Patent ID: 12246045

DETAILED DESCRIPTION

The present invention refers to a probiotic feed that comprises lactic acid bacteria, particularlyL. lactis, transformed to produce an immunostimulant cytokine, preferably type II IFN, to immunostimulate aquatic species against bacterial infections, thus achieving heterologous expression of an immunostimulant protein ofSalmo salarin lactic acid bacteria. TheL. lactisbacteria has been assigned the accession number RGM 2416, dated Oct. 22, 2017, by the INIA's Chilean Collection of Microbial Genetic Resources, Chile.

Immunostimulant proteins strongly regulate immune response. Administration of recombinant immunostimulant proteins have allowed their use as a therapeutic to increase immune response against bacterial pathogens. In aquaculture, there are no available in situ heterologous production systems for functional immunostimulant proteins for therapeutic use. Lactic acid bacteria are GRAS (Generally Regarded as Safe) organisms, that because of this classification have been used as vaccines and as therapeutic release systems.

The lactic acid bacteria of this invention can be used to express and secrete functional immunostimulant proteins in aquatic species, particularly in fishes and preferably inSalmo salarand rainbow trout. The present lactic acid bacteria allows non-invasive immunogenic protection applicable in large scale, capable of increasing current immunization systems.

Administration of the present lactic acid bacteria, that produce an immunostimulant cytokine, can stimulate in vivo the expression of genes that respond to IFN gamma and reduce mortality of aquatic species challenged withF. psychropillhumandP. salmonis

Using molecular biology tools, lactic acid bacteria was generated that produce an immunostimulant cytokine. Its immunostimulant properties were studied in species highly important for aquaculture, administrating an oral dosage of 1×107UFC per fish. Immunostimulation was assessed quantifying genes that respond to this cytokine using real time qPCR normalizing their expression against the housekeeping gene eF1a. To achieve this objective, total RNA was extracted from immunological organs (spleen and kidney), which were previously assessed studying their morphology and any signs of toxicity. The lactic acid bacteria produce and secret the heterologous protein. The protein was located mainly in the cytoplasmatic fraction. The bioassays showed that the protein is functional when stimulating the expression of immunological genes.

The invention relates, particularly, to transformedLactococcus lactisbacteria that produce interferon gamma fromSalmo salarthat includes the DNA construction that comprises pNZ8149-P1-USP45-IFNg-GGG-6×HIS where pNZ8149 is the transformed vector; P1 isL. lactisconstitutive expression promoter; Usp45 is a secretion signal; IFNγ isSalmo salarinterferon gamma (IFNγ) mature mRNA coding sequence; GGG is a sequence coding for three glycines and 6×His is s sequence coding for 6 terminal histidines. The transformed strain ofLacococcus lactisis identified as NZ3900, however, it is not limiting and other strains ofL. lactiscould be used. The bacteria is preferablyLactococcus lactisNZ3900 transformed with the genetic system that comprises the plasmid pNZ8149/P1-USP45-IFNg-GGG-6×HIS. Preferably, the recombinant strain is the one with the accession number RGM 2416, dated Oct. 22, 2017, by the INIA's Chilean Collection of Microbial Genetic Resources, Chile.

It is also part of the invention, a useful plasmid to transformL. lactisbacteria to produce interferon gamma (IFNγ) that comprising the vector pNZ8149 and the sequence P1-Usp45-IFNγ-GGG-6×HIS; where P1 isL. lactisconstitutive expression promoter; Usp45 is a secretion signal; IFNγ isSalmo salarinterferon gamma (IFNγ) mature mRNA coding sequence; GGG is a sequence coding for three glycines and 6×His is s sequence coding for 6 terminal histidines. Likewise, the invention relates to a method to prepare the transformedL. lactisbacteria that comprises the following steps:a) Digesting with restriction enzymes a plasmid that contains the reading frameof Salmo salarIFNg codon optimized forLactococcus lactis,b) purifying the digestion product, corresponding to the IFNg gene with sticky ends for restriction enzymes,c) Meanwhile, digesting with the same enzymes the plasmid NZ8149,d) Purifying the linearized plasmid from an agarose gel,e) Ligating both purified products with ligase,f) Dialyzing the ligation product,g) Transforming electrocompetentL. lactisbacteria with the ligated plasmid.

