Autochthonous vs allochthonous probiotic strains to Rhamdia quelen

Journal Pre-proof Autochthonous vs allochthonous probiotic strains to Rhamdia quelen Marcela Maia Yamashita, José Victor Ferrarezi, Gabriella do Vale Pereira, Guerino Bandeira, Júnior, Bruno Côrrea da Silva, Scheila Anelise Pereira, Maurício Laterça Martins, José Luiz Pedreira Mouriño PII:

S0882-4010(19)30014-2

DOI:

https://doi.org/10.1016/j.micpath.2019.103897

Reference:

YMPAT 103897

To appear in:

Microbial Pathogenesis

Received Date: 2 January 2019 Revised Date:

24 November 2019

Accepted Date: 25 November 2019

Please cite this article as: Yamashita MM, Ferrarezi JoséVictor, Pereira GdV, Bandeira Júnior G, Côrrea da Silva B, Pereira SA, Martins MauríLaterç, Pedreira Mouriño JoséLuiz, Autochthonous vs allochthonous probiotic strains to Rhamdia quelen, Microbial Pathogenesis (2019), doi: https:// doi.org/10.1016/j.micpath.2019.103897. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Marcela Maia Yamashita: Formal analysis, Project administration, Supervision, Writing- Original Draft, Investigation José Victor Ferrarezi: Investigation Gabriella do Vale Pereira: Writing - Review & Edition, Visualization Guerino Bandeira Júnior: Resources Bruno Côrrea da Silva: Resources Scheila Anelise Pereira: Formal analysis, Investigation Maurício Laterça Martins: Visualization, Writing - Review & Edition José Luiz Pedreira Mouriño: Conceptualization, Methodology, Funding acquisition, Supervision, Validation, Writing – Review & Edition

1

1

Autochthonous vs Allochthonous probiotic strains to Rhamdia quelen

2

Probiotic strains to Rhamdia quelen

3 4

Marcela Maia Yamashita1*, José Victor Ferrarezi1, Gabriella do Vale Pereira2, Guerino

5

Bandeira Júnior 3, Bruno Côrrea da Silva4, Scheila Anelise Pereira1, Maurício Laterça

6

Martins1 & José Luiz Pedreira Mouriño 1

7 8

1

9

University of Santa Catarina (UFSC), Rod. Admar Gonzaga 1346, 88040-900,

10

AQUOS – Aquatic Organisms Health Laboratory, Aquaculture Department, Federal

Florianópolis, SC, Brazil.

11 12

2

13

Marine Sciences, Plymouth University, Plymouth, UK

Aquatic Animal Nutrition and Health Research Group, School of Biological and

14 15

3

16

(UFSM), Santa Maria, RS, Brazil.

Department of Physiology and Pharmacology, Federal University of Santa Maria

17 18

4

19

Rod. Antônio Hell, 6800, 88318-112 Itajaí, SC, Brazil.

EPAGRI – Company of Agricultural Research and Rural Extension of Santa Catarina,

20 21

*Corresponding author – phone: (+55) 48-999120199. Adress: Rod. Admar Gonzaga

22

1346, 88040-900 Florianópolis, Santa Catarina, Brazil. Email: [email protected]

23 24

ABSTRACT

25

The aim of this study was to obtain an autochthonous probiotic candidate strain from

26

the silver catfish (Rhamdia quelen) intestinal tract, comparing its in vivo performance

27

with an allochthonous probiotic isolated from another fish, Nile tilapia (Oreochromis

28

niloticus), in a growth performance assay. The study was divided in two parts: in vitro

29

and in vivo assay followed by challange with A. hydrophila. In the in vitro assay, the

30

species-specific isolated strain Lactococcus lactis presented characteristics such as:

31

absence of hemolysis, antagonism to bacterial pathogens isolated from freshwater fish,

32

and considerable speed of duplication. In the in vivo trial, both fish supplemented with

33

autochthonous or allochthonous strains presented an increase the final concentration of

2 34

lactic acid bacteria in the intestinal tract of the fish after 60 days of dietary

35

supplementation reaching concentrations of 1 x 107 CFU g-1 and 4 x 107 UFC.g-1,

36

respectively. In addition, the autochthonous strain increased the mean corpuscular

37

hemoglobin (MCH) of the treated animals, but no significant differences were observed

38

in the other hemato-immunological and zootechnical parameters between treatments.

39

After challenge with Aeromonas hydrophila, only animals that received autochthonous

40

probiotic supplementation showed an increase in the serum total immunoglobulin

41

concentration, but not enough to observe a significant difference in the survival rate

42

between the treatments. Dietary supplementation of the probiotic allochthonous strain

43

did not demonstrate any effects superior to those of the isolated autochthonous strain.

44

Although the autochthonous strain did not present significant improvements in the other

45

parameters evaluated in this study, it was able to inhibit bacterial pathogens in vitro, to

46

increase the final concentration of LAB's and the amount of immunoglobulin after

47

experimental challenge, demonstrating probiotic potential. This study demonstrated for

48

the first time the isolation and in vivo use of an autochthonous probiotic strain isolated

49

from silver catfish, as well as its comparative evaluation with the performance of

50

allochthonous probiotic.

51

Keywords: Lactococcus lactis; Lactobacillus plantarum; silver catfish; disease

52

resistance; immunoglobulin; lactic acid bacteria.

53

1.

Introduction

54

The growing interest in the cultivation of the silver catfish Rhamdia quelen

55

(Quoy & Gaimard, 1824) in southern Brazil is due to its excellent adaptation to the

56

environmental conditions of the region as well as its growth performance and good

57

acceptance of the consumer market, being considered as one of the three main

58

freshwater fish species cultivated in the region [1-3].

59

The interest in the cultivation of this species has led to an increasingly intensive

60

production system that exposes fish to high feeding and stocking densities. Such

61

practices combined with inadequate sanitary management, culminate in production

62

losses associated mainly with bacterial infections [4]. Amongst the bacterial diseases,

63

aeromoniosis caused by the bacterium Aeromonas hydrophila is one of the most

64

common. Many species of fish are susceptible to this particular bacteriosis, such as

65

carps, goldfish (Carassius auratus auratus) and silver catfish (Rhamdia quelen), which

66

has led to high economic losses in the aquaculture sector [5].

3 67

Currently, diseases of bacterial origin have been combated in the farmings with

68

the use of antibiotics. Although costly, these chemotherapeutics are widely used to treat,

69

prevent and/or promote fish growth because of their rapid mode of action and easy

70

availability in the market. Its continuous use is detrimental to the ecosystem and may

71

create selective pressure for the emergence of resistant bacteria that could be transmitted

72

from fish to man by the food chain [6-7]. The indiscriminate use of antibiotics in

73

aquaculture may also affect the quality and commercialization of the fish, since residues

74

of these chemotherapeutics can remain in the fillet for long periods [8].

