Bacterial infections of farmed shrimp are quite common and likely the major cause of mortality in farmed shrimp. Vibriosis is one of the most severe bacterial diseases in shrimp farming caused by Vibrio spp. These bacteria from part of the natural microbiota of wild and cultured shrimp and the marine environments.  Vibrio infections have become a major constraint of the production and trade in shrimp aquaculture. They are responsible for several diseases and mortalities of up to 100% and cause crop loses globally.

Vibrio usually associated with multiple etiological agents. Vibrio has been reported in penaeid shrimp culture systems implicating at least 14 species and they are Vibrio harveyi, V. splendidus, V. parahaemolyticus,V. alginolyticus, V. anguillarum, V. vulnificus, V. campbelli, V. fischeri, V.damsella, V. pelagicus, V. orientalis, V. ordalii, V. mediterrani, V. logei etc. However some Vibrio species have been identified as primary pathogen,  including V. parahaemolyticus and V. harveyi have been described as the main pathogenic species in shrimp. 

Clinical signs of Vibriosis

• • Redness of body
  • Antennae cut
  • Atrophy of hepatopancreas
  • White faeces
  • Delayed hemolymph
  • Luminescence in dark
  • Anorexia
  • Lethargy
  • Melanization
  • White patches in the abdominal muscle
  • Expanded chromatophores
  • Luminescence and necrotic areas in the uropods
  • Mortality

Shrimp diseases associated with Vibriosis

Vibrio can remain in the environment without causing disease, but can very easily switch from opportunistic and commensal to pathogenic when conditions change. Thus, its ability to cause disease or increase in virulence, is a complex process affected by many variables, including host, vibrio species, developmental stage, physiological conditions, environmental stress, and infection method.

They are several diseases associated with Vibriosis.

• • V.harveyi - Luminous disease
  • V.Parahaemolyticus – EMS/AHPND
  • Vibro sp. - White Faecal Disease (WFD)

Diagnosis

Diagnosis of vibrio infection is based on clinical signs and the histological demonstration of rod-shaped Vibrio bacteria in lesions, nodules or haemolymph. Excised organs and haemolymph may be streaked on a Vibrio-selective (TCBS) or general marine agar plate. Vibrio isolates may be identified by a number of methods, including: Gram stain, motility, an oxidase test, mode of glucose utilisation, growth in the presence of NaCl, nitrate reduction and luminescence.  

Vibrio threshold in shrimp farms:

There are many  vibrios maximum threshold standards in farms. Most of them are for Vibrio spp. in pond water. These are the common maximum thresholds used by shrimp farms:

• Total Vibrio Count (TVC): varies from 10³CFU/mL to 10⁴ CFU/mL. Some farmers adopt the TVC of 10² CFU/mL as the maximum threshold. Total Vibrio Count (TVC) in shrimp gut is 10⁵ CFU/g
• Vibrio colonies:10² CFU/mL for green colonies and 10³ CFU/mL for yellow colonies.
• Vibrio percentage: 5% to 10% TVC of total plate count, and 10% Vibrio green colonies of TVC.

How to prevent Vibrio problems?

Vibrios are difficult to eradicate because they adopt well to different environment conditions and can adopt state when facing adverse conditions. However, Vibriosis is controlled by rigorous water management and sanitation to prevent the entry of vibrios in the culture water and to reduce stress on the shrimps. Pond Management and robust gut health are important strategies to control vibrio, together with frequent sampling to monitor their levels in the shrimp gut and pond ecosystem.

Good site selection, pond design and pond preparation are also important. Maintain adequate water quality with low bacterial biomass, A stable phytoplankton bloom and a proper feeding program, Sterilize or filter recirculated water, Routinely monitor shrimp and pond for early diagnosis of a problem, Avoid temperature extremes or rapid variation in temperature, handling, overcrowding, and other stressors, Infections opportunistic and probably result from poor husbandry; may be secondary to other disease processes. An increase in daily water exchanges and a reduction in pond biomass by partial harvesting are recommended to reduce mortalities caused by vibriosis.

The combination of gut acidifiers, prebiotics, use of natural antimicrobials and Probiotics, Bacteriophage applications, immunostimulants and non-antibiotic substances has superior specificity against vibriosis and Luminescent Bacteria (LB) coupled with Best Aquaculture Practices (BAP) , which makes it an effective management tool for the control of vibriosis and bacterial toxicity in aquaculture.

