Amendments from Version 1

F1000Research

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F1000 Research Limited

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10.12688/f1000research.16902.2

Research Article

Articles

Borneo herbal plant extracts as a natural medication for prophylaxis and treatment of Aeromonas hydrophila and Pseudomonas fluorescens infection in tilapia ( Oreochromis niloticus)

[version 2; peer review: 2 approved, 1 approved with reservations]

Hardi

Esti Handayani

Conceptualization Data Curation Funding Acquisition Investigation Methodology Resources https://orcid.org/0000-0002-4660-7532 1 Nugroho

Rudy Agung

Methodology Validation Writing – Review & Editing https://orcid.org/0000-0001-9006-7329 a 2 Kusuma

Irawan Wijaya

Data Curation Investigation Visualization 3 Suwinarti

Wiwin

Methodology Resources Validation 3 Sudaryono

Agung

Formal Analysis Project Administration Validation 4 Rostika

Rita

Methodology Project Administration Validation 5 1Microbiology Laboratory, Department of Aquaculture, Faculty of Fisheries and Marine Science, Mulawarman University, Samarinda, East Kalimantan, 75123, Indonesia 2Animal Physiology, Development and Molecular Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Mulawarman University, Samarinda, East Kalimantan, 75123, Indonesia 3Laboratory of Forest Product Chemistry, Faculty of Forestry, Mulawarman University, Samarinda, East Kalimantan, 75123, Indonesia 4Department of Aquaculture, Faculty of Fisheries and Marine Science, Diponegoro University, Semarang, Central Java, 50275, Indonesia 5Department of Fisheries, Faculty of Fisheries and Marine Science, Padjajaran University, Bandung, West Java, 40600, Indonesia

a rudysatriana@gmail.com

No competing interests were disclosed.

13 2 2019

2018

1847

7 2 2019

2019

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: The combination of some plant extracts to prevent and treat bacterial infections is gaining momentum, because of effectiveness against certain bacteria. This study aims to describe the antibacterial and immunostimulant abilities of Boesenbergia pandurata (BP), Solanum ferox (SF) and Zingiber Zerumbet (ZZ) plant extracts to treat and prevent Aeromonas hydrophila and Pseudomonas fluorescens infection on Tilapia ( Oreochromis niloticus).

Methods: Tilapia (initial weight 15±2 g) were injected intramuscularly (0.1 ml/fish) with a combination of A. hydrophila and P. fluorescens at a density of 1×10 ⁵ CFU ml ^-1 of each bacteria. Treatment trials were performed at day 7 post-injection with each combined extract, while the prevention trial was performed by including the combined extract into the commercial diet for six and seven days prior to injection. Various extract combinations were 60 mg SF extract/kg feed with 40 mg ZZ/kg feed (SF60/ZZ40), SF50/ZZ50, BP90/SF10, and BP50/SF50. Haemato-immunological parameters were performed for four weeks.

Results: In prevention trials, tilapia fed SF50/ZZ50 showed a significant increase of white and red blood cells. Similarly, significantly increased haematocrit was found in tilapia fed SF50/ZZ50 in the treatment trial but not in the prevention trial. In both trials, haemoglobin of tilapia was not affected by any combined extracts but decreased the number of bacteria. Phagocytic index, respiratory burst, lysozyme activity and survival rate of fish fed combined extracts were found significantly higher than controls. The amount of pathogenic bacteria in fish fed combined extracts was lower than the control at week 4 ( P<0.05). In both trials The percentage of survival rate and relative percent survival of tilapia fed SF 50/ZZ 50, showed the optimum results compared to the other combinations.

Conclusions: The combined extract in feed, especially SF50/ZZ50 has a positive effect on the tilapia's innate immune system of tilapia to treat and prevent bacterial infections.

Imunomodulator Concoction Aeromonas hydrophila Pseudomonas fluorescens Prophylaxis

Kementerian Riset Teknologi Dan Pendidikan Tinggi Republik Indonesia

121/UN17.41/KL/2018

This research is supported by the Ministry of Research and Technology of the Republic of Indonesia for the support of research funds provided through the National Strategic Research Institutions Fiscal Year 2018, contract No. 121/UN17.41/KL/2018.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Revised Amendments from Version 1

We have added short information in the abstract background. The unit of extract is changed from ml/kg to mg/kg. The method of antibody titres has been deleted, while the term phagocytic activity is changed to phagocytic index. Notations in each bar of all graphs and values in tables have been added to show significantly differences. Discussion has been developed by adding some important relevant explanation. Footnotes in the graph have been revised in order to be much clearer. We have added some information regarding Borneo herbal plant extracts as a natural medication for prophylaxis and treatment of Aeromonas hydrophila and Pseudomonas fluorescens infection in tilapia ( Oreochromis niloticus) based on the reviews from peer reviewers.

Introduction

Tilapia ( Oreochromis niloticus) is one of the most widely cultivated fish species in Indonesia. Tilapia is a freshwater fish that can be easily cultivated ¹. According to Pridgeon ² and Harikrishnan et al. ³, freshwater fish culture is inseparable from bacterial infections which are caused by motile Aeromonas septicaemia, furunculosis, edwardsiellosis and Aeromonas hydrophila. Further, Aeromonas species have been identified as major causative bacteria and a serious pathogen in fish ^{4,
5}. In Indonesia, particularly East Kalimantan, infection of A. hydrophila and Pseudomonas fluorescens in fish results in high mortality rates of up to 60–80%. In fish, both of these bacteria cause stresses, exophthalmia, ulcers, and watery-looking organs, particularly gallbladder rupture ^{6–
8}. In addition, combined bacterial infection in fish is also common, such as infections found in tilapia caused by Salmonella agalactiae and A. hydrophila ^{9,
10}.

