Impact of Streptococcosis on Tilapia in Brazil, AquaVac Strep Sa for Management

Introduction

Aquaculture is one of world's growing animal production segments. In Brazil, however, commercial fish production in net pens is only just starting, despite its great potential represented by 6 million hectares of water contained in weirs and reservoirs constructed mainly for generating hydroelectric energy. In the future, Brazil is likely to become one of the world's largest aquaculture producers.

Among fish species showing potential for cage farming is Nile tilapia (Oreochromis niloticus). Over the past decade, it has become the species with the largest production volume in Brazil, representing nearly 40% of the country's aquaculture. Tilapia cultivation has been developed primarily in Brazil's South, Southeast and Northeast regions, the latter representing the highest production volume. In 2004, 41% of total domestic tilapia production occurred in this region. The Northeast region, in fact, has led the country's tilapia production since 2003, with a clear, growing trend due to its climate and technological development, enabling it to serve the growing demand for tilapia both regionally and nationwide.

In recent decades, improved technology in fish production units has become a major competitive advantage. Efforts to survive and withstand an increasingly globalized market have become an evident need. In this context, and despite efforts to improve fish quality through the implementation of health programs and novel technologies, tilapia production success depends on innovative tools.

A future challenge can now be foreseen: Mankind demands more and more products that are not only nutritious but are wholesome and pathogen-free, that promote health and that are environmentally friendly and socially fair in perfect harmony with the globalized world we live in today.

Intensive fish production, however, brings about stress, resulting in the emergence of diseases and, therefore, mortality.5 Among the major maladies affecting tilapia, Streptococcus agalactiae infection, resulting in streptococcosis, plays a very important role worldwide.6 Epidemiological studies sponsored by MSD Animal Health around the world have shown the presence of two different S. agalactiae groups or biotypes (i.e., I and II). Tilapia isolates from different regions in the world show that 26% of streptococci were identified as S. agalactiae Biotype I, while 56% were S. agalactiae Biotype II. S. agalactiae Biotype II is the world's most prevalent biotype, found mostly in China, Indonesia, Vietnam, the Philippines and Latin America. In Brazil, serological studies show 100% positive serology to S. agalactiae Biotype II.

The largest economic impact due to S. agalactiae in freshwater-farmed fish species occurs in Nile tilapia. The geographic distribution of S. agalactiae includes regions with temperate, tropical weather, where warmwater fish are cultivated. So far, outbreaks have been reported in several countries including: the US, Japan, Kuwait, Israel, Thailand and Brazil.

This pathogen is responsible for high economic losses; mortality on a farm can reach 90%, typically at the pre-market age when substantial feed volumes have already been consumed. We must remember that feed is the largest component of production costs and that much has been invested in the fish by this time.

Infection occurs when infected fish dead or alive, moribund or apparently healthy release the bacterium into the water, allowing it to colonize the skin of other fish. Invasive infections can also occur, resulting in high mortality. In addition, the bacterium can survive for long periods of time in water, mud or pond/greenhouse substrates, and even on pieces of equipment used in routine operations.

If tilapia culture is to continue and proliferate, the industry will need strategies to minimize the effects of disease. The advent of S. agalactiae vaccines has brought about a new, promising tool, since some S. agalactiae field strains have already developed resistance to antimicrobials.9 Evaluation of the vaccine AquaVac Strep Sa under experimental conditions has demonstrated significantly decreased mortality in vaccinated fish, illustrating the efficacy of the vaccine for the prevention and control of streptococcosis in Nile tilapia.

Materials and Methods

In the study, 180 Nile tilapia (Oreochromis niloticus) juveniles from the same spawn and weighing ~35 g were used. Prior to the start of the study, the fish were maintained in quarantine and subjected to a prophylactic antimicrobial bath; they were then conditioned in 250-liter cages under constant dechlorinated water exchange.

Upon the completion of quarantine, fish were moved to the Aquatic Organism Experimental Infection Unit, Fish Immunopathology Laboratory (Unidade de Infeco Experimental de Organismos Aquticos do Laboratrio de Imunopatologia de Peixes, LIPPE) and conditioned in 12, 80-liter aquariums (n = 15). Aquariums received UV-sterilized, dechlorinated, running water from an artesian well. Fish acclimation lasted 7 days, which was time enough for plasma cortisol concentrations and osmolality to return to baseline levels.

