Streptococcus in Tilapia: Implications for Vaccine Development, Field Experiences from Asia

Introduction

Aquaculture is the worlds fastest growing food sector and tilapia represents much of that growth. The popularity of tilapia continues to skyrocket. Global production has nearly tripled since the beginning of the decade with an estimated output of 3.7 million tons in 2010, according to the Food and Agriculture Organization (FAO) of the United Nations. No other fish species has displayed such aggressive and sustainable growth, year after year.

There are several reasons for increased tilapia production. Tilapia can be produced in versatile locations, water systems, temperatures and salinities. They have good performance characteristics such as fast growth, a high fillet yield and low feed-conversion ratio (FCR) as well as firm, white fillet that makes them easy to market.

Tilapia can now be considered a commodity item with a stable supply, demand and price. However, the cost of raw materials is increasing, which is increasing the cost of production and reducing profit margins. Further advancements in production efficiency are required to improve profitability, and this trend is continually driving the industry toward consolidation and intensification. Indeed, FAO reports that the overall number of fish farms is decreasing, while the size of individual farms is increasing, indicating the concentration of fish farms into fewer hands. This situation fosters the emergence of production diseases and tilapia are no exception.

Experience has shown that most intensive fish farming operations suffer from between six and eight major production diseases and that these must be prevented or controlled before the industry can become truly sustainable. In tilapia, we have so far identified four major bacterial diseases: Streptococcus agalactiae, Streptococcus iniae, Flavobacterium columnare and Francisella spp.; one viral disease, iridovirus; and two major groups of parasites: the monogeneans such as Gyrodactylus and the protozoan such as Trichodina (Figure 1). Their prevalence and severity depend on many environmental factors, such as geographical location, culture system, farming intensity, water salinity and temperature, and on several biological factors such as age, genetics, nutrition and stress.

Streptococcus: An Established Pathogen

By far the most important diseases economically are those due to streptococcus. In many instances, streptococcosis does not contribute the highest mortality, but it kills large fish and, as a consequence, heavily affects the FCR, reduces marketable product and damages production and processing efficiency (Figure 2).

Note: Importance of the disease is roughly in proportion to the size of the arrow bars.

LEFT: Typical pin-point petecchial hemorrhaging caused by Streptococcus agalactiae

MIDDLE: 3-gram juvenile tilapia with bilateral exophthalmia

RIGHT: Late-stage bilateral exophthalmia and corneal opacity

In 2000, MSD Animal Health initiated extensive epidemiological surveys in the major tilapia-producing regions of Asia and Latin America. We have identified over 1,000 bacterial isolates from tilapia reared at 74 sites in 14 countries to better understand the relative importance of streptococcal pathogens to the industry. As other investigations have found, these streptococcal species were the dominant bacterial pathogens, accounting for more than half of all bacteria identified. However, it is interesting that while S. iniae is the most commonly reported pathogen of fish, our data show that S. agalactiae is the more prevalent of the two in tilapia.

S. Agalactiae Biotype Prevalence

Detailed analysis of our isolates reveals two distinct clusters that differ in a variety of biochemical and phenotypic characteristics. We refer to these clusters as biotypes and differentiate between typically beta-haemolytical classical S. agalactiae (Biotype I) and typically non-beta-haemolytical S. agalactiae (Biotype II).

S. agalactiae Biotype II is considered the most globally significant of the biotypes, with chronic mortality in many Asian and Latin American countries, whereas S. agalactiae Biotype I is limited to Asia and displays acute mortality peaks, often associated with higher temperatures.

In our epidemiological surveys to date, 26% of all streptococcal isolates of tilapia were found to be S. agalactiae Biotype I and 56% were identified as S. agalactiae Biotype II (Table 1).

Vaccine Development and Biotype Significance

Vaccines to protect against S. agalactiae have been described by various authors, but it is difficult to conclude from these studies if the vaccine and challenge strain were from the same, or different, biotypes.

To determine if our classification of the fish pathogenic S. agalactiae has consequences for the development of vaccines to control this devastating disease, we conducted a laboratory challenge to determine the ability of biotype-specific vaccines to protect against lethal challenge with S. agalactiae Biotype I or Biotype II strains.