75

In recent years, in an attempt to avoid this problem by minimizing the use of

76

chemotherapeutic agentes, researches are being focoused on the use of food additives,

77

vaccines and/or probiotic bacteria that can improve the immune system of fish and, at

78

the same time, contribute to the prevention of diseases [9-10].

79

Probiotics are microorganisms that can colonize and multiply themselves in the

80

intestine and exert various beneficial effects, including immunomodulation, influencing

81

various host body systems [11]. Its efectiveness to fish was assessed in several studies,

82

resulting mainly in better growth performance and immune enhancement [12-15].

83

Most of the probiotics used in aquaculture are composed of allochthonous strains

84

that is, strains isolated from another animal other than the target species. Its use can

85

present good results and a positive role in animal welfare [16-17] but to the fish may

86

also present some disadvantages, such as: the insertion of exogenous microorganisms

87

into the culture environment, the lack of knowledge of its effects on the intestinal tract

88

and of the its interaction with the others microorganisms which makes up the intestinal

89

microbiota of the target species [18].

90

Thus, it is necessary to isolate and develop autochthonous probiotics (species-

91

specific strains), that are adapted to the micro-habitat of the intestinal tract of the

92

cultivated target species [19]. Among the autochthonous probiotics, there seems to be a

93

general consensus that lactic acid bacteria strains (LAB's) are more likely to have the

94

properties and characteristics necessary to colonize the intestine and bring benefits to

95

host health [20]. Lactic acid bacteria use carbohydrates in the host's gut to ferment

96

various compounds, producing lactic acid, which inhibits the survival of pathogenic

97

bacteria [21-22]. In addition, other metabolic products such as enzymes and proteins

98

assist in the digestion increasing host growth performance [23] and can stimulate

99

immune responses in fish [24].

4 100

Many studies attest to the beneficial effects of LAB's probiotic supplementation

101

on host health [12, 25, 26, 27, 28]. Other studies have also shown positive results from

102

the use of autochthonous lactic acid strains on growth performance, immune parameters

103

and resistance to diseases [29, 30, 31, 32].

104

LAB's are constantly identified as stable components of the intestinal microbiota

105

of fish [33]. The stability of this microbiota is an important issue and the development

106

of strategies to manipulate its composition, such as food additive applications in the

107

diet, can help stabilize beneficial microbial communities, bringing benefits to fish health

108

and consequently improving farming productivity [33].

109

According to the above, and in order to contribute to the development of the

110

production of this native Brazilian species, the present work sought to isolate and

111

characterize an LAB autochthonous strain with probiotic potential to be used as food

112

additive in silver catfish farming, comparing its in vivo performance against the

113

allochthonous probiotic strain Lactobacillus plantarum isolated from tilapia

114

(Oreochromis niloticus).

115

2. Materials and Methods

116

This experimental design was approved by the Ethics Committee on the Use of

117

Animals of the Federal University of Santa Catarina (CEUA-UFSC), being registered

118

under nº 7170170516.

119

2.1 Isolation and in vitro characterization of the autochthonous strains with probiotic

120

potencial and catalase activity

121

For the isolation of the autochthonous strain, 30 healthy (asymptomatic) fish

122

(Rhamdia quelen) with an average weight of 20 ± 0.52 g were obtained from two

123

distinct regions of the state of Santa Catarina-Brazil. The animals, fasted for 24 hours,

124

were euthanized by anesthetic deepening in eugenol solution (75 mg. L-1) and their

125

intestines were excised aseptically and washed in a solution of phosphate buffered

126

saline (PBS-Oxoid®, England) for removal of bacteria not adhered to the intestinal

127

wall. The withdrawn tissue was homogenized in PBS, the supernatant removed and

128

spreaded using streak plate method on Man Rogosa's Sharpe Agar (MRS-HIMEDIA®,

129

India) plates containing 1% aniline blue. The plates were incubated at 35 ° C for 48 h.

130

After growth of the blue colonies (lactic acid producing bacteria), they were re streaked

5 131

on the same culture medium at 35ºC for 48h to assure purity and to confirm its

132

morphology by the Gram method.

133

In addition to the Gram staining and with the aim of selecting lactic acid

134

bacteria, the activity of this enzyme was evaluated by the addition of 10% hydrogen

135

peroxide on the colonies of the strains previously cultivated in MRS Agar [34].

136

2.2 Molecular identification of the selected autochthonous strains

137

The selected bacteria were identified molecularly by the amplification of the 16S

138

rRNA genes by PCR, using the genomic DNA extracted from each strain. PCR products

139

were purified and aligned using an ABI 3500 Genetic Analyzer automatic sequencer

140

(Applied Biosystems). The partial sequence of the 16S rRNA gene, obtained from the

141

primers, was collected in a single sequence, combined from the different fragments

142

obtained and compared to those deposited in GenBank [35].

143

2.3 In vitro inhibition of pathogens

144

After molecular identification, a probiotic candidate strain was defined. To

145

determine its inhibitory capacity, the following pathogenic strains isolated from

146

mortality outbreaks were used: Aeromonas hydrophila (CPQBA 228-08 DRM isolated

147

from hybrid surubim) and Streptococcus agalactiae (GRS 2035 isolated from Nile

148

tilapia), assigned by the microbiology sector from AQUOS/NEPAQ/UFSC laboratory

149

and beyond of these A. hydrophila (MF372510 and MF372509), Citrobacter freundii

150

(MF565839) and Raoltella ornithinolytica (MF372511) isolated from symptomatic

151

silver catfish and assigned by Federal University of Santa Maria (UFSM-Brazil). For

152

this assay the pathogenic strains were reactivated, cultured and maintained in Brain

153

Heart Infusion (BHI-HIMEDIA®, India) culture medium at 30° C for 24 h.

154

The inhibitory capacity of the selected autochthonous strain was evaluated

155

according to the Disk Agar Diffusion (WDA) technique described by Vieira et al. [36].

156

The isolated strain was inoculated at 109 CFU. mL-1 in Petri dish containing MRS agar

157

and incubated at 35° C for 48 h. After this period 6 mm diameter discs were removed

158

from this plate. Pathogenic strains were seeded (1 x 109 CFU. mL-1) in Petri dishes

159

containing the Trypic Soya Agar culture medium (TSA-HIMEDIA®, India), and the

160

discs removed from the MRS plates were superimposed on the surface of the same

161

newly seeded plaque with the pathogen. Plates were incubated at 30° C for 24 hours.

162

The antagonistic activity was expressed by the diameter (mm) of the zone of inhibition

6 163

formed around the discs of superimposed agar in triplicate. Positive antagonism was

164

considered to be the mean values of inhibition halos above 8 mm.

165

2.4 Tolerance to biliary salts, haemolytic assay and sensitivity to antibiotics

166

To evaluate the resistance of the autochthonous strain to biliary salts, it was

167

incubated at 35° C for 24 h in tubes containing 10 mL of MRS broth added with 5% bile

168

salts (Bile Salts Mixture - HIMEDIA®). This test was performed in triplicate always

169

containing an extra tube for the positive control, that is, without addition of bile salts.

