In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii

Abstract

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1 h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.

Introduction

The giant freshwater prawn Macrobrachium rosenbergii is an economically important farmed inland crustacean species that inhabits a wide range of salinity levels (0‰ to 18‰) during its life cycle [1]. Its farming has dramatically increased within the last decade when shrimp culture suffered a major setback due to repeated attack by viruses [2]. The intensification of prawn aquaculture linked with deteriorated pond environment and resultant stress as well as poor quarantine resulted in viral epizootics in this comparatively disease-resistant crustacean species. Many prawn hatcheries and farms from West Indies, China, Taiwan, India and Thailand have witnessed the epizootics of M. rosenbergii nodavirus (MrNV) [3], [4], [5]. Recently, the prawn farms in India have also suffered a major setback due to occurrence of appendage deformity syndrome (ADS) related mortality in juveniles and adults of M. rosenbergii [6]. Besides, several disease outbreaks have occurred due to bacterial pathogens [7].

Although Aeromonas spp. are not generally considered to be the major threat to the commercial cultivation of M. rosenbergii [8], they have sometimes been linked to disease outbreaks in this species [9]. Aeromonas-related epizootics in M. rosenbergii in Taiwan [8] and Brazil [10], [11] have been reported. Moreover, pathogenic isolates of Aeromonas have been isolated from hepatopancreas of healthy looking prawn [8].

Several quantitative, rapid and easy procedures are used to evaluate the expression of the immune response of the prawns. For example, the cellular mechanisms viz., total and differential haemocyte counts, phenoloxidase (PO) activity, phagocytosis or bacterial clearance efficiency and humoral mechanisms viz., measurement of agglutinin levels have been considered as potential markers [12], [13], [14]. It is well established that, in arthropods, the defence of the host against invasive or opportunistic microorganisms is effected principally through the phagocytic, encapsulating and agglutinating activities of the circulating haemocytes [15] as well as by antimicrobial factors in the plasma [16]. The proPO system seems to participate in host defence by enhancing phagocytosis and initiating nodule or capsule formation [17]. In crustaceans, circulating haemocytes are involved in the production of melanin via the prophenoloxidase (proPO) system, which plays an important role in defence reactions [18], [19]. Based upon the recent classification of M. rosenbergii haemocytes [20], large ovoid haemocytes and undifferentiated round haemocytes might be carrying out the functions of the proPO system, like semigranular and granular haemocytes in other crustaceans [18]. Conversion of proPO to PO is through proPO activating enzyme (ppA), a serine protease [19]. ppA is activated by several microbial polysaccharides, including β-1, 3-glucan from fungal cell walls [21]. The activity of phenoloxidase has already been reported in M. rosenbergii [12], [13], [22], [23]. Reduction in total haemocyte count (THC) and PO activity in M. rosenbergii has been demonstrated to be correlated with increase in susceptibility to Lactococcus garviae infection [22]. The defence against bacterial pathogens in shrimp depends on a number of cellular and humoral activities interrelated in a complex way. Thus, it is advantageous to measure bacterial clearance efficiency rather than only phagocytosis as a summative measure of cellular and humoral defence capacity [14].

In experimental challenge studies of the penaeid shrimp Sicyonia ingentis using different strains of bacteria Bacillus cereus, B. subtilis, Pseudomonas fluorescens and Vibrio alginolyticus, Martin et al. [24] observed that all four strains were rapidly eliminated in 1 h, in particular, with reduction of THC. Goarant and Boglio [25] challenged Litopenaeus stylirostris with V. alginolyticus and observed a significant decrease in THC 2 days after injection. M. rosenbergii injected with Enterococcus showed increased THC during the first 6 h and the lowest THC after 42 h [26].

Little is known, however, of the mechanism of nodule formation or bacterial clearance pattern from the system in M. rosenbergii. Although presence of opsonic factors in vertebrates is clearly established, its presence in M. rosenbergii is poorly understood. The presence of opsonic factors, however, has been reported in crayfish Parachaeraps bicarinatus [27], lobster [28] and shrimp Penaeus monodon [29]. In the present study, prawns were challenged with opsonized or non-opsonized A. hydrophila and cellular reactions, distribution of bacteria and clearance efficiency were studied besides measuring the defence role of haemocytes in the early phase of infection.