Tilapia vaccinated with experimental S. agalactiae Biotype I vaccines were protected against lethal challenges with virulent S. agalactiae Biotype I strains; however, no protection was observed in tilapia that received the Biotype I vaccine when they were challenged with virulent Biotype II strains. Similarly, fish vaccinated with S. agalactiae Biotype II vaccines were protected against lethal challenge with Biotype II, but not with a virulent Biotype I strain. Thus, vaccination with biotype-specific bacterin vaccines induces biotype-specific protection against mortality caused by S. agalactiae.

Laboratory studies also demonstrated that AquaVac Strep Sa, a Biotype II vaccine ultimately developed for commercial use, protects against S. agalactiae Biotype II for at least 30 weeks (Figure 3).

Experimental challenge with a virulent heterologous S. agalactiae Biotype II isolate RPP = relative percent protection

Field Assessment of Prototype Vaccine

Carefully controlled, field registration trials were conducted to determine if results in the lab would be the same in the field with AquaVac Strep Sa, the vaccine for S. agalactiae Biotype II.

The field trials were conducted in a lake environment using square cages housing ~10,000 fish at a large Asian tilapia farm. The trial was run in triplicate with three test cohorts including AquaVac Strep Sa, a placebo oil group and a negative, unvaccinated control. The fish were vaccinated at ~15 g after transfer to growout cages from the nursery ponds.

Extensive bacteriological and virological samplings were conducted before, during and every month after vaccination and at specific time points during the trial when mortality was recorded above normal. As per the routine growout strategy, the trial was terminated and fish processed after ~200 days (just under 7 months) when they reached approxmately 1.2 kg. Mortality, feed and water quality parameters were recorded daily along with the final harvest data.

Clear disease patterns emerged during the trial in all cages, indicating that columnaris disease or Flavobacterium columnare was responsible for high initial mortality peaks immediately after stocking. As the columnaris or saddle-back disease subsided, along with the initial transport and stocking stress, then iridovirus (a common viral disease of tilapia in this area) resulted in three clear peaks of mortality (Figure 4). This mortality pattern is often indicative of the disease and losses can routinely be as high as 30%. As the fish got larger, S. agalactiae and, to a much lesser degree, S. inae were increasingly reisolated from moribund fish in all three cohorts.

The mortality patterns indicate that although the incidence of columnaris disease and iridovirus was similar in all groups, the degree of mortality from S. agalactiae was relatively much lower in the vaccine group when compared to the placebo oil and negative controls. Data gathered at harvest substantiated that observation: Survival in vaccinates was 80% compared to 67% survival in the placebo and negative control groups, representing a 13% improvement in vaccinated fish.

Correspondingly, FCR figures were 1.86 in the vaccinates, compared to 2.06 and 2.05 in the placebo and negative control groups, respectively. This was an approximate 10% improvement in FCR (Table 2).

In this trial, vaccinated fish had improved feed intake and feed utilization; consequently, they had improved production efficiency performance, with 2.25 metric tons more fish harvested in the vaccinated population compared to controls.

This well-controlled field trial clearly demonstrates that AquaVac Strep Sa is safe and efficacious when used under field conditions. Significant improvements in both survival and FCR mean an effective, cost-efficient vaccine prevention strategy is available for the industry to cope with this devastating disease.

Cross - Protection Studies

Further controlled laboratory challenge studies, using Indonesian, Malaysian, Vietnamese, Honduran, Brazilian, Mexican and Ecuadorian strains against the relative commercial vaccine, indicate that the vaccine cross-protects against multiple, geographically diverse isolates (Figure 5).

Commercial Vaccination Experiences

The success of the first large commercialscale injection vaccination programs in tilapia to date has been variable, as expected. The laboratory environment demonstrates the efficacy of the vaccine for protection against specific S. agalactiae challenges, and well-controlled replicate comparison trials have been conducted comparing control versus vaccinated tanks. In the lab, the fish are clean and free from disease prior to, during and in general for 3 weeks after vaccination; furthermore, there are no other diseases present other than that for which the vaccine is being tested. Although the numbers are obviously smaller in the laboratory, another important consideration is that 100% of the housing unit is vaccinated.