170

Subsequently, 100 µL of the bacterial culture were seeded into sterile 96-well microtiter

171

plates (flat bottom), where absorbance readings were made on a microplate reader at

172

630 nm. The percentage of decrease of absorbance in relation to the positive control was

173

evaluated and determined the tolerance of the strain to the bile salts.

174

Ten microlitres of the inoculum of the selected autochthonous strain were

175

inoculated in triplicate on plates containing TSA culture medium plus 5% defibrillated

176

sheep blood. The plates were then incubated at 35° C for 48 h and analyzed for the

177

formation of β-haemolysis (transparent zones around the colonies), α-haemolysis (gray-

178

green zones around the colonies) or γ-haemolysis (absence of areas around the colonies)

179

[37].

180

Another important criterion in the selection of probiotic bacteria is the choice of

181

strains that do not carry antibiotic resistance genes, therefore antibiotic susceptibilities

182

were assessed by the diffusion test in Müller-Hinton Agar. The antibiotic sensitivity

183

included: norfloxacin 10 µg, tetracycline 30 µg and florfenicol 30 µg. The agar plates

184

were incubated at 35° C for 48 h and the diameters of the growth inhibition halos were

185

measured (mm).

186

2.5 Evaluation of growth kinetics

187

In selecting a probiotic candidate strain, it is important to evaluate its growth

188

kinetics, since the success of its production on a large scale will depend on its

189

performance characteristics. Thus, the isolated autochthonous strain was incubated in

190

tubes containing 10 mL of MRS broth in triplicate and maintained at 35 ° C for 24 h.

191

For monitoring their growth, every 2 h, 100 µL of the bacterial culture were seeded in

192

triplicate into sterile 96-well flat bottom microtiter plates. Afterwards, the absorbances

193

were read in a 630 nm filter. At each absorbance reading, every 2 h, the strain was

194

serially diluted, seeded in MRS agar medium and the plates incubated at 35° C for 48 h,

195

for determination of its concentration (CFU. mL-1).

7 196 197

The absorbance of the inoculum was transformed into colony forming units (CFU. mL-1) based on the standard curve made previously for the selected strain.

198 199

Then, its maximum growth rate (µmax) and doubling time (tdup) were calculated, according to the following equations [38]: μ

á

=

ln

− ln

200

Where:

201

Z= inoculum concentration (CFU mL-1)

202

Z0= initial inoculum concentration (CFU mL-1)

203

dt= culture time (hours) =

ln 2 μ á

204

Where:

205

µ máx= maximum growth rate

206

2.6 Viability of autochthonous and allochthonous strains in the diet

207

In order to maintain the concentration of autochthonous and allochthonous

208

strains (L. plantarum) in the diet of fish, their viability of permanence in the diet was

209

evaluated. For this, the strains were grown in MRS broth culture medium, at 35° C for

210

24 h. This solution was considered as inoculum to be sprinkled in the proportion of 100

211

mL for each kilo of the diet.

212

To verify the concentration of the strains after their incorporation into the diet,

213

30 minutes after spraying the inoculum, 1 g of freshly inoculated diet was diluted in 9

214

mL SSE (65 g. L-1) ando one-tenth serially diluted eight times (factor 1:10). Dilutions

215

10-4 to 10-8 were plated in Petri dishes containing MRS agar medium with 10 g L-1

216

aniline blue. The plates were incubated at 35°C for 48 h. The concentrations of the

217

strains were measured in colony forming units per milliliter (CFU mL-1).

218

2.7 In vivo evaluation of the probiotic potential of the isolated autochthonous strain

219

versus allochthonous probiotic strain

220 2.7.1

Biological material

221

The silver catfish (R. quelen) juveniles used in this stage of the study were

222

provided by the Company of Agricultural Research and Rural Extension of Santa

223

Catarina (EPAGRI – Itajaí, Brazil 26° 57’ 08’’ S 48° 45’ 39’’ W).

8 224 225

The autochthonous strain isolated from the intestinal tract of silver catfish in the in vitro stage was maintained and reactivated in tubes containing MRS broth medium.

226

The allochthonous lactic acid strain Lactobacillus plantarum (CPQBA 227-08

227

DRM) was isolated by Jatobá et al. [39] from the intestinal tract of healthy tilapia and

228

was molecularly identified by amplification of the 16S rRNA gene. This strain was

229

maintained and reactivated in tubes containing MRS culture medium and has its

230

probiotic effect confirmed by the inhibition of pathogenic bacteria, increased innate

231

immune response and its capacity to colonize the intestinal tract of tilapias [39].

232

2.7.2

Experimental design

233

One hundred and eighty silver catfish with an average weight of 8.54 ± 0.32 g

234

were homogeneously distributed in 12 circular experimental units of 70 L (15 fish per

235

tank), provided with constant aeration and heating system. The units were coupled to

236

the water recirculation system of the experimental laboratory, which has ultraviolet

237

sterilization (36 W), mechanical filters and biological reactors (aerobic and anaerobic).

238

The fish were acclimated for 15 days receiving a diet formulated ad libitum until

239

observed satiety.

240

The water parameters were monitored daily with multiparameter (model HI

241

9828 - Hanna Instruments, USA) and colorimetric kits (Labcon Test, Brazil),

242

maintaining the appropriate standards for the cultivation of the species: dissolved

243

oxygen 8.00 ± 0,75 mg. L-1; pH 7.2 ± 0.18; temperature 25.91 ± 1.40° C; total ammonia

244

0.38 ± 0.28 mg. L-1; toxic ammonia 0.005 ± 0.008 mg. L-1; alkalinity 34.00 ± 2.83 mg

245

CaCO3. L-1 and nitrite less than 0.1 mg. L-1. The salinity was maintained at 3 ppt.

246

The treatments were performed in quadruplicate and consisted of: fish fed a diet

247

supplemented with autochthonous probiotic bactéria Lactococcus lactis, fish fed a diet

248

supplemented with allochthonous probiotic bacteria Lactobacillus plantarum and fish

249

fed with unsupplemented diets.

250

The diet was given four times a day, representing 6.0% of the biomass [40]

251

however, the amount of ration to be offered the following day was increased or reduced

252

by 10% from the observation of leftovers at each meal. The assay lasted for 60 days and

253

the photoperiod was 12 h light.

9 254

2.7.3

Preparation of experimental diets

255

The diet was formulated according to the NRC [41] based on the nutritional

256

requirements of catfish (Ictalurus punctatus) as there are still no requirements for silver

257

catfish (Table 1).

258

Proximal analysis of the diet was performed at the Nutrition Laboratory of

259

UFSC (LabNutri). The analysis of carbohydrates and energy was performed using the

260

RDC 360 method [42] and fibers, mineral material, moisture and volatiles were

261

analyzed using protocol n. 108 MAPA [43] (Table 1).

