A study was carried out to determine the influence of a commercial probiotic (Aqualact) and dried leaf powder of Cassia auriculata against Vibrio parahaemolyticus infection in the freshwater prawn, Macrobrachium rosenbergii. Histological studies in the hepatopancreas and gills of prawns, during infectivity trials, showed bacterial invasion and multiplication in their lumens. Oedema and cell necrosis were the major pathological changes observed in gills and hepatopancreas. Degenerative changes in the electrophoretic pattern of muscle proteins were proportional to the concentration of vibrios infected. Infected prawns fed on probiotic supplemented feed could show a regaining trend towards normal protein bands. Marked reduction in mortality could be observed in infected prawns fed on feed supplemented with probiotic as well as with powdered Cassia auriculata leaves.
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Vibriosis is a major problem in prawn culture, causing heavy mortality and severe economic loss. Though they are members of the normal microbiota of prawns, Vibrio species often act as opportunistic pathogens or secondary invaders and many induce mortality even up to 100% in the affected population (Bell and Lightner, 1988; Rosemark and Fisher, 1988). Major species causing vibriosis in prawns are Vibrio alginolyticus, V. harveyi, V. anguillarum and V. parahaemolyticus (Bell and Lightner, 1988; Ruangpan and Kitao, 1991; Nash et al., 1992; Xu et al., 1994; Chanratchakool et al., 1995; Alapide-Tendencia and Dareza, 1997). Bacterial septicemia is a common symptom of vibriosis (Mermoud et al., 1998).
These moribund prawns display a wide spectrum of clinical signs, like disoriented swimming, lethargy, weakness and abnormal colouration of body and appendages. Infections due to Vibrio species are prevalent in hatcheries and grow-out facilities, causing mass mortalities and shell deformities (Lavilla-Pitogo et al., 1990).
Generally farmers apply antibacterial compounds to combat microbial infection. A wide range of antimicrobial drugs (oxytetracyclin, ciprofloxacin, chloramphenicol) are used in hatcheries and farms of freshwater prawns and marine shrimps in India (Karunasagar et al., 1994; Hameed and Balasubramanian, 2000). Indiscriminate use, or the large scale prophylactic application of antimicrobial compounds leads to the development of antibiotic resistant microorganisms which may cause multiple antibiotic resistance-transfer to other pathogenic bacteria (Frappalo and Guest, 1986; Moriarty, 1997). Transfer of resistance to human pathogens and gut bacteria is also a major concern (Salyers, 1995).
Increased concern about the emergence of antibiotic resistant microorganisms has led to alternative options for disease prevention, such as the use of non-pathogenic bacteria such as probiotic biocontrol agents (Austin and Allen, 1982; Murthy, 1997). Probiotics are widely referred to as live microbial feed supplements which beneficially affect the host animal by improving its intestinal microfloral balance (Fuller, 1989). The use of probiotics in aquaculture is not restricted to feed alone. It has also been used to modify the living environment of cultured-prawns (Verschuere et al., 1997; Robles et al., 1998).
Probiotics generally include bacteria, cyanobacteria, microalgae and fungi. The possible mode of probiotic-action is by the production of inhibitory compounds, siderophores, hydrogen peroxide, protease or organic acids (Vandenbergh, 1993; Sugita et al., 1997). Competition for chemicals and available energy and competition for adhesion sites are the other possible modes of antagonism (Verschuere et al., 2000). Enhancement of immune response and enzymatic contribution to digestion may also be the ways by which probiotics act (Balcazar et al., 2006).
Another alternative disease prevention method is the use of medicinal plant extracts. Besides many others, members of the genus Cassia auriculata are noteworthy for their diverse medicinal properties. The leaves and petals are traditionally used for their antihyperglycaemic action, antihaemorrhaging properties and antimicrobial activity (Chythanya et al., 2002). Studies on the effect of medicinal plants against Yellow Head Virus (YHV) and Systemic Ectodermal and Mesodermal Baculovirus (SEMBV) in Penaeus monodon are available (Ruangpan, 1998).
