Fish is one of the foods indicated in the 2030 Agendato fight against hunger [1] and aquaculture is one source to increase fish supply globally [2-3].  To meet up with the ever increasing demand for fish, aquaculturehas adopted intensive culture systems.  These intensive culture systems are associated with higher productivity per unit area as a result of higher stocking density [4]. Higher stocking density alongside fish handling, harvest and transportation has magnified stressand elevated the risk of disease outbreak in aquaculture [2].  Disease outbreakis one of the major constrains in fish aquaculture [5].  Most of the causes of diseases in aquaculture involve the presence of protozoa, fungi, bacteria and virus. Diseases when present consequently results in mortality and growth retardation in fish [6].

Over the years, chemotherapy has been used inaquaculture as preventive and curative measures to improve quality and sustainability [7]. Although chemotherapy has been used for disease prevention, treatment as well as growth promotion, their misuse caused antimicrobial resistance among pathogenic bacterial strains, residual accumulation in tissuenot safe for human consumptions as well as devalued environmental conditions [8]. To proffer solution among other things, medicinal herbs have been successfully used as an alternative to chemotherapy in fish aquaculture [2,9-12].   Medicinal herbs   could be used  as whole or in parts (leaves, flower, roots, seed, fruits or backs) in a crude form or as extract/compound from whole plants or parts.  A wide range of medicinal herbs have been used and reported.  For example, Gabriel et al. [13] investigated the effects of dietary Aloe vera on plasma lipid profile status, antioxidant, and hepatoprotective enzyme activities of GIFT-tilapia juveniles under Streptococcus iniae challenge; Camellia sinensis was incorporated into feed to investigate their effects on growth and resistance of Oreochromis niloticus against Aeromonas hydrophia [14]; essential oil extracted from Citrus sinensiswere investigated for growth rate and resistance against Streptococcus iniae [15-16] evaluated oil extracted from Citrus limon peels on growth and immune response of O. mossambicus challenged with Edwardsiell atarda; 

Klebsiella pneumoniae (NCBI accession number-KY003130) is a gram-negative,non-motile,encapsulated, lactose-fermenting, facultative anaerobic, rod-shaped bacterium.K. pneumoniae is a part of the Enterobacteriaceae family, which comprises other familiar pathogens such as Escherichia coli, Yersinia species, Salmonella species, and Shigella species [17] In humans, K. pneumoniae causes various infections such as pneumonia and blood stream infections [18].  There are scanty reports in the literature of K. pneumoniae in aquaculture.  Das et al. [19] reported mortality in Rohu, Labeo rohita and Oliveira et al., [20] reported lesions in common carp, Cyprinus carpio due to K. pneumoniae infection. The present research was aimed to evaluate (i) the effects of short and long term feeding of Perseaamericanasupplemented diet on plasma antioxidant levels of Clarias gariepinus before K. pneumoniae infection and (ii) the prophylactic effect of the diet on plasma antioxidant levels of C. gariepinus after K. pneumoniae infection. 

Procurement and Preparation of Experimental Specimen

The research was carried out in the aquaculture centre, Department of Fisheries and Aquatic Environment, Rivers State University, (RSU) Port Harcourt. C.gariepinus were procured from Idi-onyana farms along Abua-Ahoada road in Abua/Odual Local Government Area, Rivers State.  C. gariepinus were taken to the centre, acclimated for 2 weeks and fed with commercial diet at 5% body weight. Persea americana leaves were harvested in Port Harcourt, Rivers State, Nigeria.  P. Americanawere taken to the centre, air dried to constant weight, grounded to powdered form and sieved.Five experimental dietsof 38.35% crude protein, supplemented with varied percentages (0,3,6,9 and 12%) of P. Americana representing 5 treatments were formulated (Lee et al. [21].

Experimental Procedure

After acclimation, one hundred and fifty (150) C. gariepinus (weight:117.8±1.11g and length: 25.88±0.14cm) were randomly allocated in triplicate design to fifteen experimental tanks (i.e., 10 fish/tank; 30 fish/ treatment) representing 5experimental diet treatments (0, 3, 6, 9 and 12%). Feeding commenced 24 hours after stocking. Fish were fed experimental diets at 5% body weight twice daily (Davies et al., 2006) for 12 weeks.After the first 8 weeks of feeding (pre K. pneumoniachallenge),overnight grown K. pneumonia (1.5ml of 1.9 x 10⁵cfu/ml)were injected intraperitoneally  into each fish in all treatments including the control. K. pneumoniae was injected once a week for four weeks.

Blood Collection and Analysis

Blood samples were collected via kidney puncture at the end of weeks 4, 8 and 12. The collected blood samples were analyses for catalase (CAT), Glutathione (GSH), Glutathione-s-Transferase (GST), Superoxide Dismutase (SOD) and lipid peroxide (LPO) in the laboratory using spectrophotometer (SURGISPEC SM-23D, Surgifield medical, England).

Statistical Analysis of Data

Statistical analysis was carried out on all data using the Minitab 16 for Microsoft windows.  Data were pooled by treatment and presented as means±standard deviation (SD) for n = 30 fish/treatment unless otherwise stated.  Data were analysed for treatment and time effect by two-way analysis of variance (ANOVA).  Where treatment x time showed significant interaction, one-way ANOVA was used to test for treatment effects within a time or time effect within a treatment. The Turkey post hoc test was used at 95% confidence level to provide specific information on which means are significantly different from each other.

Plasma Antioxidant Levels of C. Gariepinus After Short and Long-Term Feeding of Persea Americana Experimental Diet 

The result of the antioxidants is shown in Figure 1.  There were statistically significant treatment effect in all antioxidants measured apart from CAT and GSH in weeks 4 and 8 (Figure 1, p>0.05). For example, the GST did not show any significant difference between the treatments on day 4 but showed difference on day 8 with the control group showing the least level of GST.Also, there were significant time effect in some treatments on day 8 compared to day 4. For example, apart from treatments 3% and 9%, the other treatments showed a significant time effect on day 8 compared to day 4.

