Molecular Identification of Vibrio alginolyticus Causing Vibriosis in Shrimp and Its Herbal Remedy

Shrimp culture is one of the fastest-growing aquaculture industries in Bangladesh. A dramatic expansion of shrimp culture occurred in the 1980s significantly contributed to the economy of Bangladesh (Paul and Vogl 2011; Hossain et al. 2013). However, in the recent years, shrimp production in Bangladesh has severely been affected by the outbreak of various diseases such as black spot, softshell, external fouling, broken appendages, hepatopancreatic infection, and vibriosis (Chow dhury et al. 2015; Ali et al. 2018). Among these diseases, vibriosis is considered as one of the most important bacterial diseases in shrimp farms of Bangladesh. Vibrio sis infects both penaeids and non-penaeid shrimps (Chowdhury et al. 2015) and is responsible for high mortality in aquaculture worldwide (Lightner 1988; Sparagano 2002), and can devastate the entire shrimp farm (Wei and Wendy 2012). Several members of Vibrio genus such as V. harveyi, V. anguillarum, V. splendidus, V. parahaemolyticus, V. fluvialis, and V. alginolyticus are reported as the causative agents of vibriosis in shrimp (de la Pena et al. 1993; Karunasagar et al. 1994; Lee et al. 1996; Lightner 1996; Austin and Zhang 2006; Chatterjee and Halder 2012). No molecular level study has so far been conducted to precisely identify the causal agent of vibrio sis in shrimp in Bangladesh and the required effective management for this worrisome disease.

A number of antibiotics and chemotherapeutic agents have been used in shrimp farms to prevent and control of microbial diseases including vibriosis (Mohney et al. 1992; Hossain et al. 2012; Karim et al. 2018). But indiscriminate and careless use of antibiotics leads to the development of antibiotic resistance in microorganisms (Karunasagar et al. 1994), which is now a major health concern worldwide (Karim et al. 2018). Therefore, an alternative approach is needed for effective and sustainable management of vibriosis in shrimp. Herbal extracts could be used as safe and alternative to synthetic antibiotics for the management of vibriosis in shrimp. Although Vibrio sp. is the important causal pathogen in shrimp farms, the investigation of the herbal remedy of vibriosis in shrimp is very limited in Bangladesh. Bangladesh is rich in diversed medicinal plants (Yusuf et al. 2009) and some of them have been found effective in the management of fish diseases. It has been demonstrated that methanol extracts of A. sativum, and methanol and acetone extracts of S. aromaticum significantly reduce the mortality of tilapia fish, artificially infected with Enterococcus faecalis as both preventive and therapeutic agents (Sindermann 1990). However, no study has so far been conducted in Bangladesh for the management of shrimp diseases caused by Vibrio sp. using herbal extracts. Therefore, the objectives of this study were to (i) identify the causal agent(s) of vibriosis in shrimp through physiological, biochemical and molecular techniques, (ii) assess the antibiotic susceptibility profile in the isolated shrimp pathogenic Vibrio sp., and (iii) control of vibriosis through the treatment with herbal extracts.

Isolation, phenotypic, and molecular characterization of the pathogen causing vibriosis in shrimp. The infected shrimps were collected from the farms near Sundarbans mangrove forest. The high mortality of shrimps (approximately 65%) was observed in these farms. The infected shrimps exhibited reduced feeding and lethargic in swimming at the edges and surface of the water body. No symptoms of white spot syndrome virus disease (WSSV) were observed in the infected farms. The farms were also free from pollution. Water quality parameters of the infected farms were 7.8 ± 0.2, 30 ± 1.6, 8.6 ± 1.0, 0.2 ± 0.08, and 4.5 ± 0.5 for pH, temperature (°C), salinity (ppt), ammonia (mg/l), and dissolved oxygen (mg/l), respectively. Twenty-five bacterial isolates were randomly selected (18 from TCBS and 7 from NA) as suspected Vibrio sp. based on their colony characteristics for preliminary phenotypic identification. Among these, a total of 20 isolates (16 from TCBS and 4 from NA) were Gram-negative, rod-shaped, fermentative, motile bacteria, susceptible to vibriostatic agent 0/129, and were positive in oxidase test, catalase test, indole production, and lysine decarboxylase test but negative in acetoin and hydrogen sulfide (H₂S) production test (Table III). Isolates were able to grow in the presence of 2, 4, 6, and 8% NaCl but did not grow in the absence of NaCl. They were unable to grow at 4°C but grew well at 40°C temperature. The isolates produced acid from glucose, sorbitol, mannitol, and sucrose but did not produce acid from arabinose. Based on the colony’s morphological and biochemical characteristics, twenty isolates were phenotypically identified as Vibrio sp. (Farmer et al. 2005; Jayasree et al. 2006; Nelapati et al. 2012). The phenotypic and biochemical characteristics of these twenty isolates were very similar to the characteristics described for V. alginolyticus (Lie et al. 2004).