262 263

Table 1

264

Formulation and analysis of the proximate composition (g. kg-1 of dry matter) of the

265

experimental diet. Ingredients (%) Corn bran Soybean meal (48 % CP) Salmon residue flour (71 % CP) Fish oil Soy oil Hydroxy-Butylated Toluene (HBT) Premix vitamin and mineral 1 Dicalcium phosphate 2 Crude Energy (CE) cal. kg-1 Crude Protein (CP) Ethereal extract Total Carbohydrate Ashes Moisture

266 267 268 269 270 271

1Levels

20 32.14 40 1 1 0.05 2 2 4394.13 474.40 94.40 304.20 127.00 907.20

of guarantee per kilo of the product: vit. A - 1.250.000 UI; vit. D3 - 350.000 UI; vit. E - 25.000 UI; vit. K3 - 500 mg; vit. B1 – 5.000

g; vit B2 - 4.000 g; vit. B6 – 5.000 g; B12 – 10 mg; nicotinic acid – 15.000 mg pantothenic acid – 10.000 mg; biotin - 150 mg; folic acid – 1.25 mg; vit. C – 25.000 mg; Hill – 50.000 mg; Inositol 30.000 mg; Iron – 2.000 mg; Copper – 3.500 mg; Copper-chelated – 1.500 mg; Zinc – 10.500 mg; Zinc- chelated – 4.500 mg; Manganese – 4.000 mg; Selenium - 15 mg; Selenium-chelated – 15 mg; Iodine – 150 mg; Chrome – 80 mg e Vehicle (q.s.p).2 Dicalcium phosphate PA.

272

After incorporation of the autochthonous and allochthonous strains in their

273

respective diets, the mixture (diet + probiotic) was kept in the vacuum for 30 min in

274

sterile plastic bags and then offered to the fish so that the probiotic inoculum could

275

penetrate the feed pellets with greater efficiency. The diet of the control group was

276

sprinkled with sterile MRS culture medium in the same proportion as described

277

previously, so the only difference between the diets was the probiotic bacteria and their

278

extracellular products. This process was performed daily.

10 279

Once a week, the average concentration of probiotic strains autochthonous and

280

allochthonous was investigated.

281

2.7.4

Hemato-immunological analysis

282

After 60 days of probiotic supplementation, the blood of the animals was

283

samlped for evaluation of hemato-immunological parameters. Sixteen fish per treatment

284

were anaesthetized in a eugenol solution (75 mg. L-1) and the blood was withdrawn

285

from the caudal vessel with EDTA-containing syringe (Hemstab®, Brazil). This was

286

used to make duplicate blood samples to be stained with Giemsa/ MayGrunwald for

287

differential leukocyte counts [44] and total counts of thrombocytes and leukocytes by

288

the indirect method [45]. Blood aliquots were also used for the determination of

289

hematocrit, for the quantification of the total number of erythrocytes (RBC) in the

290

Neubauer chamber and for the quantification of blood hemoglobin according to the

291

cyanometahemoglobin method [44] With the determination of these parameters, the

292

following hematimetric indices were calculated: MCV (Medium Corpuscular Volume),

293

MCH (Mean Corpuscular Hemoglobin) and MCHC (Mean Corpuscular Hemoglobin

294

Concentration), according to the following formulas:

295 296

MCV (fL) = Hematocrit x 10/ Erythrocytes

297

MCH (pg) = Hemoglobin x 10/ Erythrocytes

298

MCHC (g. dL-1) = Hemoglobin x 100/ Hematocrit

299 300

The blood was also collected with non-anticoagulant syringes from two fish per

301

experimental unit (n = 8 fish per treatment) for a pool which remained resting for 1 h at

302

25° C for clot formation. It was then centrifuged for 1400 g for 10 min to obtain blood

303

serum and stored at -20° C for further immunological analysis.

304

Blood serum protein was measured with Total Protein kit (Lab Test®, Brazil),

305

using bovine albumin to make the standard curve. Total immunoglobulin was obtained

306

according to the method described by Amar et al. [46], where 100 µL of the serum were

307

mixed with 100 µL of 12% polyethyleneglycol solution (PEG, 10.000 MW, Sigma

308

Chemical, St. Louis, MO, USA) and the mixture incubated at 25° C for 2 h. The

309

immunoglobulin precipitate was removed by centrifugation (5000 g at 6° C for 10 min)

310

and the supernatant removed to measure the amount of total protein. The total

11 311

immunoglobulin concentration was expressed in mg. mL-1, and calculated by the

312

following formula:

313 Total Ig mg. mL

= total protein in the serum − total protein PEG treated . Vol mL

314 315

The agglutination titer activity was performed on a 96-well “U” bottom

316

microplate, where serum was diluted 1: 4 in PBS. Then, the sérum was serially diluted

317

in factor 1: 2 for the remaining wells. After that, 50 µL of the bacterium A. hydrophila

318

(MF372510), which were inactivated with 10% buffered formalin, were added at a

319

concentration of 1 x 108 CFU. mL-1 in all wells (this strain showed the best results in the

320

inhibitory assay in vitro). The microplate was incubated at 25° C for 18 h in a humid

321

chamber. The agglutination was confirmed by the observation of a precipitate in the

322

bottom of the well with the naked eye. The agglutination titer activity was considered as

323

the reciprocal of the last dilution that showed agglutination [47].

324

The antimicrobial activity of the serum was determined against the bacteria:

325

Aeromonas hydrophila (MF372510) in a 96-well flat bottom microplate, according to

326

Silva et al. [47]. The inoculum of the pathogen strain was grown in BHI at 30° C for 12

327

h, prepared at the concentration of 0.5 on the MacFarland scale and diluted in Poor

328

Broth medium (PB - HIMEDIA®, India) 100.000 times. Serum was diluted 1: 4 in PB in

329

the first well and serially diluted in factor 1: 2 to the 12th well. For the positive and

330

white control, saline solution was diluted in PB, as was done with the serum. Finally, 20

331

µL of the A. hydrophila inoculum was added to each well of the serum samples and the

332

positive control. The microplate was incubated at 28 ° C for 12 h. The growth of the

333

microorganisms was determined in microplate reader (Expert Plus Asys®) for reading at

334

550 nm. The antimicrobial activity of the serum was reciprocal to the last dilution that

335

presented bactericidal activity.