The restriction enzymes are preferably, for steps a) and c) restriction enzymes NcoI and XbaI and electrocompetent bacteria of step g) is the strainL. lactisNZ3900.

It is part of this invention a probiotic feed to immunostimulate aquatic species, preferably immunostimulate fishes. The probiotic feed comprises transformedL. lactisbacteria with a transformedLactococcus lactisbacteria producing interferon gamma ofSalmo salarthat includes the DNA construction that comprises pNZ8149-P1-USP45-IFNg-GGG-6×HIS where pNZ8149 is the transformed vector; P1 isL. lactisconstitutive expression promoter; Usp45 is a secretion signal; IFNγ isSalmo salarinterferon gamma (IFNγ) mature mRNA coding sequence; GGG is a sequence coding for three glycines and 6×His is s sequence coding for 6 terminal histidines. Likewise, the invention comprises the method to prepare the probiotic feed described above.

Additionally, this invention protects any immunomodulatory composition for aquatic species, under the broadest scope of composition. The composition can be liquid or solid and have excipients to provide stability for storage and enhance bioavailability of the transformed bacteria of this invention. Furthermore, the composition includes all combinations of useful organic molecules to administrate to aquatic species such as proteins, lipids, saccharides, in combination with the transformedL. lactisbacteria described above or with the bacteria described in the preferred conducted examples. This composition can comprise the combination of the bacteria of the invention with recombinant protein vaccines or with nucleic acid vaccines, bacterines, probiotics, prebiotics or other immunomodulators, vitamins, etc.

The use of the transformedL. lactisbacteria is for preparing feed or a useful composition for immunostimulating aquatic species, particularly, to immunostimulate fishes. The given use for these bacteria is to prepare useful feed to reduce bacterial load, preferably bacterial load ofFlavobacterium psychrophilumand orPiscirickettsia salmonis. The use of the bacteria and the feed that comprises it is to prepare a feed or a useful composition to treat or prevent infection fromFlavobacterium psychrophilumand/orPiscirickettsia salmonisin fishes.

Is part of this invention, a kit that includes a packing that comprises the transformedL. lactisbacteria with the DNA construction pNZ8149-P1-USP45-IFNg-GGG-6×HIS. Likewise, this kit can comprise instructions to be used to feed, treat or prevent bacterial diseases in aquatic species, preferably, fishes important for aquaculture. In the preferred embodiment instructions have information for the use for feeding fishes, treat or prevent diseases caused byFlavobacterium psychrophilumandPiscirickettsia salmonisin fishes.

The invention also comprises the method to reduce bacterial load in aquatic species, that comprises the administration to this species the feed prepared with transformedL. lactisbacteria, selected from any bacteria that comprises the DNA construction pNZ8149-P1-USP45-IFNg-GGG-6×HIS detailed in the examples of this invention. The aquatic species are preferably fishes, and the method treats or prevents infection withFlavobacterium psychrophilumorPiscirickettsia salmonis. The method for delivery is capable of treating or preventing a bacterial infection by reducing bacterial load in aquatic species, preferably fishes.

EXAMPLES

Example 1

Using synthetic biology the coding gene for a codon-optimized immunostimulating protein fromSalmo salarwith a secretion peptide and 6 histidine tail was designed and synthesized. The gene was clones into a food grade expression vector. Production of the heterologous protein was confirmed using Western Blot and its functionality through bioassays.

The present invention consists in a recombinantLactococcus lactisNZ3900 strain, that comprises the plasmid NZ8149 into which a synthetic DNA segment, that comprises P1 promoter, the signal peptide of protein USP45, a gene that codifies the mature sequence of type I interferon ofSalmo salarand a tail of 3 glycines and 6 histidines, denominated as pNZ8149-IFNgSS (FIG.1), was cloned.