Among twenty isolates, four (2A1a, 2A3, 2A11 and 2V21) were randomly selected for further molecular, pathological, antibiotic susceptibility and herbal disease control studies. The 16S rRNA gene sequence data of these four selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. The sequences of the isolates 2A1a, 2A3, 2A11, and 2V21 have been deposited to NCBI Gen Bank with accession numbers MG757701, MG757699, MG757700, and MG757703, respectively. In the phylogenetic tree, these four isolates shared a common ancestor and formed a cluster with V. alginolyticus (Fig. 1).

Fig. 1.

In vivo challenge of the isolated V. alginolyticus. To observe whether V. alginolyticus isolates were pathogenic to juvenile shrimp, we conducted an in vivo challenge test under laboratory conditions. All of the four V. alginolyticus isolates tested (2A1a, 2A3, 2A11, and 2V21) produced disease symptoms in the juvenile shrimp and caused high mortality ranged from 81.67 ± 2.29% to 86.67 ± 2.29% (Fig. 2). In juvenile shrimp, mortality was observed from 24 to 96 hours after inoculation and the highest mortality was recorded within 72 hours. Hepatopancreatic discoloration, the main symptom of infection, was observed clearly in the challenged shrimp within 96–144 hours. The infected shrimp exhibited feeding redundancy and lethargic swimming at the surface of the aquarium.

Fig. 2.

Antibiogram profile of V. alginolyticus isolates. To find out whether the shrimp pathogenic V. alginolyticus isolates had any resistance against commercial antibiotics, we screened them against eleven antibiotics using disc diffusion assay. V. alginolyticus isolates exhibited resistance against various antibiotics such as erythromycin, penicillin, amoxicillin, vancomycin, ampicillin, and cefradine (Table IV). However, they were found sensitive to levofloxacin, cefuroxime, azithromycin, nitrofurantoin, and gentamicin. The maximum and minimum inhibition zone was observed for levofloxacin and azithromycin, respectively.

In vitro inhibitory effects of herbal extracts. The shrimp pathogenic V. alginolyticus isolates were susceptible to crude aqueous extracts of the leaf of E. officinalis, the bulb of A. sativum, the bud of S. aromaticum, and the leaf of T. indica. The E. officinalis extracts displayed the highest antibacterial activity (Table V). The bulb extract of A. sativum and bud extract of S. aromaticum also showed high inhibitory activity against V. alginolyticus whereas, lowest antibacterial activity against the V. alginolyticus isolates was observed for the leaf extract of T. indica. The ethyl acetate extract of the E. officinalis leaf strongly inhibited the growth of V. alginolyticus with maximum zone ratio 6.4 ± 0.19 but methanol, n-hexane, and acetone extracts of the leaf of E. officinalis caused no inhibition. The ethyl acetate extract of the bulb of A. sativum also highly inhibited V. alginolyticus (Fig. 3) followed by methanol extract. Acetone and methanol extracts of the bud of S. aromaticum also strongly inhibited the growth of V. alginolyticus isolates. The acetone, n-hexane, methanol, and ethyl acetate extracts of the leaf of T. indica inhibited the growth of V. alginolyticus but the zone ratios were not satisfactory.

Fig. 3.

An in vivo effect of herbal extracts as therapeutic agents against vibriosis in shrimps. Juvenile shrimps were fed with various herbal extracts mixed feed after exposing them to the highly virulent isolate of V. alginolyticus (2A1a). Interestingly, 100 ± 0.0% of the challenged shrimp were survived when fed with ethyl acetate extract of E. officinalis and A. sativum at the rate of 10 mg/g of feed (Fig. 4). Survival of shrimp was also high (86.7 ± 5.8%) when fed with methanol extract of A. sativum at the rate of 10 mg/g of feed. Hoverer, only 16.7 ± 2.87% shrimp survived when challenged with the pathogenic Vibrio isolate 2A1a in control group 2 (normal feed without any herbal treatment). The lower rate of survival (35.0 ± 7.07%) was also observed in the shrimp when fed with acetone extract of T. indica at a rate of 15 mg/g feed.

Fig. 4.