Treatment

Use V Phages Growout immediately when various symptoms of Vibriosis as Luminescence in water, infections in shrimp and or low feed intake etc., correlated by lab test. 

Ammonia is the major toxic component produced continuously by shrimps during culture. It can also accumulate in the water due to the decomposition of organic matter as excess feed, faeces, dead shrimp and dead algae. Ammonia is the main end product of protein catabolism in crustaceans and can account for 60–70% of nitrogen excretion with only small amounts of amino acids, urea and uric acid. Of the nitrogenous compounds, ammonia is the most toxic with nitrite and nitrate being less toxic to shrimp.

Ammonia is the primary excretion product of protein metabolism. In water, ammonia is present in both ionized (NH4+) and un-ionized (NH3) state, with NH3 as the toxic form due to its ability to diffuse across cell membranes. Unionized ammonia is highly toxic to the shrimp and must be removed from the system. The relative proportions of NH3 and NH4+ depend on pH, temperature and to a lesser extent on salinity. Ammonia is nitrogenous waste produced from feed input and microbial decomposition of organic matter in water columns. Shrimp feed is usually high in nitrogen.

In vannamei culture, sometimes only 22 percent of the Nitrogen input is converted to harvested shrimp. 57 percent is discharged into the environment and 14 percent remains in the sediment.

Ammonia Toxicity in Vannamei

• High concentrations of ammonia may affect growth rates and survival, and can in extreme cases cause mortality.
• Ammonia damages the gills and reduces the ability of hemolymph to transport oxygen while increasing oxygen consumption by tissues.
• Ammonia stress could affect a variety of physiological functions including respiration, metabolism, excretion.
• Ammonia in the form of un-ionized ammonia (NH3) is very toxic to aquatic animals and cause impairment of cerebral energy metabolism and damage to gill, liver, kidney, spleen, and thyroid tissue in fish, crustaceans and molluscs.
• Ammonia may also increase the moulting frequency of shrimps. Ammonia is also thought to cause damage to the central nervous system.

Ways to prevent ammonia build-up

• Weekly measurements
• Feeding regimes
• Maintaining alkalinity
• Keeping pH steady
• Managing Salinity and Dissolved Oxygen

If you want to solve the problem of Ammonia, you should do the following:

• Complete a 25% water change and retest after a few hours
• Treat with Ammonia Remover
• Proper feed management
• Continue to regularly test your water

White feces disease is one of the most serious problems in shrimp culture. It causes lower productivity in shrimp farming. WFD becomes apparent when the digestive system of shrimp malfunctions and feces turns from normal brownish color to pale white color. WFD has caused significant economic losses to shrimp farmers, because of high FCR, slow growth, and variable sizes of shrimp at harvest. White feces syndrome usually occurs after 60 days of culture (DOC) and it may be accompanied by high shrimp mortality.  The disease can cause moderate to severe economic loss by reducing shrimp survival by 20–30 percent.

Causes

Poor water quality, unhealthy seed, high vibrio loads, Enterocytozoon hepatopenaei (EHP), and the Presence of Protozoa gregarines-like organisms in the intestine and hepatopancreas are some of the reasons for causes of the disease.

Symptoms

• White faecal strings floating on the pond surface
• White/golden brown intestine
• Reduced feed consumption
• Growth retardation
• Reduces in average daily weight gain (ADG)
• Loose shell
• Hepatopancreas becomes whitish and soft.
• Diseased shrimp tend to be darker in color and after some time their bodies will lose firmness and become soft and limp
• Mortality during moulting

Remedies 

Cause of white faeces syndrome and treatment is uncertain. However reduced stocking density, proper water exchange together with better management practices will be helpful in evading White Fecal Disease.

Using of V Phages Growout immediately on observation of few strands on white fecal matter floating on water, or on check tray. Start this treatment to recover shrimps.

EHP or Enterocytozoon Hepatopenaei is an yeast-like fungus belonging to a group called “microsporidia”, which are obligate intracellular parasites. It was first reported as an unnamed microsporidian from growth retarded black tiger shrimp Penaeus monodon from Thailand in 2004. This fungus infects the hepatopancreas of the shrimps, so it’s called as Hepatopancreatic microsporidiosis (HPM). It also has smaller spores (approximately 1 μm in length) and is currently known to infect both P. monodon and P. vannamei.