336

2.7.5

Growth performance

337

Initial, final and biweekly biometrics were performed individually for all fish in

338

this trial. Calculations for performance characteristics were determined for each

339

experimental unit. After 60 days of probiotic supplementation, were determined:

340 Weight Gain WG = final biomass g − initial biomass g Feeding Conversion FC =

diet comsuption g WG g

12

Feed Efficience FE = 34 =

1 FC

5 8 100 67

341

Where:

342

Kf = Fulton’s condition factor

343

W = weight of the fish (g)

344

l = total length (cm)

345

2.7.6

Microbiological analysis

346

For determination of the viable bacterial microbiota present in the

347

gastrointestinal tract, portions of the anterior medial tract of 03 fish from each

348

experimental unit were sampled. These portions were processed in pool, macerated in

349

porcelain grains with 9 mL of SSE (65 g. L-1) and serially diluted five times (factor

350

1:10). Dilutions of 10-1 to 10-5 were seeded in Petri dishes containing the following

351

culture media: MRS with aniline blue (10 g. L-1; for growth of lactic acid producing

352

bacteria), TSA with defibrinated sheep blood (50 mL. L-1; for growth of total

353

heterotrophic bacteria), TCBS (for growth of vibrionaceae) and Cetrimide (for growth

354

of Pseudomonas sp.). The MRS plates were incubated at 35° C for 48 h and the others

355

were incubated at 30° C for 24 h for further determination of the bacterial

356

concentrations (CFU. mL-1).

357

2.7.7

Experimental challenge

358

The pathogenic strain used in the experimental challenge was Aeromonas

359

hydrophila (MF372510) isolated from symptomatic silver catfish by Bandeira Junior et

360

al. [48], during an outbreak of mortality.

361

To determine the dose for the experimental challenge, 25 fish were distributed in

362

5 tanks (5 fish per tank) equipped with individual heaters that kept the water

363

temperature at 26.32 ± 0.20° C. The A. hydrophila strain was cultured in BHI broth at

364

30° C for 24 h and after that period it was centrifuged 4.000 g for 30 min at 4° C, and

365

resuspended in 10 mL of SSE (65 g. L-1) in the following doses: 1 × 105, 1 × 106, 1 x

366

107, 1 x 108 and 1 x 109 CFU. mL-1. All fish received 100 µL of the bacterial solution

367

intraperitoneally. Cumulative mortality was assessed every 6 h for 96 h. After this

368

period, the concentration 1 x 109 CFU. mL-1 was defined as the dose to be used in the in

369

vivo challenge because it caused the highest mortality (80%).

13 370

After 60 days of testing, the animals had a mean weight of 51.29 ± 25.11 g

371

(mean ± standard deviation) and were fasted for 24 hours prior to infection. After

372

anesthesia with eugenol (75 mg L-1), each individual received 100 µL of the bacterial

373

solution A. hydrophila (1 x 109 CFU.mL-1), intraperitoneally. Mortality was monitored

374

every 12 hours for 144 hours (6 days).

375

2.7.8

376

Immunological analysis post-challenge

The same methodology described previously for performing the immunological

377

analyzes was performed after challenge with A. hydrophila.

378

2.8 Statistic analysis

379

The design was completely randomized. To compare the means between the

380

treatments, the data were analyzed by the Shapiro-Wilks test and the homoscedasticity

381

of the variance was evaluated using the Levene test (ANOVA (P <0.05)). The values of

382

the microbiological counts were transformed to log (x + 1). When a difference between

383

the treatments was found, the averages were compared using the Tukey test (5%) with

384

the aid of Statistica® software version 13.0.

385 386

3. Results

387

Initially 10 lactic acid producing strains were selected from the MRS agar

388

culture medium plus aniline blue (blue colonies growth). Of these, only four

389

demonstrated inactivation of the enzyme catalase (LAB characteristic) and were

390

identified molecularly. Identification of the isolates revealed a potential candidate for

391

the autochthonous probiotic Lactococcus lactis (NR_113960.1). The other strains

392

isolated (n = 3) were identified as Lactococcus garviae (NR113268 / NR104722) and as

393

there are reports of their pathogenicity to fish, these were discarded.

394

In the in vitro tests performed, the autochthonous strain presented characteristics

395

that justified its use in fish diet. Lactococcus lactis presented a considerable inhibitory

396

activity against five pathogenic strains (Table 2).

397

14 398

Table 2

399

Antagonism halos (average ± standard deviation) of autochthonous strain Lactococcus

400

lactis against pathogenic bacteria isolated from freshwater fish. Pathogenic Bacteria

Antogonism halos (mm)

Aeromonas hydrophila (CPQBA 228-08 DRM)

9.0 ± 1.0

Streptococcus agalactiae (GRS 2035)

9.7 ± 0.6

Aeromonas hydrophila (MF372509)

9.7 ± 1.5

Aeromonas hydrophila (MF372510)

10.0 ± 1.0

Citrobacter freundii (MF565839)

11.0 ± 1.0

Raoltella ornitinolytica (MF372511)

7.3 ± 1.4

401 402

The isolated autochthonous strain presented reduction of growth in bile salts

403

(78.69 % ± 5.83 (mean ± SD)), maximum growth rate of 0.354 (h- 1), doubling time of

404

1.96 (h), mean values of inhibition halos of: 11 mm, 30 mm and 25 mm for norfloxacin,

405

tetracycline and florfenicol, respectively, showing to be sensitive to the three antibiotics

406

tested [49] and abscense of hemolytic activity (γ-hemolysis).

407

For the accomplishment of the in vivo step of this study, the incorporation of

408

autochthonous and allochthonous strains in the fish diet was followed. After 24 h of

409

growth, the inoculum of the autochthonous strain L. lactis had a mean concentration of

410

2 x 109 CFU.mL-1 and after incorporation into the diet, its average concentration was: 1

411

x 107 CFU. g-1. The inoculum of the allochthonous probiotic strain Lactobacillus

412

plantarum, after 24 h of growth, presented a mean concentration of 1 x 109 CFU.mL-1

413

and, after incorporation into the diet, its average concentration was: 4 x 107 CFU.g-1.

414

After 60 days of dietary supplementation, no significant differences in

415

haematological parameters were observed between the animals treated fed with the

416

probiotic strains (autochthonous and allochthonous), and those treated without probiotic

417

supplementation (Table 3), with exception of the amount of mean corpuscular

418

hemoglobin. This amount was higher in the animals that received autochthonous

419

probiotic supplementation in relation to the animals of the control group.