To reduce high mortalities of cultured fish, aquaculturists and researchers use antibiotics to prevent and treat infection. Nevertheless, due to concerns for maintaining eco-friendly environments, the application of antibiotics should be avoided, because they may enhance antibiotic-resistant pathogens, increase the accumulation of drugs in fish tissue and trigger immunosuppression ¹¹. Methods of controlling these infections should be developed as soon as possible because the pathogen disease type has significantly increased ¹², while the type of pathogen that leads to edema in the cultivation area still cannot be overcome. One of the effective and safe methods for disease control in aquaculture is by improving the defence system of the fish through the provision of natural immunostimulants ¹³, through the use of several plant extracts.

Recently, the popularity of plant extracts as natural immunostimulant is gaining in demand and importance in medical purposes. Various plant extracts, such as Indian almond leaves ( Terminalia catappa), oats ( Avena sativa), oyster mushroom ( Pleurotus ostreatus), nettle ( Urtica dioica), sea grass ( Cymodocea serrulata) and beetroot ( Beta vulgaris) have been used as alternatives to antibiotics ^{5,
14–
16}. Plant extracts also contain levamisole ¹³ and saponin ¹⁷ which can enhance the work of nonspecific immune systems and increase the activation of phagocytosis ¹⁴. Plant extracts could optimize the fish blood function, by enhancing the number of white blood cell to prevent the bacteria ⁵. Further, the plant extracts of Boesenbergia pandurata (BP) and Zingiber zerumbet (ZZ) from East Kalimantan have in vitro and in vivo antibacterial activity against A. hydrophila bacteria, while Solanum ferox (SF) has been found to be an antibacterial agent for P. fluorescens bacteria. Similarly, for the prevention and treatment of bacterial infections in tilapia, BP and ZZ are also effective for treating A. hydrophila and P. fluorescens infection ^{8,
18}.

The incorporation of some extracts for the prevention and treatment of bacterial infections is likely to increase the effectiveness because some materials can work synergistically, so that the infection of both bacteria in the fish body can be controlled optimally. However, research regarding the combination of plant extracts to treat and prevent bacterial infection is limited. This study therefore aims to determine the effectiveness of the combination of three extracts (BP, ZZ and SF) to prevent and treat bacterial infections of A. hydrophila and P. fluorescens in tilapia.

Methods Fish and bacteria

In total, 450 Tilapias (Initial weight 15 ± 2 g, age ±2.5 months, random sex) were obtained from Teluk Dalam Village in Tenggarong Seberang, Kutai Kartanegara, Indonesia. The fish were randomly distributed and assigned into five aquariums in triplicates, representing four treatments and one control. The fish were kept in the laboratory for two weeks for acclimatization in the aquarium (60×40×30 cm). Each aquarium was filled with 60 l of freshwater and the water was changed by as much as 50% every 2 days to remove remaining faeces and inedible feed. The average temperature of the water was 27°C. The feed given in the acclimation phase was a commercial feed (PT Rama Jaya Mahakam, Kutai Kartanegara East Kalimantan-Supplier, floating pellets, containing 31–33% protein and 4–6% fat) at a rate of 5% of the body weight of the fish per day. The bacteria used for the challenge test were A. hydrophila (EA-01) and P. fluorescens (EP-01), which was provided from the Aquatic Microbiology Laboratory, Faculty of Fisheries and Marine Sciences, Mulawarman University, Indonesia. To bring about bacterial challenge, a combination of bacteria at density of 10 ⁵ CFU ml ^-1 of each bacteria was used. Each fish was injected intramuscularly with 0.1 ml of the suspension of the bacteria.

Plant and chemical materials

The plant materials, BP, SF and ZZ, were collected from a traditional market in Samarinda City, East Kalimantan, Indonesia. The plants were cleaned, cut and dried at 40°C for 48 hours in the oven, finely powdered and stored at -4°C for the further extraction stage. Ethanol solution (95%) was used to extract the plant materials, following a method described by Limsuwan & Voravuthikunchai ¹⁹. All chemicals used in this research was obtained from commercial sources (Sigma Aldrich, Inc. USA).

Experimental design and challenge test

This treatment and prevention trials were carried out for 28 days. The treatment experiments were conducted with five combination treatments with the following stages: tilapia (average initial weight 15 ± 2 g, n = 30 fish per group, random sex) were injected intramuscularly (0.1 ml) with a mixture of A. hydrophila and P. fluorescens bacteria, each bacteria at density10 ⁵ CFU ml ^-1. At day 7 after injection, the fish were fed with feed combined with extract as follows (mg per kg feed): P1, 60 mg SF extract/kg feed with 40 mg ZZ extract/kg feed (SF60/ZZ40); P2, SF50/ZZ50; P3, BP90/SF10; P4, BP50/SF50; and P5, fed with no additional extract (control). All fish were fed twice a day ad satiation. The remaining feed was siphoned out before the next feeding.

Meanwhile, the prevention trial was performed by providing the same feeding combination and procedure for 6 days prior to intramuscular injection of the fish with 0.1 ml of mixed bacteria at day 7. After injection, feeding combination was continued until the 4 ^th week. Haematological and immunological parameters were measured every week after the injection with bacteria until week 4.