About 25 species of Macrobrachium were found in India, mostly inhabit in freshwater (Soundarapandian and Kannan, 2008). M. rosenbergii culture is spreading fast to all Indian states due to its large size attainment, tolerance water quality changes, stress and ability to feed on unconventional feeds (Yathavamoorthi et al., 2010). Vibriosis in Macrobrachium rosenbergii is a less explored area and disease management through biocontrol agents is yet to emerge. Hence, this investigation is intended to assess the inhibitory activity of a commercial probiotic (Aqualact) and dried leaf powder of Cassia auriculata against Vibrio parahaemolyticus infection in M. rosenbergii.
MATERIAL AND METHODS
Pathogenic bacterial strains: Bacterial strains of V. parahaemolyticus were collected from P. monodon with black gill disease. V. parahaemolyticus used in this study were identified using standard morphological, physiological and biochemical tests (Holt et al., 1994). Optimal growth was noticed between 28-35°C in Zobells marine agar, Citrate agar and MacConkey agar media (Himedia, Bangalore). Up to 8% sodium chloride concentration in nutrient agar medium favored the growth of bacteria. V. parahaemolyticus were maintained in TCBS agar medium (Himedia, Bangalore).
Diet formulation: Pelleted feed was formulated for prawns following the guidelines of Boonyaratpalin and New (1993) and its percentage composition by weight is given below: Trash fish (28.35), Soyabean meal (11.34), fish meal (2.84), Cornmeal (22.68), Di-Calcium phosphate (0.57), Vitamin and mineral mix (0.20), Broken rice-boiled (8.5), Chicken feed (21.6), Shrimp shell meal (4.25).
Probiotic: A commercial shrimp-farm probiotic, Aqualact manufactured by Biostadt Agrisciences (Wockhardt, India) was used in the present study. The composition of the probiotic per kilogram of the substance is given below: Lactobacillus sporogenus (45,000 million CFU), Lactobacillus acidophilus (45,000 million CFU), Bacillus licheniformis (30,000 million CFU), Bacillus subtilis (30,000 million CFU), Saccharomyces cerevisiae (1,25,000 million CFU), sea weed extract (100 g), enzymes: amylase (24,000 IU), phytase (22,00,000 IU), protease (4,00,000 IU), cellulase (150-250 IU), beta-galactosidase (800-1000 IU), lipase (50-100 IU); Vitamin C, Vitamin B1 and B6. The regular feed of prawns was enriched by the probiotic at 1 and 3% of feed weight using agar as the binding agent. Serial dilution and pour plate methods of bacterial enumeration could confirm the viability and density of the component microflora of Aqualact.
Plant material: Cassia auriculata is a perennial shrub growing wild, reaching up to 60 cm height. Leaves were collected, dried in shade, ground into fine powder and stored in closed containers. The leaf powder was added at 1 and 3% by weight as supplement to the feed at its formulative stage using agar as binding agent.
Experimental animal and experimental design: Healthy, freshwater prawns M. rosenbergii were collected from a private farm at Perambalur district in Tamil Nadu, India which had no prior history of vibriosis. Before acclimatization they were washed for few a seconds in 0.1% benzalkonium hydrochloride to clear the adhering bacteria. Animals were washed three times in sterilized water, after benzalkonium chloride treatment. They were acclimatized in 60 L PVC troughs for a week, where optimum hydrological conditions were maintained. The prawns were fed daily at three intervals with formulated feed at 3% body weight.
The experimental animals were divided into six sets, each set in two groups comprising of 10 animals each. First set was kept as control set, fed only with normal formulated feed and instead of injecting pathogenic bacteria, appropriate quantities of physiological saline were injected to them. Second set of animals, fed on normal formulated diet, were injected with pathogenic bacterial suspension as detailed later. Third and fourth sets of animals were fed with 1 and 3% bacteria-incorporated feeds, respectively. These groups were challenged with pathogenic bacteria through injections. Fifth and sixth sets of animals were fed respectively with 1 and 3% Cassia auriculata leaf powder incorporated feeds and challenged with vibrio. Before the pathogen challenge, prawn sets were kept on their respective diets for 14 days.
