Diversity of virulence-associated genes in pathogenic Aeromonas hydrophila isolates and their in vivo modulation at varied water temperatures
Introduction
In India, fishery is one of the most important economic sectors with varied resources and potential for immense growth. However, aquatic species are particularly susceptible to diseases, which is a significant threat to successful aquaculture. Several infectious agents like bacteria [[1], [2], [3]], viruses [[4], [5], [6]], fungi [7], and parasites [8] cause diseases in fish. Among all pathogens, bacteria are one of the most important sources of diseases in fishes [2] with a detected incidence rate of 12.79% in Indian aquaculture [8]. Among bacterial infections, aeromonads group account for 66.66% of disease, and the major disease causing species among them remains Aeromonas hydrophila infection in both India and as well as globally [[8], [9 ], [10]]. It is an important opportunistic fish pathogen in aquaculture systems [[9 ], [10], [11]]. It is often observed that rohu (Labeo rohita) is more susceptible to aeromoniasis [12] caused by this pathogen. Rohu is an important carp species and the major producing countries of this species are India, Thailand and Laos, Bangladesh and Myanmar [13]. Rohu can grow in a wide range of temperatures but does not exhibit good growth at a temperature below 14 °C.
A. hydrophila usually causes problems in fish under environmental stress, with pre-existing physiological abnormalities or co-infected by other pathogens. The ability of this bacterium to cause disease, depends largely on the expression of virulence factors viz., haemolysins, aerolysin, outer membrane proteins, cytotoxin, type 3 secretion system (T3SS) and elastase, which help them to invade the host, produce pathological effects and avoid host defences [10,[14], [15], [16], [17], [18], [19], [20], [21]]. Bacterial haemolysin and aerolysin are cytolytic and hemolytic toxin proteins, respectively, considered to be important virulence factors, and most commonly found in A. hydrophila isolates [21,22]. Similarly, T3SS plays a crucial role in host-pathogen interactions and influences virulence in this bacterium [23]. Outer membrane proteins (Omp TS) also play a key role in the pathogenicity of bacteria and are associated with the transport of molecules and ions across the cell membrane [24]. The presence of polar flagella in association with other virulence factors such as T3SS like apparatus and entero-toxins is strongly linked with the virulence of A. hydrophila [19,25,26]. Similarly, the lipase gene, by changing the plasma membrane of the host cell, affects its permeability along with its accessibility to toxins [27,28]. It has also been reported that environmental factors like time of incubation, media and culture temperature can affect the expression of virulence factors of A. hydrophila [29]. Most importantly, fluctuation in water temperature also plays a significant role in the pathogenesis of A. hydrophila infection [30].
Temperature is one of the important factors for influencing virulence gene expression in pathogenic bacteria. Besides, a rise in the water temperature increases metabolism and exerts stress on fish. This stress induces increased in the production of corticosteroids which in turn enhances the susceptibility of fish to infection [31]. According to Schumann [32], the effect of temperature affects membrane-associated functions and leads to changes in bacterial gene expression. Many bacterial gene regulatory systems are controlled by temperature. Specific and distinct regulation systems of bacteria develop to regulate the expression of particular genes in response to moderate temperature shifts [[33], [34], [35]]. For instance, A. hydrophila can grow at temperatures ranging from 4 to 42 °C [36]. However, it has been noticed from previous studies that temperature below the optimum (TBO) is an inducer of virulence gene expression in A. hydrophila. At 25 °C, this bacterium shows an up-regulation of proteins related to T3SS. TBO also influences the structure of outer membrane components and the virulence of this pathogen directly [37]. Similarly, a report shows 28 °C is the best condition for biofilm production of these bacteria [38]. The correlation between temperature and disease is a rising concern in present times because of climate change observed worldwide and further predicted changes attributed to global warming [39].
On many occasions, although this bacterium is frequently isolated from fish, further in vivo virulence studies with the same bacterium at similar doses fail to reproduce the infection, which is a necessary step to confirm it as the primary etiological agent. This implies that besides the environmental stressors, the presence or absence of virulence-associated gene(s) in A. hydrophila also probably plays a role in modulating the infection process in a host. The factors that govern virulence of a particular strain in the same host are poorly understood. This makes it essential to explore the effect of variation in water temperature on the expression pattern of virulence-related genes in the same bacterial strain within a host. Therefore, the present experiment was carried out to evaluate the effect of variation in water temperatures on the presence and level of virulence genes in vivo upon exposure of L. rohita to selected bacterial isolates that could be translated into causing mortality in fish.
Section snippets
Sample collection and bacterial isolation
The infected fish samples were collected from different states (Odisha, Andhra Pradesh, West Bengal and Jharkhand) of India from various locations during the time of mortality or at time of bacterial infections in the pond culture systems. The infected fishes showing clinical signs of red patches and ulcers over body surface, and fin and tail rots were considered for this study. Total of seven isolates [Isolate 1 (CB105), Isolate 2 (B2C2), Isolate 3 (AH), Isolate 4 (OKTI), Isolate 5 (PK),
PCR amplification of virulent genes of A. hydrophila
All nine virulent genes viz., haemolysin, cytoen, aerolysin, lipase, elastase, flagellin, T3SS, Omp TS and β-haemolysin were successfully amplified from the genomic DNA of the seven A. hydrophila isolates with expected fragment sizes of 130, 232, 309, 382, 513, 608, 710, 1008 and 1600 bp, respectively (Fig. 1). It was observed that Isolate 1 and Isolate 3 of A. hydrophila showed maximum numbers of virulent genes, whereas Isolate 7 was having moderate numbers of virulent genes followed by
Discussion
A. hydrophila is one of the most studied bacterial pathogens in fish which is mostly responsible for causing acute septicaemia in Indian major carps and often noticed in disease outbreaks [22]. The ability of pathogens to infect host is mainly regulated by virulence genes [41]. In recent years, dozens of virulence genes of aquatic pathogens have been identified [42,43] including several virulence genes of A. hydrophila [44]. Further there is evidence of existence of a wide serotype diversity of
Conclusion
This investigation concluded that water temperature does play a crucial role in governing virulence gene expression within a host, and a temperature of 28 °C would be considered as suitable for looking into the pathogenicity of A. hydrophila for conducting any challenge study with this organism in tropical environment.
Author statement
Sabyasachi Pattanayak: Methodology, Investigation, Writing - Original Draft, Swatismita Priyadarsini: Investigation, Writing - Original Draft, Anirban Paul: Writing - Review & Editing, Supervision, P. Rajesh Kumar: Methodology, Investigation, and P. K. Sahoo: Conceptualization, Methodology, Writing - Review & Editing, Supervision, Validation, Project administration, Funding acquisition, Resources
Declaration of competing interest
There is no conflict of interests involved in this manuscript.
Acknowledgements
The authors wish to thank the Director, ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India for providing necessary facilities during this study. The funding support received from NFDB-DAHDF-NBFGR, India through the National Surveillance Programme for Aquatic Animal Diseases (G/Nat. Surveillance/2014-15/29.12.2014) is duly acknowledged.
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