Since vaccine efficacy against S. agalactiae and its ability to cross-protect against multiple geographically diverse isolates has already been clearly demonstrated in the lab, the aim of a commercial vaccination program is to discover the best way to implement the vaccine effectively under local conditions.

The initial field trials and large-scale vaccination programs have clearly taught us lessons on some fundamental factors to implement before any operation can or should embark on a vaccination program.

1. Correct diagnosis: Before a commercial producer commits to a vaccination program, the underlying cause of mortality needs to be determined by a fish health specialist.
2. Antimicrobial susceptibility analysis: If a bacterial disease is confirmed, a diagnostic laboratory should perform an antimicrobial susceptibility test to determine if the bacteria is susceptible to therapeutic remediation and, if this is the case, which antibiotic is most suitable to control the outbreak.
3. S. agalactiae biotype confirmation: If Streptococcus agalactiae is confirmed as the primary disease, a more detailed biotype analysis is necessary to ensure the right vaccine is chosen for the right reason, because we now know that immunity is biotype-specific.
4. Healthy fish: Healthy fish means that there is no sign of clinical or subclinical disease. Fish should be free of disease and stress before, during and for the first 2 to 3 weeks after vaccination. That means climatic (water quality) and housing conditions (biomass, handling, etc.) at this stage are crucial.

The best results are obtained in operations where the stages of production are separated: The hatchery, nursery, pre-growout and growout are located in different areas with separated housing unit employees and equipment. The hatchery and nursery phases should, preferably, be biosecure, well-controlled and disease-free. This not only gives juvenile fish the best start in life but also allows vaccines to be administered in a controlled environment.

There are four key components to the vaccine program.

1. Correct administration: The aim is to administer the vaccine in a consistent and proper manner so there is minimal stress on the animal and to ensure that the correct dose is given to each and every individual.
2. Sufficient immune response: Correct vaccination and maintenance in a healthy state under suitable housing conditions prior to transfer will trigger a sufficient immune response before exposure to the challenge environment.
3. Population protection: To achieve population protection, it is important that the entire population is vaccinated, not just a fraction of that population. The aim is to achieve blanket protection in the shortest time frame possible; this is much easier in an all-in/all-out production system, where 100% of stocked fish can be vaccinated, as opposed to gradually replacing susceptible fish with protected fish.
4. Shift the balance: It is important to note that even if a fish has been vaccinated, it may still be susceptible to disease and infection depending on its health, nutritional and stress status. Therefore, a combination of good management strategies, biosecurity, housing, water quality, nutrition, sanitation, immune stimulation and vaccination will tip the balance toward disease control.

A good vaccination program will result in a rapid onset and full duration of protection. Over time, the vaccination program will have a self-perpetuating or snowball effect; with fewer sick and dying fish, there will be less bacterial shedding in the water. Less bacterial shedding in turn lowers total challenge pressure, and coupled with increasingly more protected individuals, there will be overall better population performance.

Conclusion

MSD Animal Healths extensive epidemiological surveys of streptococcus infections in tilapia have revealed some startling insights about the complexity of the disease.

Detailed analysis of our tilapia S. agalactiae isolates suggests the presence of two biotypes that have a variety of different biochemical and phenotypic characteristics. These biotypes are geographically diverse, with Biotype II being the most prevalent in Asia and South America. To our knowledge, there are no obvious geographical, physiological or environmental explanations for the regional distribution of S. agalactiae Biotypes I and II. Consequently, it would be prudent to consider the possibility that the distribution of the biotypes may change over time, probably through trade in live fish.

S. agalactiae vaccines are biotypespecific and do not cross-protect. One commercially available vaccine, AquaVac Strep Sa, has been developed to help protect tilapia against S. agalactiae Biotype II. The vaccine has been fully tested in the lab and in field situations and has clearly been demonstrated to be safe and effective.

If all aspects of the production system are aligned toward good biosecurity and there is a specific health management plan with vaccination as its cornerstone, tilapia farms will be able to control streptococcosis and attain consistent production volumes.

November 2012