Haematology and phagocytic index

At days 14, 21 and 28 following bacterial challenge, haematological profiles of fish (n=3 per treatment group) were observed. Fish were anesthetized using 50 mg l ^-1 MS 222 (Sigma Aldrich, USA) / dm ³ water. The fish blood was taken through the caudal vein, using a 1 ml syringe rinsed with 10% trisodium citrate anticoagulant (fish were kept alive after blood withdrawal). Total red blood cells (RBC) (10 ⁶ per mm ³) and white blood cells (WBC) (10 ³ / mm ³) were determined manually using an improved Neubauer counting chamber. The number of WBC was calculated using the method of Blaxhall and Daisley ²⁰. Haemoglobin (Hb) was measured spectrophotometrically at 540 nm using the cyanmethemoglobin method ¹⁷. The haematocrit (Htc %) was counted using the microcentrifuge and heparinized was used as a standard solution. Meanwhile, phagocytic index was determined using a modification of previous methods by adding Turk solution into suspension of fish blood and bacteria to remove red blood cells. Thus, the number of white blood cells can be easily counted ^{20–
22}.

Respiratory burst and lysozyme activity

Respiratory burst activity test was performed using nitro blue tetrazolium (NBT) reagent, using the method outlined by Secombes and Olivier ²³. Meanwhile, lysozyme activity was performed using a microtiter plate ELISA reader at wavelength of 520 nm, following the method described by Soltani and Pourgholam ²⁴.

Total Plate Count

To perform the total plate count (TPC), a blood sample of each fish from each group was homogenized and diluted in physiological saline solution of 0.85%. The dilutions were then transferred to bacterial counts. The TPC was conducted following the method of Turkogfu et al. ²⁵.

Disease resistance

Both A. hydrophila and P. fluorescens (the pathogenic bacteria) were used for challenge testing (n = 10 fish per aquarium, in triplicates per group). The survival rate (SR) and relative percent survival (RPS) of the fish were recorded on a daily basis for 4 weeks ²⁶.

Statistical analysis

Results are expressed as means ± standard error (SE) and the data were analysed using SPSS version 22 (SPSS, Inc., USA). The data of WBC, RBC, haematocrit, Hb, TPC, phagocytic index, respiratory burst and lysozyme activity were subjected to ANOVA, followed by Duncan’s post hoc test to evaluate significant differences among the groups of treatments. The percentage of fish survival were arcsine-transformed. All tests were significant at P < 0.05.

Results Haematological profile

The present results revealed that the total WBC count of tilapia in the treatment and prevention trials were significantly increased ( P<0.05) from weeks 2–4 post-administration with combined extracts. The highest increase of WBC was found in tilapia fed with SF50/ZZ50. Similarly, total RBC and haematocrit of tilapia fed SF50/ZZ50 in the treatment trial showed a significant increase after week 2, while tilapia fed SF60/ZZ40 in the prevention trial led to a positively enhanced result from weeks 2–4. Further, haemoglobin of fish both in treatment and prevention trials were not affected by any various combination of extracts ( Table 1).

Table 1. Hematological profile of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment and prevention trials.

Variables	Trials	groups	Weeks
Variables	Trials	groups	2	4
WBC (10 ⁴ cell/mm ³)	Treatment	A	1.68±0.1 ^a	2.07±0.2 ^b
		B	3.60±0.1 ^b	8.85±0.2 ^c
		C	1.88±0.5 ^a	2.10±0.1 ^a
		D	1.98±0.5 ^a	2.20±0.1 ^b
	E	1.35±0.2 ^a	1.34±0.1 ^a
	Prevention	A	1.85±0.15 ^a	2.4±0.5 ^b
		B	3.9±0.2 ^b	7.96±0.2 ^c
		C	2.0±0.2 ^a	2.4±0.3 ^b
		D	2.0±0.3 ^a	2.5±0.1 ^b
		E	1.4±0.5 ^a	1.3±0.1 ^a
RBC (10 ⁶cell/mm ³)	Treatment	A	5.4±0.3 ^a	4,2±0.1 ^b
		B	7.7±0.2 ^b	8.8±0.2 ^c
		C	6.3±0.1 ^b	7,2±0.2 ^a
		D	6.8±0.2 ^a	7.0±0.1 ^b
		E	2.7±0.1 ^a	2.7±0.1 ^a
	Prevention	A	6.42±0.25 ^a	6.0±0.5 ^b
		B	6.97±0.3 ^a	7.0±0.2 ^a
		C	6.23±0.1 ^a	5.53±0.2 ^a
		D	5.67±0.1 ^a	7.0±0.1 ^a
		E	2.7±0.2 ^a	2.47±0.1 ^a
Hematocrit (%)	Treatment	A	27±0.1 ^a	34±0.1 ^b
		B	27±0.2 ^b	36±0.2 ^c
		C	27±0.2 ^a	31±0.2 ^a
		D	27±0.2 ^a	30±0.2 ^a
		E	15±0.3 ^a	15±0.2 ^a
	Prevention	A	27±0.1 ^a	27.7±0.1 ^a
		B	27±0.2 ^a	34±0.1 ^a
		C	27±0.1 ^a	31.5±0.1 ^a
		D	27±0.1 ^a	30±0.2 ^a
		E	14.7±0.2 ^a	15±0.1 ^a
Hemoglobin (g dL ^-1)	Treatment	A	10±0.3 ^a	10±0.1 ^a
		B	10±0.3 ^a	10±0.1 ^a
		C	10±0.2 ^a	10±0.1 ^a
		D	8±0.2 ^a	10±0.1 ^a
		E	8±0.1 ^a	6±0.2 ^a
	Prevention	A	10±0.1 ^a	8±0.2 ^a
		B	10±0.1 ^a	10±0.1 ^a
		C	10±0.1 ^a	8.3±0.1 ^a
		D	8±0.1 ^a	8±0.2 ^a
		E	8±0.1 ^a	4±0.1 ^a

Note: Mean±standard deviation followed by different superscript letters (a,b,c) in the same column in each variable and each treatment or prevention trial showed significantly different at P<0.05. WBC = White blood cell, RBC = Red blood cell, A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg per kg feed. BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment and prevention trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence.

