Fish Disease- Vibrio Vaccine for spotted grouper

The European model of using vaccination as a primary prophylactic tool was applied successfully to orange-spotted grouper juveniles. This demonstrates a new direction for health management in grouper culture in Asia.

Asian aquaculture is characterised by an enormous diversity of species, with several dozen marine fish species being farmed. Consequently, more resources are needed to understand the basic epidemiology of diseases in these species. In Asia, some disease-causing agents have been described but comparative studies between isolates from different geographical locations and fish species are generally not available (Tan et al., 2006). Epidemiological data is scarce, in the same way as is basic data on the immune systems of Asian fish species. This hampers the development of effective preventative strategies for disease control. Furthermore, most farming is operated on a small scale and technical support, including disease diagnosis and training, is often lacking at the farm level.

Learning from the salmon industry

Salmon has been considered as the model species of modern aquaculture, especially for cage farming. In the last 20 years, this industry has developed dramatically and now produces nearly 1.5 million tonnes annually (FAO, 2006). Produced largely by two countries (Norway and Chile), in marine cage culture, the focus is on only one family of cultured fish, namely salmonids. Therefore, most resources available are used for the optimisation (including disease control) of its culture. This is in stark contrast to the above-mentioned situation in Asia. A survival rate lower than 95% in salmon farming is a sign of disease outbreak, whereas, a survival rate of 50% is often acceptable in Asia. The intensification of salmon production has led to the separation of fry production (hatcheries) and on-growing sites, optimised feed and feeding strategies, good quality fingerlings (that are virtually disease free) and good farm management.

The Asian challenge

In Asia, aside from producing different species of fish at the same site, there is no segregation in year classes. In contrast this is obligatory for salmon in Europe. In Asian farms, trash fish is widely used as feed, fry are often wild caught or derived from wild-caught brood stock and the culture techniques per species are as yet undefined. Furthermore, legislation and implementation regarding farm licenses and zoning policies are non-existent in most Asian countries.

This often results in too many fish and too many farms in a concentrated area, a situation that promotes the spread of diseases. The combination of all these factors, together with the diversity of organisms in tropical waters, leads to a truly challenging disease situation with a variety of entry points for pathogens.

Disease is undoubtedly recognised as one of the biggest constraints to the production, development and sustainable expansion of marine fish aquaculture in the Asian region. As most farms operate on a small scale and with limited technical support, disease diagnosis and training is usually lacking at the farm level. Even if fish suffer from disease and overall survival is low, epidemiological data is rarely collected, reported and analysed. This situation is in the process of being corrected.

In past few years, more and more attention has been given to the identification of aetiological agents involved in fish disease epidemics.
Pathogens can be classified into bacterial, viral, parasitic and fungal groups and the following studies give a solid indication of the diversity of major pathogens affecting the fish farming industry in Asia (Bondad-Reantaso et al., 2005; Komar et al., 2005; Labrie et al., 2005; Leong et al., 2005, 2006 and Tan et al., 2003).

In Thailand, the diversification of culture is evident in recent years (Rimmer et. al., 2004) and includes the orange-spotted grouper Epinephelus coioides and other coral trout or grouper species of high economic value (de Silva and Phillips, 2007). Diseases of bacterial origin are the most significant causes for economic losses affecting fish culture (Austin and Austin 1999) and in cultured marine fish, Vibrio spp. are the pathogenic microorganisms most frequently isolated during epizootics. In particular, many epizootics have been attributed to Vibrio cholerae. The economic losses caused by these microorganisms indicate the need to develop prophylactic strategies based on the European model for salmonids (Smith et al., 1999), such
as vaccination, in order to prevent disease.

A Vibrio vaccine

Vibriosis is the most common disease affecting marine finfish grow-out culture in the Asian region. It is responsible for catastrophic economic losses, in particular in the culture of the snapper, pompano and most recently in higher value species such as grouper. Conventional treatments using antibiotics have failed due to a lack of technical expertise in their administration and use which has led directly to the spread of bacterial resistance and hence to lack of efficacy. The aim of this study was to assess the effectiveness of an injectable bivalent Vibrio vaccine against Vibrio species in Thailand affecting farmed orange-spotted grouper and to assess its possible impact on grouper farming practices in Thailand.

Fish samples

In this study, 840 juvenile of the orange-spotted grouper weighing approximately 5.75g and of approximately 4.31 cm length were used. These fish were purchased from a private farm. Fish were acclimated in 60 litre plastic aquaria for 14 days prior to initiation of the experiment. Three replicate tanks with 20 fish each were used in all immunisation and infection experiments. A light and dark period of 12:12h was maintained and aeration was supplied using an air stone. Water exchange was 50% every alternate day. The fish were fed daily to satiation with frozen Artemia. To verify the Vibrio sp. free status of the fish, two fish were sacrificed and samples were obtained for bacterial culture by passing an inoculation loop into spleen, kidney and liver. The samples were streaked directly on TCBS and blood agar and were subsequently incubated at 30°C for 24-48 hours.

