Lippia alba essential oil promotes survival of silver catfish ( Rhamdia quelen ) infected with Aeromonas sp .

In vitro and in vivo activity of the Lippia alba essential oil (EO) against Aeromonas sp. was evaluated. In the in vitro assay the minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of EO for Aeromonas cells were determined using the microdilution method. Twenty five strains of Aeromonas sp. isolated from infected fish obtained from local fish farms were used. MIC and MBC values were 2862 and 5998 μg mL for L. alba EO and 0.5 and 1.2 μg mL for gentamicin, respectively. In the in vivo assay silver catfish juveniles (Rhamdia quelen) (7.50 ± 1.85 g and 10.0 ± 1.0 cm) with typical injuries associated to Aeromonas infection were divided into four treatments (in triplicate n=10): untreated fish (negative control), 10 mg L of gentamicin, and 20 or 50 μL L of EO. Fish were maintained in aerated 20 L plastic boxes. After 10 days survival of silver catfish infected with Aermonas sp. and treated with essential oil (50 μL L) was greater than 90%.


INTRODUCTION
Various drugs have been used for treatment or prevention in aquaculture diseases.The abuse of these drugs is associated with several problems, such as the presence of illegal residues in meat and transmission of resistant bacteria to the environment, animals and man, a potential risk to public health (Schmidt et al. 2000).As an alternative to conventional drugs, medicinal plants have been widely used in veterinary and human medicine and nowadays also have a significant role in aquaculture as prophylactic and therapeutic agents against fish pathogens, presenting antiviral, antibacterial, antifungal and antiparasitic potential (Citarasu 2010).Essential oils and extracts from plants could provide a viable alternative to conventional drugs minimizing the economic losses resulting from infections.These natural products contain a rich blend of highly functional molecules some of which are beneficial but others which are not, presenting toxicity.However, in the environment, this toxicity is very low compared with those of synthetic pesticides or drugs (Park et al. 2011).
Outbreaks of bacteria of the genus Aeromonas in fish are associated with changes in environmental conditions and/or stress factors like parasitic infections (Barcellos et al. 2008).Usually considered a secondary pathogen, bacteria of Aeromonas genus can also act as a primary pathogen in some environments, causing high mortality in fish farms (Nielsen et al. 2001).
The aim of the present study was to find a natural alternative to prevent/treat infections caused by bacteria of Aeromonas genus in Rhamdia quelen, a native species suitable for fish culture in southern Brazil.Lippia alba was chosen because of its antimicrobial and anti-inflammatory potential (Pascual et al. 2001, Glamočlija et al. 2011).

ESSENTIAL OIL
Lippia alba (Mill.)N. E. Brown was cultivated in São Luiz Gonzaga, Rio Grande do Sul, Brazil.The plant material was identified by botanist Dr. Gilberto Dolejal Zanetti, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria (UFSM), and a voucher specimen (SMDB Nº. 10050) was deposited in the UFSM herbarium.Lippia alba essential oil (EO) was extracted from the fresh leaves of the plant by hydrodistillation for 2 hours using a Clevenger type apparatus (European Pharmacopoeia 2007).The EO samples were stored at -4 °C in amber glass bottles and EO analysis was performed by gas chromatography-mass spectrometry (GC/MS).The major components were linalool and 1,8-cineol (Table I) (Barros et al. 2009).

IN VITRO ASSAY
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EO for Aeromonas cells were determined using the microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute, document M31-A3 (CLSI 2008).The EO was previously diluted in ethanol to obtain the initial concentration of 176100 μg mL -1 and twofold dilutions were performed.Twenty five isolates of Aeromonas sp. from infected fish obtained from local fish farms were used.Reference samples (Aeromonas hydrophila ATCC 7966, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922) were used as quality control.The inoculum was prepared in saline solution from cultures grown in Mueller-Hinton agar (1x10 8 CFU mL -1 ; 0.14 OD 600 nm) and 10 μL (1x10 5 CFU) was added to each microplate well containing EO.The microplates were incubated at 35 °C for 24 hours under aerobic conditions.The positive control was performed with gentamicin antibiotic.

IN VIVO ASSAY
Silver catfish juveniles (7.50 ± 1.85 g and 10.0 ± 1.0 cm) were obtained from a fish culture and transported to the laboratory where they were kept in continuously aerated 250 L tanks (23 ± 1°C, pH 6.6 -7.0, dissolved oxygen levels 5.8 -7.2 mg L -1 ).Fish arrived at the laboratory with typical injuries associated to Aeromonas infection.Bacterial infection of fish by Aeromonas was confirmed by morphological and biochemical characteristics analyzed in accordance to Quinn (1994).
To evaluate silver catfish survival, juveniles were divided into four treatments (in triplicate n=10): untreated fish (negative control); 10 mg L -1 of gentamicin (Belem-Costa and Cyrino 2006), and two EO concentrations (20 and 50 µL L -1 ) equivalent to 16 and 40 mg L -1 , respectively, because the density of this EO is approximately 0.80 g mL -1 .Fish were maintained for 10 days in aerated 20 L polyethylene boxes.These concentrations were chosen because L. alba EO in concentrations above 100 µL L -1 induce deep anesthesia in R. quelen (Cunha et al. 2010).Fish were fed once a day to satiety, and 30 min later feces and food remains were removed by siphoning.To avoid dilution of the EO the water volume withdrawn during each siphoning (250 Lippia alba ESSENTIAL OIL AGAINST Aeromonas sp.mL) was not replaced until eighth day, when all water was replaced.The methodology of this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria (Process N. 046/2010).
Dissolved oxygen and temperature were measured with YSI oxygen meter.The water pH was verified with a DMPH-2 pH meter.Total ammonia nitrogen (TAN) levels were determined by Nessler method in accordance to Eaton et al. (2005).Unionized ammonia (NH 3 ) levels were calculated as per Colt (2002).
Fish survival was compared by Kaplan-Meier survival analysis with Logrank Test (SPSS 18 Software).The minimum significance level was set at P < 0.05.