Phagocytic index

All combination extracts fed to fish in the treatment ( Figure 1) and prevention ( Figure 2) trials increased the phagocytic index. The phagocytic index of fish fed SF50/ZZ50 in the diet, in both in treatment and prevention trials, were significantly higher than control and increased from the 2 ^nd to 4 ^th week of the post-challenge test.

Figure 1. Phagocytic index (%) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment trials.

BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence. Infected fish fed various combination and ratio of extract, namely: A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg kg ^-1 feed. * = significantly different between week. Different letter above the bars in each week showed significantly different at P<0.05.

Figure 2. Phagocytic index (%) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in prevention trials.

BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence. Infected fish fed various combination and ratio of extract, namely: A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition.. Extract ratio is in mg kg ^-1 feed. * = significantly different between week. Different letter above the bars in each week showed significantly different at P<0.05.

Respiratory burst

The respiratory burst activity of infected fish fed with combination extract increased from week 2 to week 4 in the treatment trial ( Figure 3). In addition, SF50/ZZ50 (mg per kg feed) combination extract resulted in a significantly different respiratory burst to other combinations of extracts and the control. Meanwhile, in the prevention test, infected fish fed SF50/ZZ50 combination extract in week 4 were significantly higher than control and other combinations of extracts ( P<0.05) ( Figure 4).

Figure 3. Respiratory burst activity of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment trials.

Figure 4. Respiratory burst activity of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in prevention trials.

Lysozyme activity

This study revealed that lysozyme activity of infected tilapia fed SF60/ZZ40, BP90/SF10 and BP50/SF50 combinations of extract did not show a significant increase ( P<0.05) at weeks 2 and 4 in the treatment test. However, starting from weeks 2–4, the addition of SF50/ZZ50 combination extract in the diet of fish resulted in significantly better lysozyme activity ( Figure 5). Meanwhile, in the prevention test at weeks 2 and 4, the lysozyme activity of tilapia fed SF50/ZZ50 was significantly higher ( P<0.05) ( Figure 6) than in other combinations.

Figure 5. Lysozyme activity (μg mL -1) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment trials.

BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence. A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg kg ^-1 feed. * = significantly different between week. Different letter above the bars in each week showed significantly different at P<0.05.

Figure 6. Lysozyme activity (μg mL -1) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in prevention trials.

BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence. Infected-fish fed various combination and ratio of extract, namely: A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg kg ^-1 feed. * = significantly different between week. Different letter above the bars in each week showed significantly different at P<0.05.

TPC

The overall combination of extracts administered to treat and prevent infection by A. hydrophila and P. fluorescens may decrease the number of bacteria in the fish body until the 4 ^th week of observation ( Table 2). The bacterial density, in both the treatment and prevention trials was lower than in the control. Total bacteria of A. hydrophila and P. fluorescens in tilapia fish fed combination extract in the treatment trial decreased from weeks 2–4. Further, the lowest bacterial density in tilapia was obtained from the fish fed SF50/ZZ50 combination extracts in their diet, which was also significantly different ( P<0.05) compared to the control.

Table 2. Total Plate Count (10 6 CFU/mL) in Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment and prevention trials.

Trials	Groups	Week
Trials	Groups	2	4
Treatment	A	17.4±5 ^a	10.86±10 ^c
	B	22.4±15 ^a	3.05 ±10 ^d
	C	55±10 ^b	16±15 ^c
	D	47±10 ^b	4.82±10 ^d
	E	42.85±15 ^b	30.9±5 ^b
Prevention	A	7.8±15 ^a	5.71±15 ^a
	B	9.16±5 ^b	3.15±10 ^d
	C	6.65±10 ^a	4.6±5 ^d
	D	11.15±11 ^c	8.27±10 ^a
	E	12.68±11 ^c	15.3±10 ^c

Note: Mean±standard deviation followed by different superscript letters (a,b,c,d) in the same column in each treatment or prevention trial showed significantly different at P<0.05. A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg kg ^-1 feed. BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. CFU = Colony forming unit. In treatment and prevention trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence.

Survival rate

The administration of extract with different combinations on tilapia injected with A. hydrophila and P. fluorescens bacteria increased the SR and RPS when compared to those not given the extracts ( Table 3 and Table 4). The percentage of survival of tilapia in treatment and prevention trials with combination extracts of SF 50/ZZ 50 had the highest SR compared to the other combinations of extract.

Table 3. Survival rate (%) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment and prevention trials.

Trials	Groups	Week
Trials	Groups	4
Treatment	SF 60/ZZ 40	76.67 ^a
	SF 50/ZZ 50	87.00 ^a
	BP 90/SF 10	76.67,30 ^a
	BP 50/SF 50	83.33.00 ^a
	No extract	33.00 ^b
Prevention	SF 60/ZZ 40	86.67 ^a
	SF 50/ZZ 50	90 ^b
	BP 90/SF 10	80 ^b
	BP 50/SF 50	76.67 ^a
	No extract	27 ^c

Note: Mean±standard deviation followed by different superscript letters (a,b,c) in the same column in each treatment or prevention trial showed significantly different at P<0.05. A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in mg per kg feed. BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment and prevention trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence.

Table 4. Relative Percent Survival (RPS) of Tilapia ( <italic toggle="yes">Oreochromis niloticus</italic>) fed different extract combination in treatment and prevention trials.