Plasma Antioxidant Levels of C. Gariepinus After K. Pneumonia Infection

At the end of week 12 (AKPI), antioxidant level was highest in the control compared to the other treatments (Figure 1).  Most of the antioxidant measured showed statistically significant decreased levels with increasing percentage of P. Americana in experimental diets (Figure 1).  For exampleat week 12 (AKPI),   GSH levels decreased with increasing percentage of P. Americana in experimental diets, values in decreasing order were mean±SD (% P. Americana experimental diet treatment): 

Figure 1: Effect of Dietary P. Americanaon Plasma Antioxidant (a) CAT, (b) GSH; (c) SOD; (d) GSH and (e) LPO Levels of C. Gariepinus Challenged with K. Pneumoniae

14.67±0.60 (0%) >11.33±0.58 (3%) >8.33±0.59 (6) >6.09±0.10 (9%)>4.60±0.10 (12%).

There were statistically significant time dependent difference in most of the treatments for all antioxidant measured atweek 12 (AKPI) compared to weeks 4 and 8.  For example for the GSH all treatment showed a time-dependent significant effect onweek 12(AKPI) compared to week 4 and 8apart from the 9% P. Americana experimental diet treatment. The 9% P. Americana experimental diet treatment did not showstatistical time dependent differences in week 12 (AKPI) compared to week 4 or week 8 in for GSH, SOD and LPO (Figure 1).

Data are means±SD, n = 30.  Different letters within the experiment duration indicates a significant treatment effect (ANOVA, p<0.05). # indicates a significant time effect within treatment compared to the previous experiment duration (ANOVA, p<0.05). * represents  a significant time effect compared to week 4 (ANOVA, p<0.05).  Note: 12 (AKPI) indicates week 12 after K. pneumonia challenge.

DISCUSSION 

Oxidative stress occurs in fish when reactive oxygen species (ROS) presence in the fish is above the considerable limit [12]. Some of the situations that can cause oxidative stress in fish and other animals include: environmental pollution, diet, diseases, pathogen presences etc [7,22]. The first line of defence against ROS is the production of antioxidants such as CAT, GSH, GST, SOD and LPO [23].

In the present study, the analyzed antioxidants such as CAT and GSH were not significantly different in all treatment including the controls at the end of days 4 and 8. However, the other antioxidants (SOD, GST and LPO) measured, showed significant differences across the treatments at the end of weeks 4 and 8, this is an indication that outside the presences of the herbs in the diets there might be other things as a result of manufacturing processes that may have interfered with the antioxidant levels of the fish [22,24-25]. Apel and Hirt [26] also reported that the environment of the cultured species can affect the level of stress impose on the fish. 

At the end of week 12 (AKPI), antioxidant level was highest in the control compared to the other treatments (Figure 1).  Most of the antioxidant measured showed statistically significant decreased levels with increasing percentage of p. americana in experimental diets (p>0.05). There were statistically significant time dependent difference in most of the treatments for all antioxidant measured at week 12 (AKPI) compared to weeks 4 and 8. There were no statistical time dependent differences in week 12 (AKPI) compared to week 4 or week 8 in the 9% experimental diet treatment for GSH, SOD and LPO (Figure 1). Although no work on the antioxidant activities of C. gariepinus as a result of K. pneumonea infection has been reported to the best of our knowledge, this report is similar to the findings of Tkachenko et al. [26] who reported increase in these anti-oxidants except SOD when unprotected Onchorhynchusmykss (rainbow trout) was exposed to Aeromonas spp.

The significant increase in SOD in the control group after K. pneumoniainfectionmight be an indication of the presence of super oxide radical (O^2-) as earlier reported [28-29] which depicts the presence of harmful pathogen such as the K. pneumonia in the fish [28,30]. The increased SOD in the fish fed control diet (unprotected fish) causes the dismutation of O^2- to less reactive molecular oxygen (O₂) and hydrogen peroxide (H₂O₂) (Weydert and Cullen, 2010; Aziz et al, 2019). The presence of H₂O₂and other free radicals as a result of the O^2- disputationstimulates the fish organs to produce more CAT to the blood system to remove the H₂O₂ and free radicals that will induce stress in the fish in the control group [31]. The significant presence of CAT in the plasma of the unprotected fish could also be as a result of excess oxidation of fatty acids to produce undue H₂O₂ [30]. The significant presence of LPO in the unprotected fish after the infection could be as a result of the lipid peroxidation of Omega-3 and Omega-6 fatty acids [32], and it is an indication of stress presence in the fish [33], a process that can lead to poor flesh quality in the fish [34]. The increase presence of plasma GSH in the unprotected fish after the infection depicts the presence of undue free radicals in the fish cells ([35], and this further explains the higher presence of GST in the fish plasma since they are co-factors [35].  Prolong stress situations as seen in the increased antioxidant activities can lead to depletion of these antioxidants and eventual mortality [36].

The decrease in levels of all the antioxidant in the fish fed p. Americana experimental diets, expresses its antibacterial activities which could also be as a result of vitamin C and other phytochemicals present in the P. Americana [37-38], which prevents stress [39].

This research has shown that short and long term feeding of P. Americana experimental diet to C.gariepinus did not induce stress, and P. Americana is a good prophylactic herbs for K. pneumonia with the 9% p. Americana supplemented diet showing the best prophylactic effect.

Acknowledgements

The authors acknowledge the immense contribution of Prof. U. U. Gabriel (of blessed memories) to the success of the research.

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