp are genetically improved stocks known to be free of one or more specified pathogens and will ensure that seed shrimp are not the conduit for introduction of pathogens (Lotz, 1997). However, SPF status is a temporary condition which isn´t passed on genetically and is lost once the SPF broodstock are transferred to a commercial facility.  

Immunostimulants 

Immune-stimulants of shrimp are widely accepted technology that promotes the immune response, have had success in reducing shrimp mortalities associated with vibriosis. Shrimp possess a non-specific immune system without antibodies, they are not enabled to specifically “remember” exposure to pathogens, which is the basis of vaccination. Consequently, the efficiency of response on subsequent encounters may be limited.  

A study conducted to evaluate the efficacy of autolysis yeast (Levabon® Aquagrow) to control the effect Vibrio. Challenge test was used V. parahaemolyticus (EMS strain). Autolysis yeast was supplemented continuously and pulsed to know the best administration method with inclusion rate 3 g/ton and 5 g/ton. 

Mortalities were happened in all treatments due to the virulent pathogen (Figure 2). Highest survival in Levabon® Aquagrow supplemented continuously 53%. Lowest survival was in control 37% and pulsed application offered some protection survival 43%. 

Survival rate (%) of L. vannamei dietary supplemented Levabon® Aquagrow (continues and pulse application), after V. parahaemolyticus challenge.
Figure 2. Survival rate (%) of L. vannamei dietary supplemented Levabon® Aquagrow (continuous and pulse application), after V. parahaemolyticus challenge.
Source: BIOMIN
Probiotics 

Probiotics are another means of disease control which have found use in aquaculture. Probiotics are administered directly into the water or via feeds. 

The mode of action of the probiotics is rarely investigated, but possibilities include competitive exclusion, that is, the probiotics actively inhibit the colonization of potential pathogens in the digestive tract by production of bactericidal substances, competition for nutrients and space, and modulation of the immune-system. The stimulation of host immunity and exclusion of pathogens may provide greater non-specific disease protection as a result of both immunity enhancement and competitive exclusion (Rengpipat et al., 2000).  

There is accumulating evidence that the prophylactic use of beneficial bacteria is effective at inhibiting a wide range of fish pathogens. According to recent analysis from BIOMIN Research Center, certain probiotic species seems to be better than others in inhibiting the growth of the pathogenic V. parahaemolyticus. As Figure 3 shows, probiotic strains such as Lactobacillus reuteri, Pediococcus acidilactici, Enterococcus faecium and B. subtilis (proprietary BIOMIN probiotics) were shown to inhibit V. parahaemolyticus. This shows that not every menace can be targeted with Bacillus bacteria. Within the Bacillus family, one out of six strains was able to inhibit the growth of virulent V. parahaemolyticus

Varying effectiveness of probiotic bacteria against pathogenic V. parahaemolyticus.
Figure 3. Varying effectiveness of probiotic bacteria against pathogenic V. parahaemolyticus.
Source: BIOMIN
Anti-microbials 

Strategies to use anti-microbial to reduce the effects of the Vibrio (particularly V. parahaemolyticus) in the shrimp digestive system can help protect the animal.  

Certain essential oil mixtures and organic acid mixtures have been shown effective for their inhibitory potential towards Vibrio

These compounds can be added to the feed to have an effect in the digestive system of the animal. The acid mixture in Figure 4 inhibited V. parahaemolyticus growth by 80% to 95% at a concentration of 5000 ppm. The minimal effective dose is between 1000 and 5000 ppm. Essential oils mixtures have also been demonstrated to possess an inhibitory potential, such as the one in Figure 5 which inhibits V. parahaemolyticus growth by 80% to 85%. The minimal effective dose is between 100 and 500 ppm. 

In vitro inhibition potential of an organic acid mixture against virulent V. parahaemolyticus.
Figure 4. In vitro inhibition potential of an organic acid mixture against virulent V. parahaemolyticus.
Source: BIOMIN
Growth inhibition of virulent V. paraemolyticus after exposure to an essential oil mixture.
Figure 5. Growth inhibition of virulent V. paraemolyticus after exposure to an essential oil mixture.
Source: BIOMIN
References 

Brock, J.A. and Lightner, D.V. 1990. Chapter 3: Diseases of crustacea. In: O. Kinne (ed.) Diseases of marine animals Vol. 3, Biologische Anstalt Helgoland, Hamburg. Pp. 245-424. 

Chen, D. 1992. An overview of the disease situation, diagnostic techniques, treatments and preventatives used on shrimp farms in China. In: W. Fuls and K.L.Main (eds.) Diseases of cultured penaeid shrimp in Asia and the Unites States. The Oceanic Institute, Hawaii. Pp. 47-55.  

Jayasree, L., Janakiram, P. and Madhavi, R. 2006. Characterization of Vibrio spp. Associated with diseased shrimp from culture ponds of Andhra Pradesh (India). Journal of the World Aquaculture Society, Volume 37 Issue 4 Page 523. 

Kannapiran, E., Ravindran, J., Chandrasekar, C., Kalaiarasi, A. 2009. Studies on luminous, Vibrio harveyi associated with shrimp culture system rearing Penaeus monodon. J. Environ. Biol. 30 (5): 791-795. 

Lotz, J.M. 1997. Viruses, biosecurity and specific pathogen-free stocks in shrimp aquaculture. World Journal of Microbiology and Biotechnology 13(4): 405-413 

Matsumoto, C., Okuda, J., Ishibashi, M., Iwanaga, M., Vi Garg, P., Rammamurthy, T., Wong, H., DePaola, A., Kim, Y.B., Albert, M.J., Nishibuchi, M. 2000. Pandemic Spread of an O3:K6 Clone of Vibrio parahaemolyticus and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and toxRS Sequence Analyses. Journal of Clinical Microbiology 38(2): 578-585. 

Nash, G., Nithimathachoke, C., Tungmandi, C., Arkarjamorn, A., Prathanpipat, P., Ruamthaveesub, P. 1992. Vibriosis and its control in pond-reared Penaeus monodon in Thailand. In: M. Shariff, R.P. Subasinghe and J.R. Authur (eds.) Diseases in Asian Aquaculture 1. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Pp. 143-155.  

Phuoc, L.H., Corteel, M., Nauwynck, H.J.,  Pensaert, M.B., Alday-Sanz, V., Van den Broeck, W., Sorgeloos, P. and Bossier, P. 2009. Effect of dose and challenge route of Vibrio spp. on co-infection with white spot syndrome virus in Penaeus vannamei. Aquaculture (290): 61-68. 

Rengpipat, S., Rukpratanporn, S., Piyatiratitivorakul, S., Menasaveta, P. 2000. Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). Aquaculture 191: 271-28. 

Sindermann, C.J. 1990. Principal diseases of marine fish and shellfish, Vol. 2, 2nd edition. Academic Press, New York.  

Sizemore RK, Davis JW (1985) Source of Vibrio spp. found in the hemolymph of the blue crab Callinectes sapidus. J Invertebr Pathol .46:109–110. 

Somboon, M.,  Purivirojkul, W., Limsuwan, C., Chuchird, N. 2012. Effect of Vibrio spp. in white feces infected shrimp in Chanthaburi, Thailand. Kasetsart University Fisheries Research Bulletin. Vol. 36 (1). Pp 7-15. 

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