Bioprospecting of novel antimicrobial metabolites from Bacillus subtilis MBTDCMFRI Ba37 and Pseudomonas aeruginosa MBTDCMFRI Ps04 of tropical estuarine habitats of Cochin, India and its application in fish health management

Aquaculture is an important economic activity the world over and about 90% of the global production is contributed by Asian countries. Global aquaculture has a persistent goal to maximize the production with optimal profit. Even though the aquaculture is growing at a rapid rate, the practice of aquaculture faces many challenges in its developing path. Intense cultivation in high densities significantly affect the environment by issues of used - up farm water discharges and heavy waste accumulation through hyper nutrification due to excessive feeding and high dietary nutrient composition (Liao and Mayo 1974; Boyd 1985). This has brought stress to the rearing environment and diseases to the cultured species and subsequently decreased the overall productivity due to mass mortality which has led to significant loss to the industry (Irie et al. 2005; Cruz et al. 2012; FAO 2012). Thus, it is necessary to face the challenges and find solutions in order to make aquaculture sustainable. Disease is the result of interaction between the host, pathogen and external environment. At the onset of disease, the harmonious interplay between pathogens and non-pathogens is disturbed result in unhealthy host and thereby infection occurs (Verschuere et al. 2000; Schulze et al. 2006; Zhou et al. 2009; Nemutanzhela et al. 2014). Bacteria, fungi, viruses and parasites are the major pathogens affecting aquaculture industry. Among different aquaculture pathogens, bacteria play a major role. It can survive and flourish independently in any aquatic environment. The bacterial fish infections are mainly from the species of Aeromonas, Vibrio, Flavobacterium, Edwardsiella, Yersinia, Pseudomonas, Streptococcus, Renibacterium, Piscirickettsia, Mycobacterium etc. The common bacterial disease symptoms are external reddening and haemorrhage in the peritoneum, body wall and viscera, generically referred to as haemorrhagic septicaemia. The progress of disease leads to ulcerative lesions and mortality of untreated cultured animals (Pridgeon and Klesius 2012). Among the bacterial diseases, vibriosis is caused by a major bacterial fish pathogen Vibrio spp. which is ubiquitous in all aqua cultured species (Jayaprakash et al. 2005; Thompson et al. 2010). In tropical countries, penaeid shrimp culture is highly affected by V. harveyi which causes their mass mortality (Austin & Zhang 2006). Another Vibrio species which affects the shrimp culture are V. alginolyticus, V. damsela, V. parahaemolyticus, V. vulnificus, V. splendidus and V. penaeicida (Saulnier et al. 2000)

Being a Women: Changing Paradigm

It is true that, if a man is getting educated and empowered, only he can be benefitted however, if a woman is getting educated and empowered, the whole family and society can be benefitted. “Behind every successful man there is a woman”. Not only in the case of a man, but this saying stands for the functioning of the whole world. She is said to be the ultimate strength and support behind the success of an individual. The basic unit of society is a woman. As woman makes a family, a family makes a home and homes make a society. So we should never think that a society would come into existence without the contribution of women. She plays roles as a mother, a sister, a daughter, a wife. They play their roles with great responsibilities in the upbringing of a healthy solid society, but she is in our so called modern world, still living in chains. We all know that without education, no development is possible. Here we have forgotten that the very first and best school of a child is its mother’s lap. A good healthy society doesn’t automatically emerge on its own and stands firm but it needs to be emerged and for its emergence women play a pivotal role. From behavioural to health education women have their hands on. It’s a woman who teaches how to behave, how to speak and how to deal with different classes of people. These all are the basic fundamentals of a good society and women are the main contributors in building up a strong society

Insight into the gut microbiology of wild-caught Mangrove Red Snapper, Lutjanus argentimaculatus (Forsskal, 1775)