CAUSES

• Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by enteric fungi, Enterocytozoon hepatopenaei (EHP).
• EHP can be transmitted directly from shrimp to shrimp by cannibalism and cohabitation.
• Shrimps become infected by ingesting spores from the water, from sediment, from eating EHP-infected live feeds (polychaetes, molluscs, frozen artemia biomass etc).

SYMPTOMS

• The target organ of EHP is hepatopancreas and affects its digestive and absorptive functioning resulting in poor growth and immunity.
• EHP infected shrimp may have a thin cuticle, white muscle as a stress response, black spots on their eyestalks, in their muscle tissue and along the hind gut.
• It is associated with severe growth retardation in P.vannamei exhibits high size variability.
• Causes chronic mortality in severe cases.
• Associated with White Fecal Disease.

DIAGNOSTIC MEASURES

• Infection can be checked by microscopic examination ( at x100 oil immersion) of the hp and the gut of the shrimp.
• Infection can also be confirmed by molecular testing of the hp by PCR.

PREVENTION

Regular health assessments are recommended. If there is a large difference in the size of PL, if PL feeding activity is less than expected, the number of lipids in the hp drops, growth and moulting slows, then these are also good indications of infection. The number of swollen tubules may provide an indication of how advanced infections are.

Some important insights on practical solutions for managing EHP is described below:

HATCHERY

• The best approach for maturation and hatchery facilities to avoid EHP is to never use live animals (e.g., live polychaetes, clams, oysters, etc.) as feeds for broodstock. Use only SPF live polychaetes, SPF squid and other fresh feeds like Mussel (subjected to -20 degrees freezing for more than 48hrs) in maturation systems.
• Feed breeders with Redi-Mate in maturation system, a biosecure diet that could replace up to 50% of live polychaetes in commercial trials & 60% fresh feeds (without live polychaetes). 
• Adopt ultrafiltration system in the hatchery system with 0.1micron size or lower to exclude spores.
• Recommended to remodel maturation system by expanding maturation system using imported PPL and accept lower spawn size without the use of live feeds.

 NURSERY

• Stressors can provoke infection of EHP in ponds. Nursed PL can make animals robust and grow faster due to compensatory growth.
• Nursery cuts down culture period and reduce animals exposure to pathogens & stressors
• Adopt water filtration system in shrimp nurseries that could exclude spores.
• Use PL Raceway 40-9, a high quality nursery feeds proven to achieve FCR 0.8 at density 1-12pcs/Liter

GROWOUT

• The spores of EHP have thick walls and are not easy to inactivate. Even high levels of chlorine alone are not effective. Infected ponds should be treated with hot limes. Burnt lime CaMgO and Hydrated (Ca(OH)2) can increase pH and knock down spores at 6mt/ha. Need to moisten the pond prior to application. It needs water to react and activate to increase pH to 14. 
• Stock EHP PCR-negative post larvae 
• Treat all incoming water with 40ppm of 65% active chlorine or 15ppm KMnO4
• Ponds with sandy loam soil and high sedimentation should be PE lined. High suspended solids makes shrimps susceptible to EHP spores through oral ingestion. 
• Manage water quality with the use of effective probiotics. Shrimps infected with EHP and challenge with Vibrio (mixed infection) after exhibit white feces.

Biosecurity in shrimp farming can be defined as the preventive measures for the exclusion of specific pathogens from cultured shrimps. Nowadays, Disease outbreaks in shrimp culture is considered as the highest level of threat. Based on the previous history of the last few years since the development of shrimp culture, mass disease outbreak has caused havoc throughout the shrimp producing regions. Apart from ensuring Pathogen-free status as SPF seeds, farmers should give equal attention to horizontal transmission of diseases from pond to pond, farm to farm, and village to village.

This is done by following precautionary measures, through proper coordination among farmer’s associations regarding judicious use of water resources, seed stocking density, periodic treatment and disposal of sludge, drainage after harvest, closure, and disposal of crops due to viral diseases, etc.