Trials	Groups	Week
Trials	Groups	4
Treatment	SF 60/ZZ 40	65 ^a
	SF 50/ZZ 50	80 ^b
	BP 90/SF 10	65 ^a
	BP 50/SF 50	75 ^b
	No extract
Prevention	SF 60/ZZ 40	82 ^a
	SF 50/ZZ 50	86 ^b
	BP 90/SF 10	73 ^a
	BP 50/SF 50	68 ^a
	No extract

Note: Mean±standard deviation followed by different superscript letters (a,b,c) in the same column in each treatment or prevention trial showed significantly different at P<0.05. A = SF60/ZZ40, B = SF50/ZZ50, C = BP90/SF10, D = BP50/SF50, E = No extract addition. Extract ratio is in ml per kg feed. BP = Boesenbergia pandurata), SF = Solanum ferox, and ZZ = Zingiber Zerumbet. In treatment and prevention trials, fish was infected with Aeromonas hydrophila and Pseudomonas fluorescence. Extract ratio is in mg kg ^-1 feed.

Discussion

The number of infectious diseases caused by pathogenic bacteria such as A. hydrophila have become a pivotal concern in fish culture, causing high economic losses owing to high mortality rates ⁵. The use of plant-based extracts as immunodulators has been applied to increase survival and immune system of fish to prevent or cure bacterial pathogen. Several plant extracts that contain active phytochemicals have been found and used as supplements in the feed of fish ^{26–
29}.

The current study found that the WBC of tilapia infected by both bacteria in the prevention and treatment trials increased significantly ( P<0.05), while the RBC of tilapia infected by both bacteria in the prevention and treatment trials decreased significantly ( P<0.05). This result is similar to those of a previous study, which stated that the WBC increased in order to tackle the infection, while the RBC was decreased in tilapia infected with Streptococcus agalactiae bacteria ³⁰, S. iniae ¹⁰, A. hydrophila and Pseudomonas sp. ⁷. In contrast, tilapia fed with a combination of extracts SF60/ZZ40 showed a similar RBC value both in treatment and prevention trials. In addition, tilapia fed SF50/ZZ50 in treatment trial resulted the highest RBC at the end of the trial. The Hb and Htc values were unchanged during the first week of all treatments including control; the decrease in Htc and Hb values occurred in controls without extract from weeks 2–4 post-infection in the prevention and treatment trials. This result indicated that the combined administration of the extracts was capable of improving the performance of the fish immune system by producing more WBC, thus making the fish more able to suppress the growth of bacteria in the body.

RBC, WBC, Hb and Htc can be used as an indicator of the blood profile in fish with respect to the innate immune defence and regulation of immunological function ³¹. WBC are particularly responsible for providing protection or resistance to disorders caused by infectious pathogens and non-infectious factors (nutrition, temperature and handling) ³². Total value of WBC also describes the health status and immune system of the fish. In addition to haematological statues, the Hb content decreases due to RBC swelling and poor Hb mobilization of the spleen and other haematopoesis organs ³³.

Besides blood profiles, the phagocytic index, respiratory burst and lysozyme activity are good indicators for immunological status of fish during infection periods. The present results revealed that infected fish treated with a compound extract of SF50/ZZ50 showed the highest IP and increased from weeks 2–4 post-injection. These results are in line with the results of a previous study, which found that fish treated with immunostimulants usually show enhanced phagocytic cell activities ³⁴. Fish have several types of phagocytic leukocytes, which are part of WBC, in the peritoneal cavity, and various tissues. The phagocytic index is also associated with the production of oxygen free radicals by using respiratory bursts, which are important events in bactericidal pathways in fish ^{35,
36}.

According to Isnansetyo et al. ³⁷, the phagocytic index is an important indicator of the enhancement of the immune system, which is showed by increasing the function of phagocytes. In addition, the increasing of phagocytic activity is affected by a complement that enhances the frequency of antigen-antibody, attaches to the receptor of phagocytosis cells, and stimulates phagocytes cells to make a forward migration to the site of infection Further, the forward migration to the site of infection has a close relationship with phagocytosis index. In addition, Secombes and Olivier ²³ revealed that the release of superoxide anions, hydrogen peroxide and hypochlorous acid into the phagosome and extracellular space during the respiratory burst can be also considered as the pivotal mechanisms involved in the bactericidal activity of macrophages.

Total lysozyme level is a tool to measure the humoral component of the non-specific defence mechanism (innate immunity), which can be used to detect infections or injections of foreign material, including bacteria ^{38–
40}. The present findings determined that tilapia fed SF 50/ZZ 50 had significantly higher ( P<0.05) lysozyme activity. This finding is in line with past research, stating that the lysozyme activity of Jian carp ( Cyprinus carpio var. Jian) ⁴¹ and large yellow croaker, Pseudosciaena crocea ^{41,
42} were increased after being fed with traditional Chinese medicine formulated from Astragalus root ( Radix astragalin seu Heydsari) and Chinese Angelica root ( R. angelicae Sinenesis).

Past research stated that the increasing survival rate and relative percent survival related to the increasing immune function of the fish which is affected by plant extract supplementation. Plant extracts containing important phytochemicals may increase monocytes, granulocytes, macrophages and neutrophils in fish, improving non-specific immune responses ⁵. Moreover, activated macrophages and neutrophils in the blood of fish also increase the number of reactive oxygen and nitrogen intermediates (ROIs and RNIs), which are toxic to bacteria ²².

Conclusion

A combination of plant extracts was found to affect the health status of tilapia when compared with control. A combination of extracts of SF and ZZ (50:50 mg/kg of feed) provides the optimum protection against bacterial infections of A. hydrophila and P. fluorescens in both prevention and treatment assays.

Data availability

Raw data for Tables and Figures can be accessed on OSF, DOI: https://doi.org/10.17605/OSF.IO/A42JB ⁴³.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Acknowledgments

The research team would like to thank the Department of Aquaculture, Faculty of Fisheries and Marine Sciences Mulawarman University, East Kalimantan, for the support of facility and equipment during the research.