LD50 test: V. parahaemolyticus stock suspension was diluted at 1:100000, 1:1000, 1:100, 1:10 with sterile physiological saline. Prawns were injected with 0.1 mL of each dilution of bacterial suspension. Mortality was recorded for two weeks and the LD50 was determined by employing probit analysis (Statistical package SPSS-10). Enumeration of vibrios at the LD50 concentration was carried out by standard pour plate technique using Nutrient Agar medium, with 8% NaCl.
Pathogen challenge test: After feeding the animals for two weeks with the probiotic (3rd and 4th set) and leaf powder supplemented food (5th and 6th sets), prawns in each group were challenged with V. parahaemolyticus. All the prawns were injected with 0.1 mL V. parahaemolyticus suspension in physiological saline intramuscularly at the LD50 dosage between their 3rd and 4th abdominal segments. The number of bacteria in the suspension was standardized by adjusting its absorbance in a spectrophotometer at 600 nm. The injected animals were observed for behavioural changes. The mortality/infectivity percentage was estimated after Sung et al. (1994):
where, A is the number of prawns dead in first day after injection due to administration stress.
Randomly selected animals from control and from experimental groups of animals were dissected using sterilized instruments. For histological studies, tissues were fixed in Davidsons fixative (Bell and Lightner, 1988), embedLavilla-Pitogoded in paraffin wax, sectioned at 5 micron thickness and stained with haematoxylin and eosin.
Electrophoresis of muscle proteins: The 12% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) modified protocol (Laemmli, 1970) was used at present. Muscle proteins were identified by running molecular mass reference standards (Bangalore Genei Cat No: PMW-M) containing Phosphorylase 97.4, Bovine Serum Albumin 66, Ovalbumin 43, Carbonic Anhydrase 29, Soyabean Trypsin Inhibitor 20 and lysozyme 14.4 kDa). Electrophoresis was carried out on a constant voltage (50 V) at room temperature for 4 h.
Protein detection by Coomassie Brilliant Blue (CBB) stain: Once the gel electrophoresis was completed the gel was rinsed with distilled water for 2 min and stained with 0.5% CBB R-250 in a solution of 40% methanol and 10% acetic acid at room temperature, for 2 h. The stained gel was destained in a solution containing 40% methanol and 10% acetic acid, until appropriate background was obtained. The gel was washed with distilled water and stored in refrigerator. The lanes selected for electrophoresis are explained in the legends for electrophorogram (Fig. 2).
Histopathology of the hepatopancreas of infected animals showed an invasion of its tubular lumens by vibrio and a reduction in lipid vacoules. Hepatopancreatic cells lining the tubules were walled off by haemocytes around the thickened basal lamina. The interstitial tissues around these invaded tubules were swollen, owing to oedema. Moreover, granulomatous lesions were formed; the thickened basal lamina underwent coagulation and a small number of bacteria remained among cellular debris (Fig. 1).
Gills of infected animals showed multifocal lamellar fusion, hyperplasia of epithelial cells and adhesion of the lamellar tips. Cyst like structures formed between lamellae became necrotic. Multifocal epithelial cell-lifting due to oedema of the lamellar tissue could be observed with separation of epithelial cells extending the length of lamellae. Necrotic tissue was characterized by loss of cellular integrity, karyolysis and infiltration by leucocytes (Fig. 1).