Vibriosis is one of the most important bacterial diseases of shrimp caused by several species of Vibrio. This study isolated and identified (phenotypically) twenty isolates of Vibrio sp. from the infected shrimp with vibriosis symptoms collected from shrimp farms of Bangladesh. Among twenty, four virulent isolates were identified as V. alginolyticus by 16S rRNA gene sequencing (Fig. 1). These V. alginolyticus isolates exhibited a high level of virulence against juvenile shrimp (81.67 ± 2.29% to 86.67 ± 2.29% mortality) in an in vivo challenged study. Liu et al. (2004) also obtained 80% mortality in shrimp in an in vivo challenge test with V. alginolyticus (CH003). In this study, the infected shrimp in in vivo challenge test exhibited almost identical symptoms as found in the naturally infected shrimp (Lightner 1993; Anderson et al. 1998).

One of the important findings of this study is that the shrimp pathogenic V. alginolyticus isolates showed resistance against various antibiotics but exhibited the high level of susceptibility to both aqueous and organic solvent extracts of herbs such as E. officinalis, A. sativum, S. aromaticum, and T. indica (Table V). Among these, ethyl acetate extract of E. officinalis strongly inhibited the growth of V. alginolyticus strains followed by ethyl acetate and methanol extract of A. sativum, and methanol extract of S. aromaticum. However, lower inhibition was obtained for both aqueous and organic extracts of T. indica. Medicinal plants are used to treat a variety of diseases for thousands of years of civilizations (Petrovska 2012). An in vitro antibacterial activity of numerous plants against both Gram-positive and Gram-negative marine bacteria has been reported elsewhere (Castro et al. 2008; Roomiani et al. 2013). Ethanol extract of turmeric (Curcuma longa) was reported to inhibit the shrimp pathogen Vibrio sp. (Lawhavinit et al. 2011). In vitro inhibitory activities of both aqueous and organic solvent extracts of A. sativum, S. aromaticum, and T. indica against fish pathogenic Enterococcus faecalis have also been reported (Rahman et al. 2017).

The most remarkable finding of this study is that ethyl acetate extracts of E. officinalis and A. sativum remarkably protected the juvenile shrimp (up to 100%) from vibriosis by a virulent isolate of V. alginolyticus (Fig. 4). High to moderate survival rates were also obtained in shrimp fed with methanol extracts of A. sativum and S. aromaticum, and acetone extract of S. aromaticum. Inhibition of V. alginolyticus both in in vitro and in vivo conditions by organic solvent extracts of E. officinalis, A. sativum, and S. aromaticum suggests that these herbal extracts contain antibacterial secondary metabolite(s). Medicinal herbs are considered as one of the most important sources for medicine and drugs, as many secondary metabolites including antimicrobial substances are obtained from various herbs. Plants possess complex chemicals with varied biological activities, making plants suitable for the treatment of multifactorial diseases, and makes plants a suitable alternative to antibiotics with little risk for development of resistance (Gostner et al. 2012; Srivastava et al. 2014). A. sativum contains several bioactive compounds such as ajone, allicin and diallyl sulfides that possess potential antibacterial activity against different microorganisms (Naganawa et al. 1996; Ankri and Mirelman 1999; O’Gara et al. 2000). S. aromaticum contains eugenol that is reported to exhibit strong antibacterial activity against Staphylococcus aureus (Xu et al. 2016). Cinnamaldehyde and its derivatives obtained from cinnamon were reported to reduce the virulence in Vibrio sp. causing vibriosis (Brackman et al. 2008). S. cumini leaf powder also reported to increase immunity in juvenile shrimp (Litopenaeus vannamei) against V. parahaemolyticus infection (Prabu et al. 2018). Organic solvent extracts of A. sativum and S. aromaticum also reported to significantly increase the survival of Oreochromis niloticus from infection against E. faecalis (Rahman et al. 2017). Herbal extracts are also reported to stimulate immunity and develop disease resistance in shrimp (Raja Rajeswari et al. 2012; Yogeeswaran et al. 2012). Dietary administration of Gynura bicolor extract was reported to enhance the innate immunity and antioxidant enzyme activities of shrimp against V. alginolyticus and WSSV infection (Wu et al. 2015). The purified garlic compounds allicin and ajoene demonstrated immune stimulant capacity against fish pathogenic protozoa Spironucleus vortens and Ichthyophthirius multifiliis, and the bacteria A. hydrophila (Nya et al. 2010; Tanekhy and Fall 2016). Recently, Foysal et al. (2019) reported that dietary administration of garlic could modulate gut microbiota, increase recovery from streptococcus infection and upregulate the expression of immune genes in the intestinal tissue of tilapia. Since crude plant extracts contain multiple secondary metabolites, the chances of development of resistance in the pathogens against these extracts are likely lesser than those of pure antibiotics (Rahman et al. 2017). Valuable drugs could be developed form these herbal extracts to control vibriosis in shrimp and other fish diseases. The extracts of E. officinalis and A. sativum could be used as alternative therapeutic agents against vibriosis disease in shrimp.