The sequence of the IFNγ gene was obtained fromSalmo salar's genome and codon-optimized to be expressed in anL. lactishost (FIG.1). This strain expresses constitutively the IFNγ gene driven by promoter P1, fromL. lactissequence SEQ ID No:2, and in inducible by the nisin promoter present in the commercial vector (FIG.2). IFNγ is secreted to the culture media by the signal peptide Usp45, sequence SEQ ID No 3, that is merged in the reading frame of the IFN gamma sequence, SEQ ID No:4 (FIG.3). Expression of IFNγ was detected in extracts of IFNγ producingL. lactisextract compared to extracts of the strain that contains the pNZ8149 without the gene's reading frame (FIG.4).

Methodology

Design of the pNZ8149 vector. To design the lactococcal vector, the plasmid pNZ8140 (Mobitec®) was used as backbone and the plasmid pJet1.2-IFNg (Genescript®) contained the reading frame ofSalmo salar's IFNγ (GenBank: FJ263446.1). The IFNγ gene was codon-optimized in silico forLactococcus lactisNZ3900 using the online application www.kazusa.or.jp, and then modified to include in the amino terminal end the sequence that codifies the USP45 peptide and in the carboxylic end a sequence that codifies a tail of 3 glycines and 6 histidines (GGGHHHHHH) SEQ ID No:5. These sequences were added in frame with theL. lactiscodon-optimized IFNγ gene. Likewise, the P1 promoter sequence was added in silico to the 5′ end of the sequence that codifies USP45-IFNg-GGGHHHHHH. This new sequence, P1-USP45-IFNg-GGGHHHHHH was synthetized in vitro and cloned into pJet1.2 (Genescript®) (FIG.1). The identity of the genetic sequence of the construct was corroborated by Sanger sequencing and the amino acid sequence that the gene synthetizes using the web page www.bioinformatics.org.

Because the plasmids pNZ8149 and pJet1.2-IFNγ have a replication origin for propagation inE. coliand ampicillin resistance as selection marker, the MC1061 strain was used to obtain high concentrations of both plasmids. Afterwards, both plasmids were digested for 1 hour at 37° C. with enzymes NcoI and XbaI, present in the multiple cloning site of pNZ8149 and in each end of the IFNγ in pJet1.2. Both digestions were corroborated in a 1% w/v agarose gel from which the linear vector and the insert were purified using the Wizard SV Gel and PCR Clean-Up System (Promega®). The vector and the insert were ligated overnight at 4° C. and dialyzed the next day for 30 minutes prior to electroporation withLactococcus lactisNZ3900 to obtain the bacteria with vector pNZ8149-IFNg.

Detection of IFNγ inL. lactiscultures. To detect the expression of the recombinant protein, Western Blot assays were performed from cytoplasmatic extract of sonicatedL. lactis. To do so, a preculture ofL. lactispNZ8149-IFNg was cultured overnight at 30° C. in M17 media with 0.5% lactose. The following day, a 40 mL culture was inoculated with 2% inoculum, after cultivating at 30° C. up to an OD6000.4-0.6 the culture was divided in equal parts. To some cultures, nisin was added between 0 and 10 ng/mL. Cultures were then incubated for 2 hours at 30° C. Afterwards, bacteria were collected through centrifugation at 7,000 RPM for 20 min at 4° C.

The bacterial pellet was resuspended in 500 uL of PBS1× supplemented with a protease inhibitor 1 mM. Afterwards, it was sonicated 5 times for 15 seconds each with an Ultrasonic processor Sonic Vibracell VCX130 (90% amplitude), kept in ice between cycles. The sample was centrifuged at 6,000 RPM for 10 min at 4° C. and the supernatant was separated from the cellular debris, storage in a new tube and frozen at −20° C. until its use.

