An effective strategy against mass mortalities caused by Vibrio harveyi and Vibrio parahaemolyticus infections in cultured Penaeus vannamei
By Rex Ferdinand M. Traifalgar, Rowena E. Cadiz, Emelyn Joy G. Mameloco, Fredson H. Huervana, Carmelo del Castillo, Mark R Napulan, Ramir Lee and Craig L. Browdy
The farming of Penaeus vannamei is a billion USD industry. However, with the intensification of culture systems to meet a growing market demand, the farming is challenged by emerging pathogens, threatening the economic viability and sustainability of this industry. To date, the industry has been stricken with serious economic losses due to the outbreaks of bacterial pathogens that include Vibrio parahaemolyticus and luminous Vibrio harveyi. V. parahaemolyticus is known as the causative agent of the early mortality syndrome (EMS), technically known as acute hepatopancreatic necrosis disease (AHPND). This disease is caused by a strain of V. parahaemolyticus (VpAHPND) with insect neurotoxins that are highly pathogenic to shrimp. It has been associated with mass mortalities in shrimp farms across the Asia-Pacific region. Luminous vibriosis disease caused by V. harveyi is an important bacterial pathogen in cultured shrimp and is known to cause shrimp mortalities of up to 100% in hatcheries and grow-out operations.
The efficient control of bacterial fish pathogens with the application of probiotics is well established. Similarly, there have been some reports on the application of lactic acid probiotic bacteria to control APHND infections in shrimp. However, information on probiotics that are effective in controlling both pathogenic luminous V. harveyi and VpAPHND is very limited or non-existent. Also, the effectiveness of an applied probiotic consortia to control disease-causing Vibrio in shrimp has not been fully evaluated.
This article describes work conducted to evaluate the effectiveness of a mixed-species Bacillus probiotics to prevent infection caused by luminous V. harveyi and VpAPHND in cultured juvenile P. vannamei.
Feeding trial
To evaluate the efficacy of the mixed Bacillus species probiotics, a feeding trial was conducted using groups of P. vannamei that were maintained with diets supplemented with the test probiotics and a control that received no probiotic supplementation. The probiotic blend evaluated in this study was a selected mixed Bacillus species probiotic (RescueTMZeigler USA) that is known to exhibit inhibitory properties against Vibrio.
The test juveniles (mean weight, 1.0g) were obtained from a local hatchery in Panay Island, Philippines. The trial had five treatments in triplicates. Experimental shrimp were randomly distributed in 15 plastic tanks of 135L capacity at a density of 80 shrimp per tank. The treatment group includes the negative control (one group), the positive controls (two groups, one for V. harveyi exposure and one for V. parahaemolyticus exposure), and the two treated groups receiving the Rescue supplemented diets (one for V. harveyi exposure and the other for V. parahaemolyticus exposure).
The negative control group was maintained with diets without probiotic supplementation and was not exposed to the pathogens. The positive control was maintained with diets without probiotics supplements but was exposed to V. harveyi and V. parahaemolyticus pathogens. The treated group was maintained with probiotic supplemented diets and was exposed to the test bacterial pathogens.
Feeding schedule during the 21-day feeding trial.
Figure 1. Vibrio harveyi (A) and Vibrio parahaemolyticus (B) bactericidal activity of Rescue™ probiotics. Spot in Lawn Assay, Mueller Hinton Agar with 2% NaCl, 24-hour incubation.
The experimental animals were fed daily at 10% of their body weight. The feed allocation was divided equally into four feed rations given at 8:00am, 11:00am, 2:00pm and 6:00pm. Before the start of the pathogen exposure test, the treated groups (two groups) were fed with probiotics-supplemented diets (Rescue, 1g/kg, 109 CFU/kg feeds) for 6 days to initiate probiotic gut colonization. Following the 6-day pre-feeding period, the pathogen exposed groups were challenged during the 8:00am feeding only, with diets top coated with V. harveyi, and V. parahaemolyticus at a LC50 concentration of 107 CFU/g feed.
During the trial, dead shrimp were recorded and collected daily in each experimental group and the mortality rates between the control groups and the experimental groups were calculated. The trial was terminated on day 21, 15 days after pathogen exposure.
To confirm the mechanism of Vibrio inhibitory activity of the probiotic, an in vitro plate inhibition assay was also performed to evaluate the bactericidal activity against V. parahaemolyticus and V. harveyi. Direct bactericidal activity of the probiotic was evaluated as a clear zone of inhibition when the probiotic was spotted in the lawn of the pathogens plated in a solid media.
Results were analysed using SPSS16 with a significance level set at 0.05. Bacterial counts were log-transformed before being subjected to a one-way analysis of variance (ANOVA) to determine if there were differences among the treatments for each sampling time. The results with significant differences were subjected to post-hoc analysis using Tukey’s Test.
Inhibition of pathogenic Vibrio
Results from this trial confirmed that Rescue probiotics exhibited potent bactericidal activity against the test pathogens, V. parahaemolyticus, and V. harveyi. Direct bactericidal activity of the probiotics manifested as a clear zone of inhibition when the probiotic was spotted in the lawn of the pathogens plated in a solid media (Figure 1 A and 1B).