The following biosecurity measures to be followed in shrimp farming:

• Use SPF Post Larvae(PL)
• Use reservoir and water treatment system
• Minimal / Zero water exchange system
• Correct stocking density based on available infrastructure
• Use crab and dog fence
• Use bird scare lines
• Vehicle movement and disinfection
• Personal hygiene and facilities of farm workers
• Disinfection of equipments and nets after every use
• Control movement of workers, within the farm and from outside farm
• Control movement of outsiders not related to that form

Shrimp health management has become the main focus of improving production and minimizing infectious diseases in shrimp ponds for the smooth development of the aquaculture industry. To accomplish this goal, we should be concerned with the quality of post larvae especially the selection of high-health post larvae shrimp, before stocking in the pond.  

Post Larvae selection is important as it will determine the performance and production of the ponds. The objective of larvae selection is to obtain the best survival and growth in the pond. There are several characteristics of good quality post larvae, and visual observation can be used in identifying good quality or healthy post-larvae for stocking.

Some of the useful criteria are given below to help farmers identify high-health post larvae:

1. Activity:     

• Place some PL (about 100 pcs) in a basin, stir the water and check.
• Healthy PL will swim against the current. Weak PL will gather at the center.

2. Size:

• • Good and healthy shrimp Larvae should be even in size.
  • PL 10 should have a total length of 10 mm.
  • The 3 stages form one spine so PL 10 has 3 to 4 spines this is the main character of larval age finding.
  • Uneven size may be due to different Stages, underfeeding, disease, poor water quality, or over-aged Post-Larvae.

3. Disease-free:

a) Check of larvae are red/pink in color - Caused by expansion of Chromophores,it indicates

b) Any chronic or acute mortality - Indicative of advanced disease stress

c) Any limb or appendage necrosis - Indicative of bacteria infection

d) Check of fouling organism, e.g Zoothamnium  - Indicative of poor water quality Overfeeding

e) Check Hepatopancreas -

Brownish – normal

Whitish – disease problem

f) WSSV should be negative        - Accepted with PCR

4. Gut:

• • Full gut indicates good health, empty gut indicates possible stress problem

5. Molting:

• • Molting problem, indicative of nutritional disorders related to sterol, phospholipids or calcium or phosphorous

6. Pigmentation:

 (a) Shape

• Finger like – healthy
• Round Shape – unhealthy

  (b)  Color

• Rust like brown/gray / dark gray – healthy
• Yellow – average
• Blue – unhealthy

7. Muscle development:

The ratio of width of gut and body depth below the gut at the 6^thsegment,

healthy PL should be 1:4 or > 4

8. Antibiotic usage:

• • With: PL shorter, fatter and darker color sometimes with the crooked body.
  • Without: PL longer, thinner and lighter color

9. Stress test:

• • Use 200 PLs for the stress test
  • Temperature – Lower temperature to 20^oC in a short period (5-10 minutes)
  • Strong healthy PL will survive no death should occur.

10. External features:

For a healthy PL, the antennal scales of head portion are closed together whereas the tail fan (uropod) is widely opened. Closed uropods are indicative of young fry not yet suitable for stocking (

11. Feeding activity:

• • Healthy PLs have a good appetite.
  • After acclimatization.
  • PLs should feed actively.
  • When feed is available, the gut should fill up after 10-15 minutes.

• As water pH is one of the most critical chemical parameters for shrimp farming. pH is the measure of hydrogen ion concentration in the water.
• The pH of the pond water is indicative of its fertility or potential productivity.
• The optimum water pH range in the shrimp pond is 7.5 - 8.5. It is essential to stabilize the pH within this range.
• The pH value in the water is normally lowest in the early morning and highest in the afternoon.
• pH levels of the pond water will change depending on the aquatic life within the pond. Carbon dioxide produced by aquatic organisms when they respire has an acidic reaction in the water. The pH in ponds will rise during the day as phytoplankton and other aquatic plants remove CO₂ from the water during photosynthesis. The pH decreases at night because of respiration and the production of CO₂ by all organisms.
• For the best water quality, the maximum diurnal pH fluctuation should not exceed 0.5. It is important to maintain a stable pH at a safe range because it affects the metabolism and other physiological processes of the cultured shrimps.
• The growth of shrimps is retarded if pH falls below 5.0. Water with low pH can be corrected by adding lime to neutralize the acidity.
• If the pH is too high (pH values > 9.5) due to the photosynthesis of algae, we can adjust the pH by changing the water properly and controlling the algae by cultivating microorganisms. The water of excessive alkalinity may also be harmful to shrimp growth and survival.
• The measured pH level will be influenced by the time of the day the sample is taken due to fluctuations in the CO2 level.  Therefore, pH should be measured before dawn for the minimum level and in the afternoon for the maximum level. A sudden drop of more than 0.5 indicates that the water in the tank should be partially changed. 