FAO, Food and Agriculture Organization of the United Nations: Fisheries and Aquaculture Statistics. Rome, Italy.2015. Reference Source

Pridgeon

Klesius

: Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection. J Fish Dis. 2012;35(3):227–237. 22324346

10.1111/j.1365-2761.2011.01343.x

Harikrishnan

Balasundaram

Kim

: Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts. Fish Shellfish Immunol. 2009;27(3):508–515. 19616631

10.1016/j.fsi.2009.07.004

Carriero

Mendes Maia

Moro Sousa

: Characterization of a new strain of Aeromonas dhakensis isolated from diseased pacu fish ( Piaractus mesopotamicus) in Brazil. J Fish Dis. 2016;39(11):1285–1295. 26850370

10.1111/jfd.12457

Nugroho

Manurung

Nur

: Terminalia catappa L. extract improves survival, hematological profile and resistance to Aeromonas hydrophila in Betta sp. Arch Pol Fisheries. 2017;25(2):103–115. 10.1515/aopf-2017-0010

Hardi

Pebrianto

: Isolasi dan uji postulat Koch Aeromonas sp. dan Pseudomonas sp. pada ikan nila ( Oreocromis niloticus) di Sentra Budidaya Loa Kulu Kabupaten Kutai Kartanegara. J Ilmu Perikanan Tropis. 2012;16(2):35–39.

Hardi

Kusuma

Suwinarti

: Antibacterial activities of some Borneo plant extracts against pathogenic bacteria of Aeromonas hydrophila and Pseudomonas sp. Aquaculture, Aquarium, Conservation & Legislation-International Journal of the Bioflux Society (AACL Bioflux). 2016;9(3):638–646. Reference Source

Hardi

Saptiani

Kusuma

: Immunomodulatory and antibacterial effects of B oesenbergia pandurata, Solanum ferox, and Zingiber zerumbet on tilapia, Oreochromis niloticus. Aquaculture, Aquarium, Conservation & Legislation (AACL Bioflux). 2017;10(2):182–190. Reference Source

Rijkers

Teunissen

Van Oosterom

: The immune system of cyprinid fish. The immunosuppressive effect of the antibiotic oxytetracycline in carp ( Cyprinus carpio L.). Aquaculture. 1980;19(2):177–189. 10.1016/0044-8486(80)90018-6

Sumiati

Sukenda Nuryati

: Development of ELISA method to detect specific immune response in Nile tilapia O. niloticus vaccinated against A. hydrophila and S. agalactiae. Jurnal Riset Akuakultur. 2015;10:243–250. Reference Source

Raa

Rorstad

Engstad

: The use of immunostimulants to increase resistance of aquatic organisms to microbial infections. Diseases in Asian aquaculture. 1992;39–50. Reference Source

Sudheesh

Al-Ghabshi

Al-Mazrooei

: Comparative pathogenomics of bacteria causing infectious diseases in fish. Int J Evol Biol. 2012;2012:457264. 22675651

10.1155/2012/457264

3364575

Findlay

Munday

: The immunomodulatory effects of levamisole on the nonspecific immune system of Atlantic salmon, Salmo salar L. J Fish Dis. 2000;23(6):369–378. 10.1046/j.1365-2761.2000.00231.x

Baba

Acar Ü, Öntaş

: The use of Avena sativa extract against Aeromonas hydrophila and its effect on growth performance, hematological and immunological parameters in common carp ( Cyprinus carpio). Italian J Anim Sci. 2016;15(2):325–333. 10.1080/1828051X.2016.1185977

Bilen

Ünal

Güvensoy

: Effects of oyster mushroom ( Pleurotus ostreatus) and nettle ( Urtica dioica) methanolic extracts on immune responses and resistance to Aeromonas hydrophila in rainbow trout ( Oncorhynchus mykiss). Aquaculture. 2016;454:90–94. 10.1016/j.aquaculture.2015.12.010

Devi

Dhayanithi

Kumar

: In vitro and in vivo efficacy of partially purified herbal extracts against bacterial fish pathogens. Aquaculture. 2016;458:121–133. 10.1016/j.aquaculture.2016.02.035

Yuniar

Darmanto

Soegianto

: Effect of saponin-pods extract Acacia ( Acacia mangium) to hematocrit, hemoglobin at Tilapia ( Oreochromis niloticus). UNEJ e-Proceeding. 2017;67–69. Reference Source

Hardi

Kusuma

Suwinarti

: Short Communication: Antibacterial activity of Boesenbergia pandurata, Zingiber zerumbet and Solanum ferox extracts against Aeromonas hydrophila and Pseudomonas sp. Nusantara Bioscience. 2016;8(1):18–21. Reference Source

Limsuwan

Voravuthikunchai

: Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibiofilm producing and antiquorum sensing in Streptococcus pyogenes. FEMS Immunol Med Microbiol. 2008;53(3):429–36. 18631184

10.1111/j.1574-695X.2008.00445.x

Blaxhall

Daisley

: Routine haematological methods for use with fish blood. J Fish Biol. 1973;5(6):771–781. 10.1111/j.1095-8649.1973.tb04510.x

Watanuki

Ota

Tassakka

: Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio. Aquaculture. 2006;258(1–4):157–163. 10.1016/j.aquaculture.2006.05.003

Isnansetyo

Fikriyah

Kasanah

: Non-specific immune potentiating activity of fucoidan from a tropical brown algae (Phaeophyceae), Sargassum cristaefolium in tilapia ( Oreochromis niloticus). Aquacult Int. 2016;24(2):465–477. 10.1007/s10499-015-9938-z

Secombes

Olivier

: Host—Pathogen Interactions in Salmonids.In Furunculosis. Elsevier.1997;269–296. 10.1016/B978-012093040-1/50014-2

Soltani

Pourgholam

: Lysozyme activity of grass carp ( Ctenopharingodon idella) following exposure to sublethal concentrations of organophosphate, diazinon.2007. Reference Source