Total gut Bacillus counts
Results indicated that dietary supplementation of the mixed Bacillus probiotics influenced the total gut Bacillus counts in the treated shrimp group as compared to the control groups (Figure 2). Total Bacillus counts in shrimp gut in the Rescue treatment group showed a significant increase starting from day 3. The significant increase in gut Bacillus contents during the early phase of the rearing indicated colonization of the applied probiotics on the shrimp gut. Bacillus species are known to be surface colonizers and are also known to effectively colonize the digestive tract of aquatic animals.
However, in the present work, the level of gut Bacillus counts was found to be similar in all the treatment groups in the last weeks of the trial. The data suggested that endogenous Bacillus were also present in shrimp gut specifically in the negative control group. Bacillus species are known to comprise the natural gut microflora of aquatic animals and have been documented to be also present in shrimp gut. The increase in the control group gut Bacillus counts could be attributed to the growth of this normal bacterial flora in response to the nutrients coming from the feeds.
Lower total counts for V. harveyi and VpAHPND
The total V. harveyi colony counts in the gut of the probiotics treated group were lower than the counts in the positive control group in all the sampling periods (Figure 3A). However, the difference in counts was not statistically significant among the treatments. The enhanced Bacillus gut colonization in the group receiving the mixed probiotic strains could account for the trend towards lower counts of luminous V. harveyi in the shrimp gut. No V. harveyi was observed in the gut of the negative control group.
In contrast to V. harveyi gut colony counts, V. parahaemolyticus colony counts in the shrimp gut were similar in all the treatments and no clear influence of the probiotic addition could be observed (Figure 3B). However, the V. parahaemolyticus colony counts in the hepatopancreas suggested that the probiotic-treated group had significantly lower colony counts of V. parahaemolyticus in almost all the sampling periods.
Figure 2. Bacillus spp. count in the gut of Penaeus vannamei fed with RescueTM probiotics.
These data indicated that dietary application of the probiotic blend promoted the active colonization of the Bacillus probiotics in the shrimp gut and in effect might have inhibited populations of pathogenic V. harveyi and V. parahemolyticus in the shrimp gut.
Efficacy of mixed Bacillus probiotics in preventing mortality
The infection challenge test results indicated that the treated group fed Rescue probiotics, exhibited a 33% improvement (Treated, 60%) in survival as compared to the control group (Control, 46%) when exposed to infectious luminous V. harveyi. Similarly, supplementation of the mixed Bacillus probiotics showed a 40% improvement in survival (Treated, 66%) as compared to the control group (Control, 47%, Figure 4) following exposure to pathogenic V. parahaemolyticus.
This improvement in survival could be attributed to the efficient gut colonization of the Bacillus strains in the probiotic blend that might have inhibited the gut tissue entry, infectivity and or pathogenicity of luminous V. harveyi and the AHPND causing V. parahaemolyticus.
Demonstration farm in Cebu, Philippines using RescueTM top coated in shrimp feed.
The significantly higher survival observed in the treated groups could probably be associated with the lower numerical counts of the bacterial pathogens in the shrimp gut. These results might further support the hypothesis that suppression of the pathogenicity of V. harveyi and V. parahaemolyticus due to Rescue supplementation could be linked to the high gut colonization activity, indicated by higher Bacillus counts in shrimp gut. The efficient colonization of Rescue Bacillus could have reduced V. harveyi and V. parahaemolyticus gut populations inhibiting the infection and/or the pathogenicity in the hepatopancreas.
The hepatopancreas is considered the main target tissue of V. harveyi and V. parahaemolyticus. It has been known that V. parahaemolyticus manifests its pathogenicity by infecting and damaging the hepatopancreatic cells of shrimp. The efficient colonization of Rescue probiotics might have prevented the entry of these pathogens in the hepatopancreas and/or reduced their pathogenicity, hence resulting in the significantly higher survival in the treatment group as compared to the positive control group.
Collectively, the present findings showed that dietary supplementation with the mixed Bacillus probiotics could reduce the populations of pathogenic V. harveyi and V. parahaemolyticus, lessen the virulence of the pathogens, and/or enhance the resistance of P. vannamei against these bacterial infectious agents. Additional research will be needed to further elucidate the mode of action of the observed reduction in mortality. Dietary supplementation of Rescue probiotics was shown to be an effective biocontrol approach to minimise mortality and associated economic losses caused by luminous vibriosis and AHPND in the commercial farming of P. vannamei.
Figure 3. Vibrio harveyi (A) and Vibrio parahaemolyticus (B) colony counts in the gut of Penaeus vannamei fed with RescueTM probiotics.
Figure 4. Survival of Penaeus vannamei fed diets supplemented with RescueTM probiotics and exposed to Vibrio harveyi (A) and Vibrio parahaemolyticus (B) in an infection challenge test over 21 days.