To determine total protein concentration in the extract the Bradford method was used. Once the concentration was known, it was normalized to resolve and compare the amount of protein through SDS-PAGE electrophoresis. 10 μg of the cytoplasmatic extract of induced and not induced with nisin IFNγ producingL. lactisand ofL. lactisthat contained a pNZ8149 plasmid without a IFNγ reading frame as control were loaded. Samples were run in an electrophoresis for 40 min at 60 V and then for 1.5 hr at 120 V. Proteins contained in the polyacrylamide gel were transferred to a nitrocellulose membrane at 300 mA for 7 min. Afterwards, the membrane was blocked with a PBS-BSA 2% solution while shaking at 4° C. Then, the membrane was washed at room temperature 3 times for 10 minutes with PBS 1×-Tween 20 at 0.1% and incubated for 1 hour with an anti-His primary antibody at a 1:2,000 dilution in a 2% PBS-BSA solution. Next, the membrane was washed with the same procedure and then incubated for 1 hour with a 1:5,000 dilution of the secondary antibody, an anti-rabbit IgG conjugated with horseradish peroxidase. The membrane was washed again and developed by chemiluminescence with the Supersignal® West Pico Chemiluminescent Substrate Kit, incubating the solutions for 5 minutes and exposing the membrane and a digital development equipment C-Digiy model 3600 (LI-COR) for 7 minutes for imaging.

Detection of IFNγ in the supernatant ofL. lactiscultures. To detect the protein from the supernatant ofL. lactiscultures, the supernatant was collected after centrifuging the bacterial culture, then it was precipitated with 6 volumes of acetone and stored at −20° C. overnight. Afterwards, it was centrifuged at 6,000 RPM for 10 min at 4° C., then the supernatant was discarded and the precipitate was washed 2 times with double distilled water, finally, the pellet was resuspended in 50 uL of protein lading buffer. To detect the protein the Western Blot protocol described above was used.

IFNγ mRNA quantification inL. lactiscultures. To correlate the induction of protein with IFNγ messenger RNA levels generated byL. lactis(FIG.4), culture and induction conditions were replicated. After nisin induction for 2 hours, the bacterial culture was centrifuged at 6,000 RPM at 4° C. for 10 minutes, the supernatant was discarded and the bacteria was lysed in 1 mL of TRIzol to extract total RNA according to the manufacturer's recommendations. Afterwards, RNA was treated with DNase I (Promega®) to remove traces of contaminant plasmidial DNA, then this was used to quantify the number of IFNγ coding mRNA copies by qRT-PCR. The reaction mix was: 2 uL of total RNA (100 ng), 5 uL od 2× SensiMix SYBR No-ROX One-Step Kit, 0.5 uL of IFNγL. lactisFw primer, 0.5 uL of IFNγL. lactisRv primer (10 mM) (Table 2) and 2 uL of nuclease free water.

Biological activity of IFNγ produced byL. lactis. To determine if the produced protein was biologically active, cytoplasmatic extracts of the bacteria were incubated with SHK-1 cells, cells derived fromSalmo salar(Table 1) and then some transcripts involved in the response to IFNγ were quantified. Briefly, different proportions betweenL. lactisandL. lactis-IFNγ cytoplasmatic extracts were mixed with L-15 medium and incubated for 8 hours at 16° C. with SHK-1 cells to determine the contribution of the recombinant protein amount from the total protein content by the bacteria. Afterwards, total RNA from salmon cells was extracted using the E.Z.N.A Total RNA kit (OMEGA bio-tek) and treated with DNase I (Promega®). Treated RNA was used for synthesis of cDNA with Oligo(dT) with the following reaction mix: 11 uL of treated RNA, 0.5 uL of Oligo(dt) (10 mM), 1 uL of dNTPs (10 mM each), 0.2 uL of M-MLV (Promega®), 7 uL of nuclease free water. The obtained cDNA was diluted 1:2 and used for relative quantification of transcripts related to the immune response by qPCR with the following reaction mix: 2 uL of cDNA, 5 uL of 2× SensiMix SYBR No-ROX Kit, 0.25 uL of Fw (10 mM), 0.25 uL of Rv (10 mM) and 2 uL of nuclease free water (Table 2).

