Phage Therapy, A Novel Approach To Control Bacterial Fish Pathogens In Aquaculture Sector

Dinesh Kumar¹, Yogendra Prasad¹, Prabjeet Singh² and Vinod Kumar Verma²

¹Aquatic Biotechnology and Fish Pathology Laboratory,Department of Animal Science, M.J.P. Rohilkhand University, Bareilly-243006 (U.P.)

²College of fisheries, G.B.Pant University of Agriculture and Technology, Pantnagar, Uttrakhand, India.

Correspondence: dineshfish@rediffmail.com

Our freshwater aquaculture is mainly dominated by Indian major carp (IMC) comprising of Labeo rohita, Catla catla and Cirrhinus mrigala and accounts to about 80% of total inland fish production. They are main candidate fish species of freshwater aquaculture, extensively cultured and have proved to be boon in upliftment rural economy. Due to various stressful conditions in aquaculture like overcrowding, poor water quality, competition for feed etc the occurrence of infectious diseaseshas now become a common problem.

Among the infectious diseases, bacterial fish diseases are reported to infest to most of the cultivable as well as wild fish species. There are 40 - 60 bacterial fish pathogens found to be involved in fish diseases. Occurrence of Epizootic Ulcerative Syndrome (EUS) which has devastated the fish industries during nineties badly and ensured heavy economic loss is a self evident proof. Generally, fishes are prone to various microbial diseases because they live in potentially hostile world filled with bewilder array of infectious microbes which would very happily use fish as aliment and render them to a variety of ailments.

Diseases like Columnaris, Bacterial Haemorrhagic Septicaemia (BHS), Infectious Abdominal Dropsy, Furunculosis and Coldwater disease etc. are very common in fish hatchery and culture systems. Among the bacterial pathogens Flavobacterium, Pseudomonas and Aeromonas spp. are known to be great impeder of Indian aquaculture by being involved in various diseases.

To alleviate the incidences of bacterial diseases in aquaculture, different anti-microbial chemicals KMnO₄, CuSO₄, H₂O₂, Formalin, Benzolkonium chloride, strains (Crystal violet, Methylene blue and Malachite green), lime, common salt and finally antibiotics (Ofloxacin, Tetracycline, Erythromycin, Neomycin etc.) and vaccine have been used. Now, there has been sporadic application of probiotics and adjuvant to cope up with such problems.

But in general, chemicals are toxic to fish and aquatic ecosystem and some of them (like Malachite green) gets accumulated in different organs of fish. Thus it seems that chemicals are toxic to fish and the young ones can not tolerate their high concentration. Moreover, use of antibiotics for antimicrobial property has resulted in antibiotic resistance in pathogenic bacteria and posed serious problems in the treatment of infectious disease. Similarly, antibiotics not only destroy the targeted bacterium but also destroy the general micro flora in intestine of fish and also disturb the ecological balance of water body. The emerging crisis of resistance to antibiotics (fig. 1) has led to renewed interest in other methods of controlling bacterial infections. The applications of probiotics (which are beneficial microorganisms or their products) have been made in aquaculture in order to develop immunocompetance in fish to combat with bacterial diseases and also inhibit the colonization of potential pathogens in the digestive tract through competition exclusion principle. But generally probiotics are low — immunogenic in nature, temperature and salinity sensitive and cumbersome in application.

As far as vaccination is concerned, it has proved to be an excellent method in disease combating in poultry, animal and humans due to trouble-free application in them. While in aquaculture, fishes are under water, large scale cultured crop and it is just impossible to vaccinate each and every fish. Therefore, vaccination becomes a tedious job for large-scale aquaculture systems.

Therefore, the usual methods employed to control the disease caused by this pathogen are not proving effective and there is increasing trend of multiple drug resistance (MDR) development in pathogenic bacteria. Due to such reasons they have not been proved as an appropriate and suitable strategy to combat with potential pathogens in aquaculture.

In the non availability of appropriate strategy to eradicate bacterial pathogens, bacteriophages appear to be the most plausible and appropriate candidate to overcome the above problems. Bacteriophage may prove to be a good candidate to mitigate such problem due to various reasons. Bacteriophages (phages) are viruses that infect bacteria. Like all viruses, phages are obligate intracellular parasites, which have no intrinsic metabolism and require the metabolic machinery of the host cell to support their reproduction. They subsist on the bacterial cells and lead lytic and lysogenic life cycle and make the survival of host extremely difficult. They are species specific, self perpetuating and genetically flexible in nature. Bacteriophages are highly abundant in the aquatic environment ranging from 10⁴ ml^-1 to in excess of 10⁸ ml^-1. Numbers are typically 3-10 times greater than the bacterial counts, although there is substantial variation between ecosystems.