The biochemical characteristics of V. parahaemolyticus isolated from diseased P. monodon are presented in Table 1. After feeding the prawns, with supplemented feeds of 1 and 3% probiotic and Cassia leaf powder for fourteen days, they were challenged with LD50 dosage of V. parahaemolyticus (7.2x105 CFU). After three weeks of experiment about 59% mortality could be observed in vibrio challenged prawns. 33.2% mortality was observed in prawns supplemented with 1% concentration of probiotic, whereas the mortality was 46.3 in 1% leaf powder supplemented group.
|Fig. 1:||Pathological effects of Vibriosis in the hepatopancreas and gills of Macrobrachium rosenbergii|
|Table 1:||Main phenotypic characteristics of the strain Vibrio parahaemolyticus isolated from black gill diseased Penaeus monodon|
|-: Absent, +: Present|
|Table 2:||Mortality percentage of M. rosenbergii following V. parahaemolyticus challenge by intramuscular injection after supplementation with probiotic and Cassia auriculata leaf powder for three weeks|
A mortality rate of 13.1% was observed in 3% probiotic supplemented group and in 3% leaf powder supplemented group, the mortality was 23.1% (Table 2).
Electrophoretic pattern of various polypeptide fractions of the muscle tissue of the control prawn and muscle tissues of the animals treated with probiotics and infected with vibrios are shown in Fig. 2. More than 16 polypeptide fractions were seen electrophoresed in control tissue (Fig. 2, lane 2). Major fractions could be seen at 68.0, 57, 36, 32, 29, 22 and 14.3 kDa regions. Lane 3 shows the nature of polypeptide fractions of muscle tissue of prawns injected with a stock suspension of vibrio. In these prawns, muscle proteins were fused together and did not show any separation of fractions. Lane 4 shows the polypeptide fractions of muscle tissue from prawns injected with sublethal concentration of vibrio. In this lane, disappearance of some of the fractions and the decrease in intensity of few fractions (36 kDa) could be observed. In lane 5, where the muscle tissue of prawns injected with LD50 concentration of vibrio colonies, further reduction in the intensity of polypeptide fractions, could be observed. Lane 6 shows the recovery of prawns treated with 3% concentration of probiotic. This lane showed the reappearance of polypeptide fractions that disappeared in lane 5. Prominent fractions could be seen between polypeptide such as 14.3 and 68 kDa. Almost similar results were observed in lane 7 also, where the muscle tissue was from prawns treated with 1% concentration of probiotic. When compared to lane-6, bands at 18 and 20 kDa were less intense. Basically all the animals infected with vibrios showed the disappearance of polypeptide fractions at 14.3 kDa and formation of a new fraction at 82.3 kDa, irrespective of the probiotic treatment.
|Fig. 2:||Electrophorogram of proteins in the muscle of M. rosenbergii fed on probiotic incroporated diet. Lanes (1): Marker, (2): Central diet fed prawns. Vibrie injected prawns (3): Vibrie injected at stock concentration, (4): Vibrie at sublethal concentration, (5): Vibrie at LD50 concentration, (6): Vibrie challenge at prawns kept on 30% probiotic, (7): Vibrie challenge prawns kept on 1% probiotic supplemented diet|
Most bacterial diseases like vibriosis, strike when the culture organisms are in a weakened or stressed condition. Vibriosis is a serious problem in the hatcheries and farms of marine shrimps and freshwater prawns (Takahashi et al., 1985; Hameed et al., 1996). Like many other species of vibrio, like V. harveyi, V. anguillarum and V. ordalii (Austin et al., 1995), V. parahaemolyticus has also been recognized to be pathogenic to prawn larvae in saltwater and freshwater hatcheries (Sae-Qui et al., 1987; Lavilla-Pitogo et al., 1992; Tonguthai, 1992).
In the present study, histopathological manifestations of vibrios in the gills and hepatopancreas of M. rosenbergii, could be observed and the pathological changes observed were comparable with earlier studies. Numerous tubules of the hepatopancreas had reduced number of stored lipid vacuoles and many were dilated and devoid of epithelial cells. These changes suggest that the prawns under pathological stress, were probably not eating well or metabolizing feeds normally (Fig. 1). Villalon (1993) also made similar suggestion in his work on Vibrio alginolyticus infestation of Artemia. Jiravanichpaisal et al. (1994) and Esteve and Herrera (2000) could demonstrate the granulomatous appearance of hepatopancreatic tubules in vibrio-infected P. monodon. They suggested that, the host was producing inflammatory response in an attempt to destroy or wall off the injured tissues. A similar inflammatory response to vibriosis in the hepatopancreas of M. rosenbergii could be observed at present. Hepatopancreas important site of vitelline synthesis and total protein content fluctuation during the reproductive cycle in M. rosenbergii was studied by Shanju and Geraldine (2011).