Turkoglu

Ceylan

Dayisoylu

: The microbiological and chemical quality of Orgu cheese produced in Turkey. Pakistan Journal of Nutrition. 2003;2(2):92–94. 10.3923/pjn.2003.92.94

Citarasu

: Herbal biomedicines: a new opportunity for aquaculture industry. Aquacult Int. 2010;18(3):403–414. 10.1007/s10499-009-9253-7

Madhuri

Mandloi

Govind

: Antimicrobial activity of some medicinal plants against fish pathogen. Int Res J Pharm. 2013;3(4):28–30. Reference Source

Chakraborty

Horn

Hancz

: Application of phytochemicals as growth-promoters and endocrine modulators in fish culture. Rev Aquac. 2014;6(1):1–19. 10.1111/raq.12021

Sivasankar

Anix Vivek Santhiya

Kanaga

: A review on plants and herbal extracts against viral diseases in aquaculture. JMed Plants Stud. 2015;3(2):75–79. Reference Source

Seeley

Gillespie

Weeks

: A simple technique for the rapid spectrophotometric determination of phagocytosis by fish macrophages. Mar Environ Res. 1990;30(1):37–41. 10.1016/0141-1136(90)90009-D

Ballarin

Dall'Oro

Bertotto

: Haematological parameters in Umbrina cirrosa (Teleostei, Sciaenidae): a comparison between diploid and triploid specimens. Comp Biochem Physiol A Mol Integr Physiol. 2004;138(1):45–51. 15165570

10.1016/j.cbpb.2004.02.019

Harikrishnan

Balasundaram

: Antimicrobial activity of medicinal herbs in vitro against fish pathogen, Aeromonas hydrophila. Fish Pathol. 2005;40(4):187–189. 10.3147/jsfp.40.187

Lie

Evensen

SØrensen

: Study on lysozyme activity in some fish species. Dis Aquat Organ. 1989;6:1–5. 10.3354/dao006001

Sakai

: Current research status of fish immunostimulants. Aquaculture. 1999;172(1–2):63–92. 10.1016/S0044-8486(98)00436-0

Sharp

Secombes

: Observations on the killing of Aeromonas salmonicida by rainbow trout ( Oncorhynchus mykiss, Walbaum) macrophages. Diseases of Asian Aquaculture. 1992;1:379–389.

Sharp

Secombes

: The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish Shellfish Immunol. 1993;3(2):119–129. 10.1006/fsim.1993.1013

Isnansetyo

Irpani

Wulansari

: Oral Administration of Alginate From A Tropical Brown Seaweed, Sargassum sp. to Enhance Non-Spesific Defense In Walking Catfish ( Clarias sp.). Aquacultura Indonesiana. 2015;15(1). 10.21534/ai.v15i1.29

Saurabh

Sahoo

: Lysozyme: an important defence molecule of fish innate immune system. Aquac Res. 2008;39(3):223–239. 10.1111/j.1365-2109.2007.01883.x

Siwicki

Studnicka

: The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L. J Fish Biol. 1987;31(sA):57–60. 10.1111/j.1095-8649.1987.tb05293.x

Dotta

de Andrade

Gonçalves

: Leukocyte phagocytosis and lysozyme activity in Nile tilapia fed supplemented diet with natural extracts of propolis and Aloe barbadensis. Fish Shellfish Immunol. 2014;39(2):280–284. 24857767

10.1016/j.fsi.2014.05.020

Jian

: Influences of traditional Chinese medicine on non-specific immunity of Jian Carp ( Cyprinus carpio var. Jian). Fish Shellfish Immunol. 2004;16(2):185–191. 15123322

10.1016/S1050-4648(03)00062-7

Jian

: Effects of traditional Chinese medicine on nonspecific immunity and disease resistance of large yellow croaker, Pseudosciaena crocea (Richardson). Aquaculture. 2003;218(1–4):1–9. 10.1016/S0044-8486(02)00192-8

Nugroho

: Borneo Herbal Plant Extracts as a Natural Medication for Prophylaxis and Treatment of Aeromonas Hydrophila and Pseudomonas Fluorescens Infection in Tilapia ( Oreochromis Niloticus). OSF. 2019. http://www.doi.org/10.17605/OSF.IO/A42JB

10.5256/f1000research.19635.r44765

Reviewer response for version 2

Hancz

Csaba

1 Referee https://orcid.org/0000-0001-9730-063X 1Aquaculture and Fishery, Kaposvár University, Kaposvár, Hungary

Competing interests: No competing interests were disclosed.

28 3 2019

2019

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

approve-with-reservations

There is a significant weakness in the part of M&M of this article but this can be corrected after which its scientific value could surely be elevated.

Some statistical re- evaluation of the data would also be useful.

Please see my annotated copy of the article here which includes additional corrections and suggestions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Aquaculture, fish nutrition, experimental statistcs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

10.5256/f1000research.19635.r44431

Reviewer response for version 2

Isnansetyo

Alim

1 Referee 1Department of Fisheries, Faculty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia

Competing interests: No competing interests were disclosed.

28 3 2019

2019

recommendation

approve

The authors already addressed almost all reviewer commands, and overall the manuscript quality is improved significantly. However, there is one parameter that should be clarified:

It is still confusing between Phagocytic activity (PA) and Phagocytic Index (PI). The unit for PA is percent (%) and the unit of PI is cells or particles/Phagocyte. I think, the authors observed PA, instead of PI as the unit is %. Please replace the PI to PA.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Immunology, microbiology, fish diseases, natural products

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

10.5256/f1000research.19635.r44432

Reviewer response for version 2

Venugopal

Vishnu K.