TABLE 1(FIG. 5) Amount of total protein used in assays to determine the contributionof IFNγ to the immune response of SHK-1 cells. Samples with 20 ug oftotal protein amount were obtained by mixingsonicated lysate of IFNγexpressingL.lactis(L. L IFNγ) and lysate ofL.lactisthat does notexpress it (L. L empty) in different proportions. Starting from the samplecontaining 20 ug of total protein all working dilutions were prepared.L. L IFNγL. L emptyFinal protein(ug)(ug)content (ug)0.020.0202.018.0206.014.02010.010.02014.06.02020.00.020

TABLE 2Nucleotide sequence of primers used for quantification of immuneresponse genes and studied pathogens.GenePrimer5′ → 3′IFNγ salmonFWCCG TAC ACC GAT TGA GGA CTRVGCG GCA TTA CTC CAT CCT AAIL-1b salmonFWCCC CAT TGA GAC TAA AGC CARVGCA ACC TCC TCT AGG TGC AGTGF-β salmonFWAGC TCT CGG AAG AAA CGA CARVAGT AGC CAG TGG GTT CAT GGgamma IP10 salmonFWGTG TCT GAA TCC AGA GGC TCC ARVTCT CAT GGT GCT CTC TGT TCC AIFNγFWCAC ATT TGC AAA ATC TTT GGG CTL.lactis-IFNγRVCAA TCG TTG TGC TTG TCG TCTIL-6 salmonFWCCT TGC GGA ACC AAC AGT TTGRVCCT CAG CAA CCT TCA TCT GGT CIL-12 salmonFWTGA CGC TTT TTC TCA CCG GTT GTRVACG CTT TGC AGC ATG AGC TTG AeF1α salmonFWGGG TGA GTT TGA GGC TGG TARVTTC TGG ATC TCC TCA AAC CGRpoSF.FWGAA GAT GGA GAA GGT AAT TTA GTT GATpsychrophilumATTRVCAA ATA ACA TCT CCT TTT TCT ACA ACTTGASTAT1FWGAC CAG CGA ACC CAA GAA CCT GAARVCAC AAA GCC CAG GAT GCA ACC ATrDNA 16SFWAGG GAG ACT GCC GGT GAT AP.salmonisRVACT ACG AGG CGC TTT CTC A
Result Description

pNZ8149 has a food grade selection marker that allows lactose metabolism (LacF), it has a constitutive promoter P1 and a nisin inducible promoter,L. lactis' natural secretion signal Usp45,Salmo salarIFNγ codifying gene and a histidine tag. RepA and RepC are genes that allow the plasmid's replication (FIG.1). When transforming NZ3900 bacteria, which presents a chromomsomal deletion of the lactose ORF, the bacteria can use this carbon sourse and keep the plasmid. The constitutive promoter P1 allows the expression of IFNγ, this induction can be proportionaly increased with the amount of nisin added to culture media due to the pNisA promoter (FIG.2). The protein can be easily detected from the cytoplasmatic fraction by Western Blot, however the amount of secreted protein is in a much lower proportion (FIG.3). This increase in the amount of protein detected by Wester Blot can be seen when quantifying the number of transcripts of IFNγ mRNA by qRT-PCR (FIG.4).

When using the cytoplasmitic extract of sonicated bacteria on salmon SHK-1 cell line, normalizing the amount of total protein (Table 1) it is possible to detect the induction of transcipts such as STAT1, gIP10 and IL-1b, suggesting that the protein is functional in vitro because the first two are involved in the signalling cascade initiated by IFNγ and the third is involved in the natural proimmflamatory response following IFNg. (FIG.5). However, when quantifying IL-6, a pleitropic cytokine related to an induced innate immune response, it increases proportionally with the amount of recombinant protein present in culture, surprisingly suggesting that IFNγ would be inducing its expression.

Example 2: Effects of Administering IFNγ ProducingL. lactisBacteria

To assess the immunoestimulating effect of IFNγ producingL. lactisin an in vivo model an assay was performed with rainbow trouts (average weight: 20-25 g) were the effect of the probiotic was evaluated through the course of the experiment during and after feeding it as it is described inFIG.6.