420

15

421

Table 3

422

Effects of the experimental diets on the hematological parameters of silver catfish Rhamdia quelen. The data show the hematological parameters

423

of fish fed supplemented diets with autochthonous probiotic (L. lactis) or allochthonous probiotic (L. plantarum) or unsupplemented (control) for

424

60 days. Data are presented as means and standard deviation. RBC: red blood cells, WBC: white blood cells, MCV: mean corpuscular volume,

425

MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration. Treatment Control

6

Thrombocytes -1

(x 10 . µL ) 1.87 ± 0.50

3

-1

(x 10 . µL ) 201.85 ± 171.25

WBC 5

Lymphocytes -1

(x 10 . µL ) 1.43 ± 0.82

3

-1

(x 10 . µL ) 106.41 ± 53.03

Monocytes 3

-1

(x 10 . µL ) 1.39 ± 1.42

Neutrophils 3

-1

(x 10 . µL ) 12.47 ± 5.38

LG-PAS 3

-1

(x 10 . µL ) 2.07 ± 3.32

Hematocrit (%) 37.8 ± 6.72

Hemoglobin -1

(g. dL ) 6.23 ± 0.93

MCV

MCH

(fL)

(pg)

192.69 ± 45.95

34.61 ± 6.95

MCHC (g. dL-1) b a

16.87 ± 3.24

L.lactis

1.56 ± 0.48

214.65 ± 123.16

1.96 ± 1.20

174.24 ± 112.13

2.16 ± 1.56

18.68 ± 16.45

1.13 ± 1.57

38 ± 6.96

6.37 ± 1.06

228.24 ± 77.06

43.59 ± 12.05

18.49 ± 3.02

L. plantarum

1.75 ± 0.33

220.37 ± 144.92

2.40 ± 1.85

214.84 ± 164.93

1.79 ± 2.00

20.34 ± 20.14

1.29 ± 2.08

35 ± 5.94

6.35 ± 1.07

202.13 ± 42.88

35.58 ± 7.38b

17.92 ± 1.82

0.16

0.94

0.94

0.06

0.44

0.41

0.51

0.65

0.91

0.54

0.015

0.58

p value

426

RBC

Different letters indicate significant difference by Tukey test (p < .05).

16 427

The total protein and immunoglobulin, agglutination titer activity and

428

antimicrobial activity of serum did not show a significant difference between animals

429

not supplemented and supplemented with probiotic strains, before the experimental

430

challenge. Additionaly, no statistical differences were observed between treated animals

431

in terms of final weight and length, final condition factor, weight gain, feed conversion

432

and feed efficiency.

433

The increase of lactic acid bacteria concentration viable counts was observed in

434

the intestinal tract of animals receiving probiotic supplementation (autochthonous or

435

allochthonous), in relation to those not supplemented. After probiotic supplementation,

436

the concentrations reached in the intestinal tract of the animals treated with the

437

autochthonous strain Lactococcus lactis and the allochthonous strain L. plantarum were:

438

higher when compared to the control group (Fig. 1).

439

440 441

Fig. 1. Effects of the experimental diets on the intestinal microbiota of silver catfish

442

Rhamdia quelen. The data are transformed in log 10 CFU.g-1 intestinal tract and show

443

the intestinal microbiota of fish fed with supplemented diets with autochthonous

444

probiotic (L. lactis) or allochthonous probiotic (L. plantarum) or unsupplemented

445

(control) for 60 days. THB: Total heterotrophic bacteria, LAB: Lactic acid bacteria.

446

Bars indicate the standard deviation.

447

*Significant difference as determined by Tukey test (p < .05).

17 448 449

Survival rates after experimental challenge did not differ statistically between

450

treatments. Animals supplemented with autochthonous probiotic strain presented higher

451

immunoglobulin values after challenge when compared to the control group, and the

452

group supplemented with allochthonous probiotic strain (Table 4).

453 454

Table 4

455

Effects of the experimental diets on the immunological parameters of silver catfish

456

Rhamdia quelen. The data show the immunological parameters of fish fed

457

supplemented diets with autochthonous probiotic (L. lactis) or allochthonous probiotic

458

(L. plantarum) or unsupplemented diet (control) for 60 days, after challenge with

459

Aeromonas hydrophila (MF372510). Data are presented as means and standard

460

deviation. Treatment Control L. lactis

Total Protein Immunoglobulin Agglutination Antimicrobial (mg.mL-1) (mg.mL-1) (log (x)) (log (x+1)) 68.44 ± 24.91

31.69 ± 1.72b

69.65 ± 15.95

a

L. plantarum 66.52 ± 12.87 p value

461 462 463

0.98

9.67 ± 1.15

5.0 ± 0.0

45.31 ± 3.04

9.67 ± 0.58

5.30 ± 0.58

35.12 ± 2.86b

11.33 ± 0.58

5.70 ± 0.58

0.0017

0.07

0.30

Different letters indicate significant difference by Tukey test (p < .05).

4. Discussion

464

The isolation and selection of autochthonous strains leads the development of

465

production of native species, because it can contribute to the improvement of the growth

466

performance, immune system and protection against diseases in situ. The present study

467

selected a species-specific strain L. lactis that demonstrated in vitro characteristics that

468

justified its use as a feed additive for R. quelen juveniles. Its in vivo probiotic effect

469

have demonstrated better immunomodulation after experimental challenge when

470

compared to an allochthonous strain L. plantarum.

471

The intestinal microbiota of a kind of catfish (Ictalurus punctatus), was observed

472

by Larsen, Mohammed e Arias [50] who attested that Fusobacteria was the dominant

473

phylum (94.8%) and Firmicutes represented only 0.05% of this total. Indeed this phyla

474

is a small representative group in the intestinal microbiota of this species. Therefore, the

475

low number of lactic acid strains isolated from the intestinal tract of silver catfish (R.

476

quelen) in the present study, can be attributed to the natural composition of the intestinal

477

microbiota of this species that may have the filo Firmicutes very poorly represented.

18 478

Another factor to be considered is the modulation of the intestinal microbiota by diet.

479

Grass carp (Ctenopharyngodon idellus) which received diet made with fish meal,

480

showed dominance of the genus Cetobacterium (phylum Fusobacteria), a bacterial

481

group known to be possibly linked to protein digestion [51]. This also can explain the

482

low representativeness of the phylum Firmicutes in the silver catifish of the present

483

study which also were feed with diet made with fish meal and this can have reflected in

484

the reduced number of lactic acid bacteria isolated by the authors.

485

In the present study, the search for the selection of LAB's (filo Firmicutes) as

486

probiotic candidates, although justified by the positive results observed in previous

487

studies, could have obtained more promising results if another group, more

488

representative of the intestinal microbiota of silver catfish, had been chosen in the

489

selection stage or, if wild fish had been used for autochthonous strain selection.

490

Therefore, for future studies on the selection of autochthonous probiotic strains, the

491

ideal would be, firstly, knowledge of the natural composition of the intestinal

492

microbiota of the target species, by high throughput sequencing to increase the

493

efficiency in the potential probiotic selection.

494

Among the isolated lactic acid bacteria in this study, three were identified as L.

495

garviae and discarded from subsequent in vitro and in vivo tests, because this species is

496

associated to lactococcosis, an emerging disease affecting freshwater and marine fish

497

cultures [52-53]. Although pathogenic strains have already been used as probiotics for

498

fish their use should be avoided since they can express their patogenicity and cause

499

mortalities in the fish farmings [54-55].

500

A wide range of studies have demonstrated the ability of isolated fish LAB’s to

501

inhibit the growth of pathogens [56-58]. Results similar to those found in the present

502

study were observed by Pereira et al. [10] who isolated 4 strains of Lactococcus lactis

503

subsp. lactis of the intestinal tract of pirarucu (Arapaima gigas) evaluated their

504

antagonistic capacity against freshwater fish pathogens and also observed positive

505

antagonism (halos> 08 mm). The antagonistic effect of L. lactis is probably explained

506

by the release of antibacterial compounds, such as bacteriocins, organic acids, and

507

especially hydrogen peroxide, or by competition for nutrients and/ or adhesion sites in

508

the intestinal microhabitat [59-60].