Detachment and thickening of basal lamina from hepatopancreatic tubule, reduced lumen, proliferation of blood cells into the lumen and degeneration of tubules appear to be the basic response of shrimps to toxicants (Nagesh et al., 1999). Similar observations were made in P. monodon exposed to Gusathion A (Baticados and Tendencia, 1991) and Endosulfan (Pillai, 1991) and in P. indicus exposed to Phosphamidon (Renukaprasad, 1993). Detachment of basal lamina, loss of structural integrity and reduced lumen could also be noticed in the hepatopancreas of P. monodon, exposed to Perfekthion (Vogt, 1987). The degenerative changes in the hepatopancreas of M. rosenbergii observed at present, under vibrio infestation were similar to those general changes effected by pesticides in penaeids.
Gills of vibrio-infected M. rosenbergii also showed various degenerative changes like necrosis, vacoulation of nuclei leading to karyolysis, oedema and cellular disintegration. Observations similar to those in M. rosenbergii could be recorded by Karunasagar et al. (1997) in virus-affected shrimps from the west coast of India. Egusa et al. (1988) and Mohney et al. (1994) have reported that gills of many vibriosis affected penaeids may also show evidence of bacteria and amorphous debris accumulated to the surface of the secondary gill filaments. This might be due to decreased cleaning activity by the animals or increased suspended material in the water column (Bauer, 1977, 2002; Martin et al., 2000). Loss of the regular surface structure of gill may result in gill fouling as well (Bauer, 1979).
Vibriosis has generally been considered indicative of the unhealthy status of a culture system. Antimicrobial compounds are used prophylactically and as medicines in aquaculture. There exists a possibility of transmittance of drug resistant pathogens to human beings either through consumption of seafood or via farm effluents. Although, the use of probiotics in aquaculture is not as popular as in livestock management, their use can be an effective strategy in combating diseases in aquaculture (Gatesoupe, 1999; Gram et al., 1999; Rengpipat et al., 1998; Rengpipat et al., 2000). The use of probiotic as biological control agents could be like a risk insurance under normal conditions and also highly effective during disease outbreaks. Several possible mechanisms have been proposed to explain the health-promoting action of probiotics such as competitive exclusion, supplementing the enzyme production of the host, water quality improvement, immuno-modulation and production of inhibitory compounds (Balcazar et al., 2006). Several investigations have shown that the manipulation of gut microbiota through feed supplementation by probiotics bacteria can bring about increased disease resistance and survival rate (Villamil et al., 2003; Balcazar and Rojas-Luna, 2007). Many of these beneficial strains also show immunostimulatory effect and increased phagocytic activity (Gullian et al., 2004). Lactobacillus acidophilus isolated from the gut of marine prawn P. monodon exhibited bacteriocin activity can be used as probiotic strain (Karthikeyan and Santhosh, 2009). In the present study, a commercial probiotic with Lactobacillus and Bacillus as dominant components, was administered as feed-supplement (1 and 3%) to M. rosenbergii. Although lactobacilli are widely used probiotic for terrestrial mammals, their effectiveness against vibriosis in aquatic environment has also been established (Gatesoupe, 1994; Sugita et al., 1998). Rengpipat et al. (1998, 2000) and Balcazar and Rojas-Luna (2007) have recorded the probiotic prospects Bacillus strains in the health and immunity enhancement of aquatic organisms such as fish and shrimps. In probiotic administered M. rosenbergii, a noticeable reduction in mortality could be observed (Table 2). Survival percentage of prawns were directly proportional to the dosage of probiotic supplemented. This might be indicative of the need for maintaining an ideal level of health promoting gut microbiota, through high level probiotic supplementation on a regular basis. The present results were comparable to those of Vaseeharan and Ramasamy (2003) for their study on the antagonistic effect of Bacillus subtilis against V. harveyi infected P. monodon. Enhanced survival rate, protein content with supplemented feed was observed in M. rosenbergii (Davassi, 2011). Inoculation of Bacillus S11 strain and a mixture of Lactobacillus spp. also have showed improved survival rate in the juveniles of P. monodon (Gatesoupe, 1994; Rengpipat et al., 1998; Rengpipat et al., 2000; Otta and Karunasagar, 1999). Ziaei-Nejad et al. (2006) also reported the use of a commercial probiotic to increase the survival and enhance the growth in Fenneropenaeus indicus.