The approach consisted in acclimating the fish for 7 days, then feeding them for 7 days with a)L. lactis-IFNγ, b)L. lactiswith an empty vector and c) commercial feed. At day 0 (before treatment), 2 and 6 and 3 fish were sampled. Afterwards, treatment was suspended and all fish were given commercial feed. Then, 3 fish were sampled from each group at days 1, 3, 5 and 7 days after probiotic feed. While sampling, during probiotic feed (d.p.f) and after probiotic feed (a.p.f), 100 uL of blood was extracted from 3 fish, which were then sacrificed prior to spleen and kidney extraction. Blood was used to quantify lysozyme enzymatic activity in serum by means ofMicrococcus luteussuspensions. The assay consisted in measuring the decrease of absorbance at 450 nm in 180 uL of aM. luteusstock incubated with 20 uL of serum, using as a positive control 200-400 units of lysozyme/mL.

The following formula was used to calculate the amount of lysozyme:

units/ml⁢enzyme=(Δ⁢A4⁢5⁢0/min⁢Sample-Δ⁢A4⁢5⁢0/min⁢Blanc)(0.001)⁢(0.02)

To determine if stimulation of the immune response at a transcript level occurs, total RNA from spleen and kidney was extracted with TRIzol following the manufacturers recommendation. Then, cytokines IL-1β, IL-6, IL-12, TGF-β, IFNγ, gamma IP10 and STAT1 were quantified by relative quantification. Treated RNA with DNase I was used as template for cDNA synthesis with Oligo(dT) following this reaction mixture: 11 uL od treated RNA, 0.5 uL of Oligo(dt) (10 mM), 1 uL of dNTP (10 mM each), 0.5 uL of M-MLV (Promega®), 7 uL of nuclease free water. The obtained cDNA was diluted 1:2 and used as template for relative quantification of the transcripts related to the immune response by qPCR with the following reaction mix: 2 uL of cDNA, 5 uL of 2× SensiMix SYBR No-ROX Kit, 0.25 uL of Fw (10 mM), 0.25 uL of Rv (10 mM) and 2 uL of nuclease free water (Table 2).

To assess if the observed immunostimulation is able to create protection against pathogens relevant to salmon farming, a challenge in rainbow trouts (average weight: 15-20 g) was set up usingF. psychrophilum(1×108bacteria/fish) as model. Fish were acclimated for 7 days prior to treatment; then, a 7 day special feeding period began and 5 days later the challenge was performed. Afterwards, mortality was registered during the following 18 days to asses immune response and bacterial load.

In this assay the following treatments were studied: a) Fish fed for 7 days withL. lactis-IFNγ(1×107bacteria/fish) and then infected, b) Fish fed for 7 days withL. lactiswith an empty vector (1×107bacteria/fish) and then infected and c) Fish fed for 7 days with commercial feed and then infected. Mortality was registered for 17 days after infection, time during which spleen of dying fish was extracted and from fish that survived the experiment for bacterial load quantification

To determine if higher doses or a prolonged diet could increase survival, a second challenge in rainbow trout (average weight: 30-40 g) was performed withF. psychrophilum(1×108bacteria/fish). In this assay, fish were acclimated for 7 days, then special feeding began for 7 days and 5 days afterwards the challenge was performed. The following conditions were studied: a) Fish fed for 7 days with a doses 3 times higher than the original ofL. lactis-IFNγ(3×107bacteria/fish) and then infected, b) Fish fed for 13 days withL. lactis-IFNγ (1×107bacteria/fish) and then infected, c) Fish fed for 7 days withL. lactis-IFNγ (1×107bacteria/fish) and then infected, d) Fish fed for 7 days withL. lactiswith and empty plasmid (1×107bacteria/fish) and then infected and e) Fish fed for 7 days with a commercial feed and then infected. To determine if probiotic administration changes the physical condition, length and weight of surviving fish was recorded.