Water quality parameters were monitored. Temperature was measured daily. The pH, hardness, ammonia, nitrite and alkalinity were determined daily using a commercial test kit (AQUA-VBC, Thailand). The salinity was measured weekly with a refractometer (Table 1). Water quality parameters appeared to be normal although ammonia did give cause for concern.Table 1. Means of water quality parameters.

Bacterial preparation

V. cholerae isolated from moribund sea bass (Lates calcarifer) were provided by the Veterinary Medical Aquatic Animal Research Centre. (VMAARC), Faculty of Veterinary Science, Chulalongkorn University, Thailand. V. cholerae was plated onto Tryptic Soy Agar supplemented with 1% NaCl and harvested after 24 hours growth. Bacteria were identified using API® 20E (BioMerieux, Madrid, Spain) and the API profiles compared with the API database (Apilab Plus, version 3.3.3.; BioMerieux). The pure isolate was kept in stock media at room temperature.

Virulence assays/Dose response curve

Virulence assays were performed with 540 juvenile orange-spotted grouper via intra-peritoneal injection. Bacterial doses ranging from 10³ to 10 ¹⁰ CFU/fish in a volume of 0.1 ml, injected intra peritoneally (IP) were used to establish pathogen doses that would kill 50 and 100% of injected fish within 3 days. Each dose had 3 replicates with 20 fish in each replicate. Control groups were challenged with sterile 0.9 % (v/v) saline.

After injection of the pathogen, fish would be observed daily. Mortalities were recorded on a daily basis, and symptoms associated with the ensuing pathogen attack were recorded and photographed. Samples of liver, spleen, heart, kidney, digestive tract and brain were preserved for histological examination and a photographic record was maintained. The mean lethal dose (LD) was calculated by the Reed and Muench (1938) procedure for establish optimal pathogen dose to be used during the efficacy stage of the trial.

Methods of vaccination

Vaccine manufacture

The vaccine was a bivalent Vibrio vaccine composed of 2 virulent strains of V. harveyi and V. parahaemolyticus isolated from diseased, moribund fish located in a farm near Phuket, Thailand. Cell concentration was 1x10¹¹CFU/ml for each strain, which were then formalin killed. A small amount of adjuvant was added to enhance vaccine potency. The sterile vaccine was contained in 500ml polyethylene bottles with a rubber stopper with an aluminium foil cap.

Injection vaccination

Fish were not sedated. The vaccine (NovaQsol Holdings Inc., Philippines), was administered intraperitoneally to each fish. Injection volume was 0.05 and 0.10ml/fish of 5g average body weight. Fish are then returned to their respective tanks.

Vaccination protocol and establishing vaccine safety

In this part, 240 juvenile orange-spotted grouper were separated into 4 groups of 60 fish/group. Each group was further sub-divided into 3 groups of 20 according to the following schedule;

a) Negative control (NC), no injection.
b) Positive control (PC), 0.1 ml saline injection.
c) Test (T1), Injection vaccinated 0.05 ml injection.
d) Test (T2), Injection vaccinated 0.10 ml injection.

Immunised and control fish were held for 21 days before the challenge. All groups were monitored for mortality on a daily basis. In this manner, vaccine safety could be clearly established.

Efficacy test of vaccination

At 21 days post-vaccination, the positive control and all test groups received a 0.1 ml aliquot of a 100% lethal dose of pathogen administered intraperitoneally. The positive controls were administered an injection of 0.1 ml sterile saline. The fish were monitored for mortality for 3 days post-challenge.

Fish mortalities were observed and recorded on a daily basis and symptoms of the ensuing pathogen attack were recorded and photographed. Tissue samples of liver, spleen, heart, kidney, digestive tract and brain were preserved for histological examination, again with an accompanying photographic record. The percentage mortality rates and relative percentage survivals (RPS) of each vaccine dose were calculated against the relevant controls. In this way, the efficacy of both vaccine doses was clearly established.

Dead fish were removed twice daily and upon post-mortem examination, specimens were obtained aseptically from spleen, kidney and liver tissues for examination of V. cholerae infection. Specimens were culture directly onto TCBS at 30°C for 24 hours and biochemically identified by API 20E.

Vibrio sp. was not isolated from the two randomly selected fish, indicating that fish held at the start of the experiment were clear of actual and potential Vibrio infection.