The devastating effect of vibriosis was evident in the electrophorogram of the muscle proteins of M. rosenbergii (Fig. 2, Lane 3). A structural disintegration was evident due to the heavy dosage of V. parahaemolyticus. With LD50 and sublethal dosages of Vibrio, the effect of pathogens on the structural assembly of polypeptides was not so intense. However, when the prawns were maintained on a probiotic supplemented diet, the effect of vibriosis on the polypeptide fractions was less evident. As there was no difference was exhibited in food and feeding habits of two species of Macrobrachium the formulated feed supplemented with probiotic can be used for all commercially important species (Bello-Olusoji et al., 2006). In general, it may be concluded that probiotics help host animals to resist pathogen-induced biochemical changes, although, its specific mechanism needs further investigation.
Another effective alternative for chemotherapeutic control of microbes could be the herbal products which enhance the growth and also elicit antimicrobial activity (Karunasagar et al., 1994; Hameed and Balasubramanian, 2000). Herbal products are presumed to be devoid of toxic chemical substances and hence, considered environment-friendly. Antimicrobial activity of two medicinal plants against common pathogens were reported in our earlier studies (Balasundaram et al., 2011). Enhanced growth and increased survival rate against vibriosis have been reported in prawns when supplemented with the extracts of terrestrial plants and seaweeds (Ramesh et al., 2002; Immanuel et al., 2004). Vibriosis resistance has also been observed when Chinese medicinal herbs and the extracts of several seaweeds have been used as feed supplements (Jian and Wu, 2003; Selvin and Lipton, 2004; Huang et al., 2006). Impact of three medicinal plants on biochemical parameters and combat against Aeromonas hydrophila in Labeo rohita was observed in our earlier studies (John et al., 2011). In Tilapia rendalli fed with plant diets enhance growth performance and higher protein efficiency in turn resist diseases (Hlophe and Moyo, 2011). Pharmacological properties of Cassia nigricans Vahl, against human and veterinary diseases was reported by Attitalla (2011). Use of Cassia auriculata leaf powder as feed supplement in the present study also confirmed the health promoting property of the herbal extract, expressed as increased survival rate against V. parahaemolyticus infection (Table 2), although not to the extent as effected by the probiotic. However, as seen in the case of probiotic-use, increased supplementation (3%) with Cassia auriculata showed a proportionate reduction in mortality. Disease resistance potential of Cassia auriculata leaf powder, with reference to shrimps has already been demonstrated (Supamattaya et al., 2005). Several medicinal plants were studied for their curative potential against vibriosis (Selvin and Lipton, 2004; Immanuel et al., 2004). Experiments conducted so far using medicinal plants and their extracts, including the present study, underscore the effectiveness of many phytic principles in the mitigation of diseases common in cultured crustaceans and fishes.
In the present study, even though both probiotic and leaf powder supplemented prawns showed increased survival rate, leaf powder has been used in a crude form. Thus, it could effect only reduced survival rate compared to that of probiotic supplemented group. If the active compound from the plant is identified and used in the purified form, its effect on vibriosis may be further impressive.
Authors are grateful to Ministry of Earth Sciences, Ocean Atmospheric Science Technology Cell (MOES-OASTC), Government of India for providing financial support.
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