Documenting bacteria present in healthy individuals forms the first step in understanding the effects of microbial manipulation in aquaculture systems. Among the commensal microflora, gut microbiota has attracted extensive attention owing to their role in host metabolism and health maintenance. Basic knowledge on normal gut microbes within a particular host species is thus essential to determine how successfully these microbes can be manipulated and engineered for sustainable aquaculture systems. In spite of the good aquaculture potential of Mangrove red snapper, Lutjanus argentimaculatus, the information on microbial communities associated with the gut of this fish, and their contribution towards digestive efficiency and disease resistance is scarce. Therefore, an attempt was made to elucidate the abundance and diversity of cultivable gut microbes of wild caught L. argentimaculatus along with their digestive exoenzyme profiles and prohibitory effect against fish pathogens. Results on abundance showed similar gut bacterial loads as that of other marine fish imposing the less contribution of microflora to the volume of gut materials in fish. Eleven distinct bacterial species including two proposed novel vibrios were identified. An incidental observation of Morganella morganii throughout samples is an alarming signal, emphasizing the need for immediate de-gutting to avoid histamine intoxication. Abundance of digestive enzyme producers and excellent enzymatic potential of some isolates suggested the contribution of digestive enzymes may supplement to the symbiosis between gut flora and host and the information is of interest to aquaculture nutritionists/commercial industries. Interestingly, some isolates demonstrated estimable co-aggregation with aquatic pathogens, indicating their involvement in disease resistance and the results correlated well with gut microbial diversity. These findings highlight the significant role of gut microbes towards nutritional physiology and disease resistance of this aquaculture candidate in natural ecosystem. The culturable microbiota profiles of wild fish generated in the study can be applied for measuring the quality of husbandry routines in aquaculture facility of this marine fish. Overall, the present study fetches insights on the gut microbiome of healthy L. argentimaculatus which forms a platform for follow-up studies. The study may also help in the development of “functional” fish feeds for L. argentimaculatus. The investigation also demonstrated some potential digestive enzyme-producing isolates having probiotic applications in commercial aquaculture

Development of a novel multiplex-PCR technology for simultaneous detection of five major aquaculture pathogens

Early and precise pathogen identification and corresponding disease management are primary concerns in aquaculture. Here, we attempted at diagnostic methods that can simultaneously identify multiple pathogens, where many samples, several pathogens, and concurrent infections are to be handled. Hence, a multiplex PCR assay targeting five major aquaculture pathogens, viz. Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi was developed for the first time. The primers targeting toxR of V. parahaemolyticus, amiB of V. anguillarum, col o

Farm evaluation of formulated diets on the growth and body composition of Etroplus suratensis reared in cages in low-saline coastal ponds

A 90-day on-farm feeding experiment was carried out in the installed cages in brackishwater ponds to evaluate the efficiency of three formulated diets on the growth performance and body composition of Etroplus suratensis. Two diets DI and DII were in-house formulations based on optimum macronutrient requirements (protein and fat) derived from an indoor nutritional evaluation of E. suratensis, and diet three DIII was a commercially available pellet meant for pearl spot fish. A sum of 900 fishes were randomly distributed with an average weight of 18.2±0.02g into three different treatment groups each in triplicate, and each replicate had 100 fishes stocked in cages with dimensions 2×2×1.5 m. At the end of the feeding trial, the fish were weighed to assess growth parameters in terms of % gain in weight, feed conversion ratio (FCR), and specific growth rate (SGR). The diet (DI) displayed significantly (p<0.05) higher % weight gain, and SGR than (DII) and (DIII). The FCR of diet (DI) was significantly (p<0.05) lower than diets (DII) and (DIII). Diet (DI) showed better growth when compared with other feeds. The study emphasized the need for species-specific diets for application at the farm level for an effective return on investment

Antimicrobial resistance and virulence characteristics of Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi from natural disease outbreaks of marine/estuarine fishes

Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio harveyi are the causative agents of the most severe diseases of marine and brackish aquaculture systems. These are also associated with serious ailments in humans. The present paper unravels the virulence features/genes and antimicrobial resistance (AMR) phenotypes/genes of these Vibrio spp. isolated from natural disease outbreaks of marine/estuarine fishes of India for the first time. Results on virulence features showed that V. vulnificus infected fish can pose public health risk. While, it has been found clinically that V. parahaemolyticus without tdh and trh genes are pathogenic to fish, even though they are reported to be, not associated with human diseases. It was significant to note that virulence features of V. harveyi were induced at increased salinity. Analysis based on the percentage prevalence of susceptible isolates and variation coefficient of zone diameters categorized 17 antibiotics in terms of their efficiency against each fish pathogenic species. Multiple antibiotic resistance index (MARi) of the isolates ranged between 0.058 and 0.47. Results on MARi and percentage of multidrug resistance strains indicated that >50% of the isolates were from low antibiotic usage area. The study generated tetH sequence from V. parahaemolyticus for the first time, and the sequence revealed high identity to that of clinical strains. Presence of tetB/ tetH gene was identified as the predictor for the resistance against the first generation tetracycline, the most commonly used antibiotic against Vibrio spp. in aquaculture practices. The data on associations between AMR features predicted certain cross-resistance between antimicrobials within Vibrio spp. Altogether, the paper serves as the baseline for epizootic tracking of public health significant vibrios from diseased fishes, to devise practical guidelines for antibiotic use and to formulate efficient control measures against three Vibrio spp. in aquaculture, targeting final applications in the implementation of national green and healthy aquaculture practices

Hemolymph of healthy Doclea rissoni Leach, 1815: A pool for Vibrionaceae?