Water temperature was measured daily (27 C/81 F 1.5). Hydrogen ion potential (7.1 0.3) and dissolved oxygen (5.5 mg/L 1 mg/L) were measured weekly. All values remained within welfare-recommended levels.

Throughout the experiment, ad libitum feeding was provided twice daily (09:00h and 17:00h) at the rate of 5% of biomass.

Experimental Design

Fish in the 12 aquariums represented three repetitions for each treatment group (Table 1).

Vaccination

Vaccination against S. agalactiae was performed at the end of the acclimatization period at the laboratory. Fish weighing ~35 g (medium bodyweight) were submitted to the vaccination process as recommended by MSD Animal Health. The fish were anesthetized by immersion in 1% eugenol (Biodinamica) containing 50 mg/L water. A single 0.05 mL dose of AquaVac Strep Sa was injected intraperitoneally in the anterior-medial aspect, using a sterilized insulin syringe/needle.

Challenge

Challenge was performed 25 days after vaccination. For inoculum preparation, live streptococcus isolates from naturally infected tilapia were used. Isolates were previously classified as Lancefields B group, using the Slidex Strepto Kit latex agglutination test (BioMerieux, France), then characterized as S. agalactiae based on phenotype characteristics as determined by the ApI 20 Strep Microtest System (BioMerieux, France.)11 The selected S. agalactiae strain was seeded in brain/heart infusion broth, incubated for 24 hours at 29 C (84.2 F) under aerobic conditions. The challenge dose (106 colony-forming units/mL) was calculated on the basis of a lethal dose killing 50% of the fish population (LD50). Fish were observed for 15 consecutive days after challenge for clinical signs and death; the findings were recorded daily and subjected to statistical analysis (Tukey's test), with a 5% level of significance.

Results

Table 2 shows survival among fish in various treatment groups. No significant mortality occurred when comparing groups T1 (vaccinated/challenged) and T2 (vaccinated/unchallenged) with group T4 (not vaccinated/unchallenged), demonstrating the vaccines safety. None of the fish that died in those groups showed clinical signs compatible with streptococcosis, but deaths occurred within 48 hours after challenge, suggesting that the cause of death was handlingassociated stress.

Animals in group T3 (not vaccinated/ challenged) that died showed clinical signs compatible with streptococcosis, mostly after 7 days post-challenge. Clinical signs included lethargy, reduced appetite, body-color darkening, uni/bilateral exophthalmia, abdominal distention and erratic/circular swimming. Necropsy findings among these fish included ascites, enlarged livers and diffused hemorrhages in the central nervous system. Upon microbiological analysis, S. agalactiae was re-isolated, particularly from the brain, which means generalized infection.

The appearance of clinical signs after 7 days post-challenge represents the natural evolution of the infection. Neurological signs suggest meningoencephalitis, and it represents a clinical sign consistent with streptococcosis. The mean number of deaths among repetitions supports the LD50 calculated for this particular study. Variations in the numbers of dead fish among repetitions can be the result of innate, individual resistance variability.

Vaccination with AquaVac Strep Sa protected animals against experimental challenge with S. agalactiae, since mortality in group T1 (vaccinated/challenged) was significantly lower (P < 0.05) than mortality in the T3 (not vaccinated/challenged) fish (Figure 1). In this context, the relative protection percent (RPP) was 84%. RPP was determined using the following equation: RPP = (1- (vaccinated fish deaths/control fish deaths)) x 100.

Conclusion

Results from this study demonstrate that AquaVac Strep Sa induced effective protection in Nile tilapia experimentally challenged with S. agalactiae. We, therefore, conclude that AquaVac Strep Sa (MSD Animal Health) is a safe and highly efficacious vaccine against the disease caused by S. agalactiae Biotype II. A single dose of AquaVac Strep Sa, administered as directed, can be an important tool in the prevention and control of streptococcosis in Brazil, since serological results so far show only the presence of Biotype II in this country.

August 2012