509

Once the probiotic potential of a strain is attested in vitro, its evaluation is

510

followed by in vivo growth assays where the strain studied is incorporated into the fish

511

diet. Thus, it is necessary that the evaluated probiotic strain is able to survive the

19 512

passage through the stomach and colonize the fish intestine this will include its

513

exposure to bile salts which have antimicrobial properties [61]. Therefore, one

514

prerequisite for the selection of a probiotic candidate strain is the evaluation of its

515

resistance to biliary salts [62-63]. There is no consensus on the ideal concentration to be

516

used in in vitro to determine the tolerance of a strain to biliary salts [57]. Some authors

517

used concentrations of up to 10% salts for to evaluate the tolerance of a strain [64-65].

518

However, Balcázar et al. [57] suggest that the concentration of bile in the

519

gastrointestinal tract of fish is between 0.4% and 1.3%. Therefore, the survival of lactic

520

acid strains (L. lactis, L. plantarum and L. fermentum) in concentrations between 2.5%

521

and 10% bile, obtained optimum survival results at all concentrations tested [57]. In the

522

present study, we observed that the autochthonous strain isolated from the intestinal

523

tract of silver catfish L. lactis had a very reduced growth, in approximately 78% in the

524

presence of 5% of bile salts. It is important to emphasize that in our study, in addition to

525

the concentration of biliary salts (5%) has been high, the exposure time to salts (24 h)

526

was higher when compared with studies made by Balcázar et al. [57] and Burbank et al.

527

[66] which was only 1,5 hours. Therefore, the low resistance of the autochthonous

528

probiotic strain L. lactis found in this test may have been a consequence of the extreme

529

conditions used in vitro (high biliary salts concentration and time of exposure) to

530

simulate the gastric environment of silver catfish.

531

Other important characteristics to be considered in the selection of a probiotic

532

strain are its maximum growth rate and doubling time (h). The higher the growth rate

533

and the shorter the doubling time, the more efficient the use of the strain on a

534

commercial scale; which can also mean higher in vivo competitiveness [67]. In this

535

study, L. lactis showed a higher maximum growth rate and a shorter replication when

536

compared to studies that isolated and selected lactic acid probiotic strains of the

537

intestinal tract of bullfrog Lithobates catesbeianus and shrimp Litopennaeus vannamei

538

[35, 68].

539

According to Merrifield et al. [26], another essential selection criteria in

540

choosing a probiotic candidate strain is that it should be free of antibiotic resistance

541

genes. The main reason for this is to avoid the transfer of this resistance both to

542

pathogenic bacteria that could inhabit the gastrointestinal tract of the host, and to the

543

host itself [69]. Considering that the aquaculture sector makes indiscriminate use of

544

chemotherapeutics, that the food chain is the main route of transmission of bacteria

545

resistant to humans, and that antibiotics used in aquatic environments contribute to the

20 546

emergence of resistant bacteria in the environment, sensitivity to antimicrobials is a

547

very important criterion in the selection of probiotic candidates [70]. In the present

548

study, Lactococcus lactis did not show resistance to the three antimicrobials tested,

549

corroborating Pereira et al. [10], which evaluated the presence of resistance genes in

550

strains of L. lactis subsp lactis isolated from the intestinal tract of pirarucus, observed

551

absence of resistance genes to erythromycin, chloramphenicol and tetracycline.

552

In the process of selection of a probiotic strain, another criterion to be

553

considered is the hemolytic activity of the strain. Lactococcus lactis presented negative

554

hemolysis (gamma), evidencing another positive characteristic as a probiotic candidate.

555

This result corroborates Pereira et. al. [10] who also observed gamma hemolysis in

556

strains of L. lactis isolated from pirarucu (A. gigas). The probiotic use of a strain with

557

positive hemolytic activity is not considered safe since it may cause pathogenicity to the

558

cultured organisms [71].

559

The ability of probiotic strain remains in the diet in a concentration that

560

guarantees its arrival to the gastrointestinal tract of the fish is also an important step in

561

the selecting process. The L. lactis final concentration in diet was similar to the

562

concentration of the allochthonous strain L. plantarum, which already has a proven

563

probiotic effect by Jatobá et al. [39]. Other authors also obtained concentrations similar

564

to the present study, incorporating L. lactis in the fish diet. Dawood et al. [72] and Kim

565

et al. [73] reached concentrations of 1 x 106 CFU. g-1 and 1 x 106 to 1.25 x 108 CFU. g-1,

566

respectively, achieving promising results in growth performance and immune response.

567

Usually, probiotic supplementation improves the general health status of animals

568

although, its immunostimulatory capacity in fish is still under investigation. In humans

569

for instance, the ability of these supplements to stimulate the production of blood cells,

570

particularly RBC and WBC, is already well known [74]. In this study, probiotic

571

supplementation of autochthonous strain L. lactis presented significantly higher mean

572

corpuscular hemoglobin (MCH) values than those observed in the control group and in

573

the group treated with allochthonous probiotic strain L. plantarum. This result

574

corroborates Munir et al. [74] who, by supplementing Channa striata for 16 weeks with

575

probiotic Lactobacillus acidophilus, also observed higher MCH values in the treated

576

group compared to the non-supplemented group. The inclusion of Lactobacillus

577

acidophilus (3.01 x 107 CFU g -1) in the diet of African Catfish, Clarias garepinus, also

578

contributed to the improvement of hematological parameters of fish after 12 weeks of

579

supplementation [75]. It is known that MCH is a mass unit that measures the amount of

21 580

hemoglobin within each erythrocyte and in humans this index is used to identify the

581

type of anemia. In the present study, probiotic supplementation may have contributed to

582

a greater amount of hemoglobin per erythrocyte, meaning greater transport of oxygen to

583

the tissues, and consequently, animals more prepared to transport oxygen to the tissues

584

affected by some type of stressor agent.

585

The probiotic supplementation during 60 days didn’t present significant

586

differences between treatments regarding immunological parameters. However, after

587

challenge with A. hydrophila, the fish supplemented with Lactococcus lactis had higher

588

values of total immunoglobulin when compared to the other treatments. The increase in

589

immunoglobulin concentration after challenge may be due to the induction of the

590

immune response by autochthonous probiotic supplementation. Elevation of

591

immunoglobulin levels has already been observed in other studies after probiotic

592

supplementation in fish [75-77]. This increase may mean greater protection of the

593

intestinal mucosa against pathogens since, along with other factors related to the

594

immune system, such as lectins, mucins and antimicrobial peptides, these proteins form

595

the first defense barrier that lines the intestinal epithelium [78].