• The pH will vary in the pond environment depending on a number of factors

  • Acid sulfate soil (acidic alum soil, acidic soil), acidic source of water
  • Rate of rainfalls in pond areas
  • Poorly buffered water
  • Stocking density of shrimps
  • Feeding & rate of sludge formation in the pond bottom.
  • Presence of micro/ macro-organisms.
  • Existence of phytoplankton in pond water.
  • Rate of carbon dioxide production in pond water.
  • Quantity of respire by aquatic species in the pond water.

Effect of pH in Shrimp Culture:

• If pH changes significantly, it can make shrimp shocked, weakened, and stop eating.
• In high or low pH extends for a long time, it will make shrimp grow slowly, stunting growth and susceptible to diseases. It can cause stress, less survival, low production and leads to poor growth.
• Signs of sub-optimal pH include increased mucus on the gill surfaces, black gill disease, damage to the eye lens, abnormal swimming behavior, loose shell, soft shell, irregularity in molt, and poor phytoplankton and zooplankton growth.
• The mortality may occur for culture shrimps in the below pH 4 and above pH 10 range respectively. The low pH levels will cause the shell of the shrimp to become soft. This is due to the shell of the shrimp being composed of calcium carbonate which reacts with acid.

Water Quality is vital for a successful crop. Availability of good quality water is required quantities is one of the most important prerequisites for sustainable aquaculture. While locating the farm site, careful study should be made on the source of water, a quantity of water available during the different seasons, and the quality of water. All instructions and precautions should be followed strictly to get the best out of your farm.

Some Key Points to follow are,

• Maintain Pond to Reservoir in 3:1 ratio
• Pump water with proper mesh filtration (20, 40, 60 and 80 mesh in stages)
• Do Sedimentation and Chlorination sufficiently.
• Establish optimum bloom.
• Provide sufficient aeration.
• Follow all biosecurity measures to keep the carriers away from pond.
• Use Probiotics for managing the water quality

Optimum water quality parameters for Vannamei

Maintenance of water quality within the optimal levels during the culture is very essential for ensuring good growth and survival of shrimps are presented below. 

Lithopenaeus vannamei is highly susceptible to a number of viral pathogens. Certain viral pathogens of shrimp, notably White Spot Syndrome Virus (WSSV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), are transmitted from broodstock to postlarvae that are then stocked with the infection, increasing the likelihood of a serious profit-limiting disease outbreak. In order to eliminate the presence, the virus in the seed, Specific Pathogen Free (SPF) stock has been developed by producing a number of generations in highly biosecure facility with continued surveillance of pathogen presence.  

SPF status refers to the absence of specific pathogens from a population of shrimp. SPF shrimp are selected through a multigenerational process that includes strict quarantine and endless sampling – typically polymerase chain reaction and histopathology – for the presence of the pathogens of concern. The primary advantage to using SPF animals relates to the ability to control disease. One can expect to see other advantages over years to come, including real disease resistance, enhanced growth and stress resistance.

The specific pathogen free (SPF) Litopenaeus Vannamei has capacity to produce quality seeds with faster growth and higher survival rates for commercial farm. The status of SPF signify that the shrimps have passed through a rigorous quarantine and disease screening process that determined them to be free from specified pathogens of concern to culturists. They are repeatedly bred under controlled conditions to maintain their freedom from specific pathogens and the SPF designation itself is tested on a regular basis. Offspring of SPF shrimp are not considered SPF unless they are produced and maintained at an SPF facility.

Many farmers have been led to believe that SPF animals can solve all of their problems. For farmers thoroughly disen­chanted with the problems associated with trying to rear non-SPF P. vannamei, the success they enjoy by the shift to SPF P. vannamei has been nothing short of miraculous. However, this does not mean they can become complacent and ignore basic biosecurity rules.