Historically, they were discovered by Twort (1915) and D’ Herelle (1917) in the pre-antibiotic era. Now it has been proved that phage therapy decline the bacterial population below threshold number to a level where the host defense can take care of remaining bacteria.

There are two types of bacteriophage reproduction: (1) Lytic - The virus attaches to a host cell and injects its nucleic acid into the cell, directing the host to produce numerous progeny. These are then released by a fatal bursting of the cell, allowing the cycle to begin again. (2) Lysogenic - The nucleic acid of the virus becomes part of the host genome and reproduces genetic material (prophage) in the host cell. An induction event, such as a physiological stressor, can trigger this reproduction to switch to lytic. In general, the replication of phage in the bacterial cell occurs in five steps: adsorption, penetration of genetic material, replication, maturation and lysis. During adsorption, the phage gets attached to the cell in order to infect its genetic material in to the host cell. Penetration involves the actual infection of the genetic material. In replication, the viral genetic material takes over the host metabolic machinery for its own replication. While the phage becomes mature and goes into it's infectious state (maturation) and releases its progeny through lysis. Lysis occurs when the phage particle releases lytic enzyme (Lysin) that causes the cell wall to loosen, leaving it weak enough for the break through of the matured phage.

Lysogenic bacteriophages may incorporate into the genome of the bacterium rather than being lytic. These phages are poor candidates for therapy as they do not provide the rapid growth in phage numbers. Therefore, lytic phages are good candidate for therapeutic as well as prophylactic use against pathogenic bacterial fish diseases. This is evidently confirmed in in vitro test, in which lytic phages clears the bacterial lawn in Petri plate and forms clear plaques (fig. 2). During the lytic life cycle the number of phages released (burst size) after lysis varies from 50-409 new phage particles (Yang et al., 2010). One of the advantages of phage therapy over antibiotics is that they are species specific. Therefore, they can destroy only the harmful bacteria without affecting the regular microflora of the environment. Antibiotics generally target both pathogenic and non-pathogenic microflora. Therefore, phage therapy is safer and there is no need of repeated administration as phages can replicate as long as the host cells are available. On the contrary, antibiotics undergo metabolic destruction and if at stable, they need in numerous molecules to act on bacteria.

Fig. 1: Showing drug resistance activity against various antibiotics by Flavobacterium columnare.

In aquaculture the first isolation of phages against Aeromonas salmonicida, an etiological agent of Furunculosis in Coho salmon (Oncorhynchus kisutch) was made by Paterson et al. (1969) and Popoff, (1971). Some studies showed that viral particles in the marine waters are generally found at concentrations ranging from 10⁴-10⁷ particles per ml. Treatment with bacteriophage was shown to improve survival of shrimp larvae (Penaeus monodon) and it was suggested that bacteriophage have a potential for biocontrol of Vibiro harveyi of shrimp (Penaeus monodon). Good numbers of phages have been isolated against important bacterial fish pathogens, such as Edwardsiella tarda, causative agent of Edwardsiellosis; Yarsinia ruckeri, causative agent of red mouth disease in Salmo gairdnerii; Aeromonas hydrophila causative agent of abdominal dropsy; Lactococcous garvieae, causative agent of serious disease in Yellow tail (Seriola quinqueradiate), Pseudomonas plecoglossicida infect ayu (Plecoglossus altivelis), Aeromonas salmonicida in brook trout (Oncorhynchus fontinalis), Pseudomonas fluorescens in Rohu (Labeo rohita), Flavobacterium psychrophilum in coldwater fishes and Flavobacterium columnare in Clarias batrachus and Labeo rohita.

Conclusion

Bacteriophages can be administered through any rout viz. im(intra muscular), ip(intra peritoneal), bath and oral. Various researchers found that no phage neutralizing antibodies were found in phage treated fish. All of these studies have demonstrated the potential of specific phages to significantly reduce the impacts of their bacterial hosts, with a resulting positive effect on fish survival. The studies emphasize the need for further investigations of the possibilities in using phages as an alternative to antibiotic treatment of other fish diseases in aquaculture and it can also be use in fish processing industry against spoilage causing bacteria. Therefore, phage therapy may be a realistic alternative approach for controlling pathogenic bacteria in aquaculture owing to its several advantages over the conventional antibiotics and other methods against pathogenic multiple drug resistant (MDR) bacteria.

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