596

The effects of probiotic supplementation for fish are not fully understood. Some

597

probiotics will act as growth promoters, while others will provide greater protection

598

against disease [79]. In this study, the autochthonous strain L. lactis did not significantly

599

alter the zootechnical indexes in relation to the animals supplemented with

600

allochthonous strain L plantarum or the control group. This result differs from the study

601

by Nguyen et al [28] that, supplementing Paralichthys olivaceus, for 4 and 8 weeks,

602

with autochthonous strain L. lactis at a concentration of 1 x 109 CFU. g-1 diet, observed

603

significant improvements in the growth performance of the animals. The result observed

604

in this study may be explained by the lower concentration of L. lactis in the diet: 1 x 107

605

CFU. g-1 which may not have been high enough to promote such benefits to animals.

606

The same is true for the results observed in the animals treated with allochthonous strain

607

L. plantarum, besides having the disadvantage of being non-specific to the target

608

species.

609

The final concentration reached by the strain in the diet is of paramount

610

importance for the success of probiotic supplementation, since it means the amount of

611

bacteria that will in fact be delivered to the fish for later colonization of the intestinal

612

tract. In this study, fish fed diets supplemented with probiotic strains (autochthonous or

613

allochthonous) showed a significant increase in the concentration of lactic acid bacteria

22 614

in the intestinal tract. A similar result was observed by Yamashita et al. [80]

615

supplementing the diet of tilapia with autochthonous strain L. plantarum at a

616

concentration of 1.81 x 107 CFU. g-1 diet, reached a significant increase of LAB's in the

617

intestinal tract of the animals when compared to the control group. The bacterial

618

microbiota of aquatic organisms consists predominantly of gram-negative bacteria [81],

619

and may vary according to the environment, lack of any nutrient or by the

620

supplementation of probiotic bacteria [82]. Probiotics inhibit the growth of other

621

bacteria by competitive exclusion, by space and/ or nutrients, or by the production of

622

inhibitory compounds, such as organic acids and hydrogen peroxide. Organic acids can

623

cross the cell wall of gram-negative bacteria causing reduction of intracellular pH and

624

consequent death of the bacteria by energy depletion, when it spends energy in the

625

expulsion of cations (H+) [83-84] this may explain the higher concentrations of LAB's

626

observed in the groups supplemented with probiotic strains in this study. In addition,

627

hydrogen peroxide is one of the main extracellular products responsible for the

628

inhibitory action of L. lactis [59] and, knowing that it acts on the lipid membrane and

629

DNA of anaerobic microorganisms [85], this may also explain the higher LAB count in

630

the supplemented animals of the present study. The concentration of LAB's found in the

631

intestinal tract of control animals (1.70 x 104 UFC.mL-1) could be explained due to the

632

prebiotic action of the MRS culture medium that was incorporated into the diet of this

633

treatment.

634

Some studies have shown that dietary supplementation with a mixture of

635

autochthonous

and

allochthonous

probiotics

for

fish

demonstrates

better

636

immunostimulation and protective effects against disease when compared to

637

supplementation with only one probiotic strain [26;86-87]. In this sense, perhaps one

638

way to contribute to the probiotic effects of autochthonous strain L. lactis, in vivo, was

639

to increase its supplementation with the allochthonous strain L. plantarum. Beck et al.

640

[88] to improve the probiotic effects of allochthonous strain (L. lactis) created a mixture

641

by the addition of autochthonous probiotic strain (L. plantarum) in the olive flounder

642

diet for 30 days and obtained significant improvements in innate immune parameters,

643

weight gain and survival after challenge with S. iniae, when compared to diets with

644

single probiotic and control. The efficiency of the probiotic strains applied in the fish

645

diet is determined by some factors, such as probiotic type, duration and dose of

646

supplementation, mode of application, besides age and size of fish [87, 89-90].

23 647

Therefore, candidate strains for a probiotic mixture need to be wisely selected to

648

maximize their combined effects.

649

In conclusion, L. lactis (NR_113960.1) isolated from silver catfish demonstrated

650

probiotic properties in vitro, such as its ability to inhibit pathogenic bacteria, but in vivo

651

experiments so far, have showed no improvement in survival after challenge or in

652

changes in the growth performance and immunological parameters. However, the

653

autochthonous strain was able to raise the concentration of lactic acid bacteria present in

654

the intestinal tract, the amount of hemoglobin per erythrocyte (MCH) and serum

655

immunoglobulin concentration after challenge with A. hydrophila. This strain could be

656

eventually used as food additive for silver catfish however, other studies about the

657

composition of the natural intestinal microbiota of this species, the evaluation of its

658

monostrain or multistrain supplementation with other probiotic strains or even new

659

methods of its inclusion in the diet at different concentrations are highly recommended.

660 661

Acknowledgements

662

The authors thank CNPq (National Council of Scientific and Technological

663

Development) for the financial support (Universal Project 447029 2014-2); grant to J.

664

L. P. Mouriño (CNPq 308292/2014-6); Bernardo Baldisserotto and Federal University

665

of Santa Maria (UFSM) for the pathogenic strains’s doation and CAPES (Coordination

666

for the Improvement of Higher Education Personnel) for the PhD scholarship to M.M.

667

Yamashita.

668 669

References

670

[1] REYNALTE-TATAJE, D. A. et al. Spawning of migratory fish species between two

671

reservoirs of the upper Uruguay River, Brazil. Neotropical Ichthyology, v. 10, n. 4, p.

672

829-835, 2012.

673 674

[2] SILVA, B.C. et al. Desempenho produtivo da piscicultura catarinense. Revista

675

Agropecuária Catarinense, Florianópolis, p. 15 - 18, 03 maio 2017.

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678

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679

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da

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[4] HAMED, Said Ben et al. Fish pathogen bacteria: Adhesion, parameters influencing

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virulence and interaction with host cells. Fish & Shellfish Immunology, [s.l.], v. 80,

682

p.550-562, set. 2018. Elsevier BV. http://dx.doi.org/10.1016/j.fsi.2018.06.053.

683 684

[5] SOUZA, Carine F. et al. In vivo bactericidal effect of Melaleuca alternifolia

685

essential oil against Aeromonas hydrophila: Silver catfish (Rhamdia quelen) as an

686

experimental model. Microbial Pathogenesis, [s.l.], v. 98, p.82-87, set. 2016. Elsevier

687

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36

HIGHLIGHTS • • • • •

A LAB was isolated from the intestinal tract of silver catfish to be used as feed additive. Isolated autochthonous probiotic strain Lactococcus lactis presented optimist in vitro results. Lactococcus lactis inhibited bacterial pathogens in vitro, presented absence of hemolysis and considerable speed of duplication. Both probiotic supplementations (autochthonous and allochthonous) increased the final concentration of LAB’s of the intestinal tract of R. quelen. Autochthonous probiotic supplementation increased the amount of immunoglobulin after experimental challenge.