1 Referee 1Centre for Marine Living Resources & Ecology, Kochi, Kerala , India

Competing interests: No competing interests were disclosed.

27 3 2019

2019

recommendation

approve

I am happy to note that the manuscript has now improved significantly from the original submission. The corrections made by the authors are satisfactory and I can now recommend this paper for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

10.5256/f1000research.18481.r41178

Reviewer response for version 1

Isnansetyo

Alim

1 Referee 1Department of Fisheries, Faculty of Agriculture, Gadjah Mada University, Yogyakarta, Indonesia

Competing interests: No competing interests were disclosed.

13 12 2018

2018

recommendation

approve-with-reservations

This article presented the finding of Indonesian herbal extracts for preventing and treating fish diseases. This article might be indexed after several major issues are addressed:

Are Boesenbergia pandurata (BP), Solanum ferox (SF) and Zingiber Zerumbet (ZZ) typical plants in Borneo? Are the plants not found in the other parts of Indonesia? If yes, please replace the "Borneo" with "Indonesia" in the title and throughout the article.

Write one sentence of background in the Abstract.

Describe systematically in the Abstract: how to prepare the extracts, design experiment, feed preparation, infection, data collecting (hematology, non-specific immune etc.) and data analysis.

Write the exact concentration for the extract in mg/kg feed instead of ml/kg. Using units of ml/kg feed is not appropriate as the exact concentrations are not known.

Write systematically the results in the Abstract as described in the Methods.

The units are written inconsistently: format (.../...., .... per ...., .... .... ^-1 ).

“Antibody titre” is a term to evaluate the effect of vaccines. To evaluate the effect of immunostimulants, we should use the term "Natural Agglutination" as we are not evaluating the specific antibody. No data are presented for Antibody titre/Natural Agglutination, even though this parameter is described in the Methods.

The authors are confused by the terms of phagocytic activity and phagocytic index. Phagocytic index is not described before either in the Abstract or Materials and Methods. However, the authors describe phagocytic activity in Materials and Methods. Phagocytic index and phagocytic activity are two different parameters. Please refer to some of the recommended references. Add deviation standard for each bar in all graphs.

Add the notation in each bar of all graphs and values in tables to show insignificant or significant difference based on DMRT test results.

Discussion: please interpret properly and add additional explanation about why the extracts affect the immune system of fish and increase the SR and RPS. Describe the possible constituents in the extracts by citing the previous publications.

Add these references in the Introduction, Materials and Methods, and Discussion: Yudiati et al. (2016 ¹), Isnansetyo et al. (2016 ²) and Isnansetyo et al. (2015 ³).

Some grammatical errors were found.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Immunology, microbiology, fish diseases, natural products

References 1

: Innate immune-stimulating and immune genes up-regulating activities of three types of alginate from Sargassum siliquosum in Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol .2016;54: 10.1016/j.fsi.2016.03.022 46-53

26993614

10.1016/j.fsi.2016.03.022

: Non-specific immune potentiating activity of fucoidan from a tropical brown algae (Phaeophyceae), Sargassum cristaefolium in tilapia (Oreochromis niloticus). Aquaculture International .2016;24(2) : 10.1007/s10499-015-9938-z 465-477

10.1007/s10499-015-9938-z

: Oral Administration of Alginate From A Tropical Brown Seaweed, Sargassum sp. To Enhance Non-Spesific Defense In Walking Catfish (Clarias sp.). Aquacultura Indonesiana .2015;15(1) : 10.21534/ai.v15i1.29

10.21534/ai.v15i1.29

Nugroho

Rudy

Mulawarman University, Indonesia

Competing interests: No competing interests

29 1 2019

Dear Dr. Alim Isnansetyo,

Thank you for your valuable comment on our article. We have added some important information based on your comment and suggestions. We really appreciate it. For further details of our responses to your comment, please download the details of our responses from this link below:

https://osf.io/qwy8z/download [direct download link]

10.5256/f1000research.18481.r41177

Reviewer response for version 1

Venugopal

Vishnu K.

1 Referee 1Centre for Marine Living Resources & Ecology, Kochi, Kerala , India

Competing interests: No competing interests were disclosed.

28 11 2018

2018

recommendation

approve-with-reservations

After checking the research article “Borneo herbal plant extracts as a natural medication for prophylaxis and treatment of Aeromonas hydrophila and Pseudomonas fluorescens infection in tilapia (Oreochromis niloticus)” by Dr. Rudy et al., I reached the following suggestions to be made for the article’s acceptance:

The overall structure of the manuscript is satisfactory, though some changes are recommended.

In the introduction the mechanism of action of plant extracts and its medical importance could have been added.

The authors didn’t mention the composition of feed.

There is a possibility of residual ethanol in the sample. How can you conclude the results with this concern?

The nature and source of chemicals (Materials) used in this experiment are not mentioned.

Footnotes can be much clearer and the legends used in the figure should be mentioned properly. Also, in some graphs standard deviation is missing.

Give enough information about the figures in figure legends.

The Discussion part can be much stronger.

In conclusion, the content of the manuscript has value for indexing. The mentioned suggestions can be considered and resubmitted.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Clinical biochemistry, Lipid chemistry, Bioactive compounds characterization

References 1

: Dietary Withania sominefera root confers protective and immunotherapeutic effects against Aeromonas hydrophila infection in Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol .2018;80: 10.1016/j.fsi.2018.06.009 641-650

29886140

10.1016/j.fsi.2018.06.009

Nugroho

Rudy

Mulawarman University, Indonesia

Competing interests: No competing interests

29 1 2019

Dear Dr. Vishnu Venugopal,

First of all, we would like to say thank you for your valuable review and comment. We have revised our article according to your review. We have also made some responses to your review - for the details of our responses, please see the link below:

http://osf.io/vzsqe/download [direct download link]