As diseases have a great impact on the population dynamics, evolution and immune biology of affected organisms, it is important to understand the modes and mechanisms of interaction among the lower animals and their microbial symbionts in nature function together for survival. To achieve this, it is important to understand the community structure of the native flora of these animals. Spider crabs are well known for their tolerance to various pollutants. Nevertheless, the microbiology of their circulatory systems and their role in disease transmission are unknown. Therefore, the bacteria associated with the hemolymph of a spider crab, Doclea rissoni in its natural ecosystem was characterized. Nine healthy crabs were collected from the coastal areas of Kochi, and their hemolymphs were collected. Enumeration of viable bacteria using various media revealed a similar count in all the media (7-10×102 CFU/ml). Characterization of representative isolates by conventional microbiological methods and 16S rRNA gene sequencing was followed. Despite using three different media, all isolates belonged to a single family Vibrionaceae. There were two genera as Vibrio (86.67%) and Photobacterium (13.33%). Among Vibrio, five species belonging to three different clades were isolated, of which two could not be assigned to any known species. These were classified as novel species (belonging to Harveyi clade and, Brasiliensis clade) and their description is underway. The order of relative abundance was V. alginolyticus>Novel Vibrio-II>P. damselae>V. furnissii / V. parahaemolyticus / Novel Vibrio-I. Of these, V. alginolyticus, P. damselae and V. furnissii are implicated in occasional disease outbreaks in humans and animals, posing an increased risk to human economic activity, notably aquaculture practices. Therefore, further investigations are required to find the basis for the mutualistic relationship of vibrios in different crabs

Pathological investigations of Vibrio vulnificus infection in Genetically Improved Farmed Tilapia (Oreochromis niloticus L.) cultured at a floating cage farm of India

An outbreak of skin ulcers with morbidity and mortality rate of 80% and 20% respectively, among Genetically Improved Farmed Tilapia (GIFT) sub-adults reared in a brackish water floating cage farm of Kochi, Kerala, India was reported. Bacterial isolation attempts from various internal organs (blood, kidney, liver, spleen and brain) of different diseased fish indicated a common bacterial etiology. Organisms were identified as Vibrio vulnificus based on conventional microbiological methods followed by molecular confirmation. Additionally, V. vulnificus marker specific for fish virulence was present in the isolates. Fish pathogenicity was confirmed by challenge tests. LD50 was calculated as 2.1 × 105.13 CFU/fish and 2.1 × 106.12 CFU/mL in injection and immersion route respectively. pilF polymorphism-based PCR and vvhA gene sequence indicated potential danger for humans. The strain was positive for siderophore, hemolysin, capsule, polar and lateral flagella. The strain could multiply rapidly in healthy fish serum. All these results indicated that the isolate could act as a primary pathogen for tilapia fish. Interestingly, serum survival ratio of the pathogen was significantly larger at higher temperatures (p ≤ .05); which may explain the increase in infective capacity of V. vulnificus at higher temperature. Multiple antibiotic resistance index of the strain was ≤0.2. In conclusion, the paper provides detailed descriptions of clinical signs, microscopic and macroscopic lesions of a natural disease outbreak among tilapia raised in brackish water cage farms of India. The concurrent attempts to identify and characterize the etiology fetches the basis for future development of effective control and preventive measures against V. vulnificus, the most infectious and lethal of all zoonotic vibrios

Glimpse towards cultivable hemolymph microbiota of marine crabs: Untapped resource for aquatic probiotics/antibacterial agents

Bacterial diseases have turned out to be the primary constraint in sustainable aquaculture production, where, probiotics can play an important role to prevent or control fish and shellfish diseases. As the autochthonous preparations would be more effective than commercial products, continued search for novel antibacterial strains native to aquatic environment are warranted against aquatic pathogens. Further, knowledge on abundance, composition and role of hemolymph microbes is also essential to predict the health status and disease diagnosis. Hence, in present study, 4 commercially significant marine crabs that are important for aquaculture were used to unravel the implication and significance of cultivable hemolymph microbes. Bacterial abundance was found to be individual- and species-dependent; and statistically significant interaction was present between growth media and abundance. Gram negative isolates represented 84% of hemolymph microbes. Vibrio was the principal genera in all species; each carrying a specific hemolymph microbiota (both in terms of abundance and diversity). The present study forms the first report of genera viz., Enterovibrio, Pantoea, Kluyvera and Enterobacter in crustacean hemolymph. Interestingly, new Vibrio species were also found. Further, the study forms the first observation on inhibitory activity of marine crab hemolymph microbes against aquatic pathogens. Overall, the results highlight marine crab hemolymph microbiota as a promising moreover, an untapped resource for probiotics/ antimicrobial agents to combat aquatic pathogens. Concurrently, the present study fetches a platform for the prediction of health and disease diagnosis of 4 potentially important aquaculture crab species