The virulence assays/dose response curve was very clear for this particular isolate of V. cholerae. Figure 1 clearly indicated both LD50 and LD100 of V. cholerae were 1x108 CFU/ml and LD100 and 1x1010 CFU/ml respectively. The LD100 dose was used to challenge 21 days vaccinated fish. Results are shown in Table 2.


Figure 1. Determination of LD50 and LD100 for V. cholerae in juvenile spotted grouper.




In this efficacy test of vaccination, the highest rate of protection against V. cholerae was observed in 0.10 ml vaccinated fish (T2), with RPS values up to 77.59% and significantly lower percentage mortality rates than in all infected groups (P<0.05). In this group (T2) there was no significant difference in the percentage mortality rate when compared to the control group (P>0.05). The 0.05 ml vaccinated fish (T1) displayed a 45.76 RPS value, which was a lower RPS value than in 0.10 ml vaccinated groups but was not significantly different from the percentage mortality rate at a (P>0.05) confidence limit. The positive control group displayed a 100% mortality rate.

Table 2. Percentage mortality of juvenile orange-spotted grouper, non-vaccinated and vaccinated by two intraperitoneally injected doses of a Vibrio vaccine and relative percentage survival (RPS)after challenge with Vibrio cholerae.




The dead fish from this challenge experiment were confirmed positive for V. cholerae infection by re-culture and re-identification using API 20E. Histopathological studies of the dead fish showed atypical symptoms such as acute peritonitis and hepatic septicaemia.



                              Acute Peritonitis


                   Septicaemia of hepatic tissue

Discussion

It is a well known fact that there are many problems associated with the use of antibiotics. In addition to developing antibiotic resistance, many antibiotics are water soluble leading to leaching from feed, and some, like oxytetracycline, will complex with calcium and magnesium ions in seawater or hard water, rendering their efficacy highly reduced.

Sick fish often do not eat and the efficiency of delivering antibiotics orally is therefore questionable. Two key technical comments should be made regarding antibiotics:

Application of antibiotics is a curative measure used to treat an existing infection. In contrast, vaccination is a preventative measure, dependent on the immune system of the animal. Vaccines can act against bacterial, viral and, at least experimentally, parasitic infections and they will usually act only against specific targeted pathogens. The duration of protection obtained with vaccines normally largely exceeds that of antibiotics. Historically, there are clear indications that the introduction of vaccines has greatly reduced the use of antibiotics in Norwegian salmon production from a high of over 50 tonnes in 1984 to less than 2 tonnes in 2004; with salmonid production in the same period rising from 25,000 tonnes to just over 500,000 tonnes.

In the past, fish vaccines were only available for salmonid species. This situation is changing with new vaccines being registered in Asia for Asian species (Grisez and Tan, 2005). Significant progress has been made in the field of vaccine research and development (Grisez and Tan, 2005). Besides yellowtail in Japan and grass carp in China, a commercial vaccine has recently been launched for use in Asian sea bass, tilapia and other species in some Southeast Asian countries (Komar et al., 2005).

It must be remembered, however, that vaccination is only one of the tools employed in good health management strategies and it is not sufficient on its own to guarantee high survival and profitability (see box).

In addition to the above, various studies have been carried out on the efficacy of various Vibrio vaccine types on survival of different grouper species. In the present study, results indicated that the 0.10 ml vaccinated fish group had a clearly higher RPS and lowest % mortality rate than the 0.05ml group. However, this method of administration is difficult to apply in routine farm management. In conclusion, the high protection afforded by the Vibrio vaccine injection in cultured orange-spotted grouper strongly suggests it should be a good prophylactic measure against mortalities caused by both pathogens.

Bottomline

As Asian aquaculture continues to grow, disease problems will inevitably become worse unless key steps are taken. Under the threat of disease epidemics and the vigilance of governments and consumers regarding food safety, the industry must undergo changes. Therefore, disease research and the implementation of new disease control concepts are inevitable. Collectively, this includes the use of healthy fry, quarantine measures, optimised feeding, good husbandry techniques, disease monitoring (surveillance and reporting), sanitation, vaccination, and the responsible use of chemicals and antibiotics when diseases occur. Overall, the emphasis must be on prevention rather than cure (treatment). This is the only way to sustain a responsible yet profitable Asian aquaculture industry.

The authors would like to convey their thanks to all the staff of VMAARC in Chulalongkorn University, Bangkok Thailand, and to the supporting staff of the Research Institute at Rayong, Rayong Province, Thailand, and finally to Drs. Gee, Na, Lin and Jim for their endless patience.

(References are available on request)