RESULTS AND DISCUSSION
In the in vitro test MIC and MBC values were 2862 and 5998 µg mL -1 for L. alba EO and 0.5 and 1.2 µg mL -1 for gentamicin, respectively (Table II).At the beginning of the experiment 85% of the fish from each treatment had signs of infection, including ulcerated fins and tissue loss between fin rays with haemorrhage, ulcerative dermal lesions, abnormal coloration (darkening) and lesions in the genital pore and anus.In the control group, these signs and lesions remained and gradually increased until all the fish died.Between the fourth and sixth day of treatment, 65% of fish treated with 50 µL L -1 EO did not show evidence of any lesions.Fish treated with 20 µL L -1 EO or gentamicin only showed the same improvement between the sixth and eighth days of the experiment.The mortality in the control group was 100% at the end of experiment.In groups treated with gentamicin (10 mg L -1 ) and 20 µL L -1 of EO survival was greater than 80%.In the group treated with 50 µL L -1 of EO survival was greater than 90% (Figure 1).The survival was not significantly different between groups treated with EO and gentamicin.
The active compounds of herbs possess characteristics that could be useful in fish and shrimp culture; various herbs can stimulate growth and appetite, increase immune system responses, and have broad spectrum antimicrobial activity (Friedman et al. 2002).Different species of Lippia have been used against Gram-positive and Gram-negative bacteria (Pascual et al. 2001).According to Ríos and Recio (2005) only extracts or essential oils that kill or inhibit bacterial growth at concentrations below 100 µg mL -1 should be considered active.Based on their statement, L. alba EO would not be interesting for future research because a MIC of 2862 µg mL -1 was observed.However, the in vivo results of the test conducted with silver catfish infected with Aeromonas sp. and treated with this EO demonstrated higher survival compared with control group.
Plasma cortisol level is widely used as a general indicator of stressful situations in vertebrates and particularly in fish (Pickering and Pottinger 1989) and high plasma cortisol levels cause secondary immunosuppression (Wedemeyer and Mcleay 1981).The observed effect of L. alba EO may be due to its effects on the fish' immune system because this EO prevented the cortisol rise in R. quelen submitted to acute stress (Cunha et al. 2010).Glycyrrhizin (Jang et al. 1995), aloe (Kim et al. 1999), and azadirachtin (Logambal and Michael 2001) enhance innate immunity in fishes.Oral administration of Quillaja saponin increased leucocyte migration in yellowtail Seriola quinqueradiata (Ninomiya et al. 1995).The use of diets supplemented with the medicinal plants Cynodon dactylon, Aegle marmelos, Withania somnifera, and Zingiber officinale increased non-specific immune responses and bacterial resistance against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio campbelli, Vibrio vulnificus, Vibrio harveyi, and Photobacterium damselae, and these compounds lead to lower mortality in tilapia (Oreochromis mossambicus) compared with control (Immanuel et al. 2009).The exact mechanism of action of herbal extracts on the fish immune system is still unclear (Harikrishnan et al. 2011).
The pathogenesis of Aeromonas genus is multifactorial and depends upon the secretion of numerous extracellular virulence factors (Yu et al. 2005).Research shows that subinibitory concentrations of EOs from plants decrease or inhibit the production of certain virulence factors in bacteria.Essential oils of plants can inhibit biofilm formation or the production of exotoxins such as haemolysins that cause erythrocytes hemolysis thus reducing the bacteria pathogenicity (Smith-Palmer et al. 2004, Cândido et al. 2010, Millezi et al. 2013).Thymus vulgaris EO significantly reduced biofilm formation in Aeromonas hydrophila.The major compound in T. vulgaris EO was 1,8-cineol (Millezi et al. 2013) which is also found in L. alba EO.Linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, which are important virulence factors in this fungus (Costa et al. 2014).
Additionally, it is desirable that antibiotic use in fish cultures be reduced and replaced by natural medicines to prevent the emergence of bacterial resistance in aquatic animals and its environment.In Brazil, antibiotics, such as oxytetracycline, are widely used in fish farms to prophylactically treat bacterial infections and as a growth promoter.Despite its widespread use, there is no regulation on this drug class in fish (Rigos and Troisi 2005).
Our results demonstrate that L. alba EO has weak in vitro activity against Aeromonas sp.However, the EO added to water at lower concentrations than the anesthetic concentration, promotes survival in fish infected with this bacterial genus.Further studies should be conducted to verify this activity against other pathogenic bacteria of interest in aquaculture and to confirm immune response involvement or its potential as a virulence factors inhibitor.

Figure 1 -
Figure 1 -Survival of juvenile silver catfish infected with Aeromonas sp. and treated with L. alba EO. (*) indicates significant difference relative to negative control.Kaplan-Meier survival analysis with Logrank test (P < 0.05).