To assess the protective effect ofL. lactis-IFNγ onSalmo salar, the intracellular bacteriaPisiricketssia salmoniswas used as a model (1×107bacteria/fish). Fish were first acclimated for 7 days and then the following treatments began: a) Fish fed for 7 daysL. lactis-IFNγ (1×107bacteria/fish) and then infected, b) Fish fed for 14 daysL. lactis-IFNγ (1×107bacteria/fish) and then infected, c) Fish fed for 7 days withL. lactiswith and empty plasmid (1×107bacteria/fish) and then infected and d) Fish fed for 7 days with commercial feed and then infected. Mortality was recorded for 23 days after infection.

Bacterial Load: Bacterial load of infected fish withP. salmonisorF. psycrophillumwas determined by absolute qPCR using total DNA extracted from the immunological organs spleen and kidney. From these organs, total DNA was extracted with the Wizzart Genomic DNA Kit (Promega®). Purified DNA was quantified by absorbance at 260 nm. 50 ng of total DNA were used for the qPCR reaction. To detectP. psycrophillumthe primers rpoS-F (5′ GAA GAT GGA GAA GGT AAT TTA GTT GAT 3′) and rpoS-R (5′ CAA ATA ACA TCT CCT TTT TCT ACA ACT 3′) were used, which amplify a 200 bp fragment. To detectP. salmonisthe primers 16SPS-F (5′ AGG GAG ACT GCC GGT GAT A 3′) and 16SPS-R (5′ ACT ACG AGG CGC TTT CTC A 3′) were used. Each amplification product showed only one denaturation peak indicating a specific amplification product. For each one of the amplicons a calibration curve was constructed with increasing concentrations of the PCR product previously cloned on pGEM-T. Through this method the number of bacteria was calculated, assuming that there is only one copy of the gene in each bacterium chromosome.

Result Description

To asses the in vivo effect, transformed bacteria was administrated with feed to healthy rainbow trout specimens, then these were analyzed during and after feeding. Two paramenters were studied from spleen and kidney: a) transcripts related to immune respone of IFNg from spleen and kidney during probiotic feeding (FIG.7) and after probiotic feeding (FIG.9Ato E) and b) lysozyme enzymatic activity from serun during (FIG.8) and after probiotic feeding (FIG.10). When observing transcript changes it is possible to see an increase of IL-12, STAT1 and gamma IP10, the first is involved in cellular response triggered by IFNg increase and the second two are related to the downstream response of IFNg. When studying the effect of the probiotic after feeding, no significant increase can be observed in the transcripts related to type II IFN, but the anti-inflammatory cytokine TGF-b is stimulated and IL-6 increases considerably from the fifth day after feeding, effect that correlates to what is observed in cell culture. When quantifying lysozyme's enzymatic activity no increases are seen during feeding, however, a strong increase in seen from the fifth day after feeding as observed for IL-6 by qPCR, which suggested that the cytokine could be involved in the levels of lysozyme in serum.

Given that estimulation of type II IFN related genes was observed, the protective effect of the probiotic was assessed, for this an experiment was set up where rainbow trout were challenged withFlavobacterium psychrophillum(FIG.11). Fish fed withL. lactis-IFNγ (Treatment a)) presented a survival percentage higher than 70%, effect that was not seen in fish fed withL. lactiswith an empty vector which only reached 35% survival (Treatment b). Fish that did not receive probiotic treatment presented lower survival (25%) (Treatment c)). When comparing bacterial load of fish that died during the experiment and surviving fish, a decline of at least 2 orders of magnitude in the number of bacterial RpoS gene copies can be observed (FIG.12). Therefore, the heterologous protein produced byL. lactiswould be protecting fish from infection ofF. psychrophilum.

When administratingL. lactis-IFNγ for a longer period of time (13 days instead of 7), it was observed higher survival (54%) than in fish fed for 7 days (42%), the same effect was seen in fish fed with a dosage 3 times higher than the original one (42%), which indicates that prolonging feeding gives a better result (FIG.13). Fish fed only withL. lactis(Treatment d)) presented a lower survival percentage (29%), very close to the 25% observed in fish fed with commercial feed (Treatment e)), behaviour also observed in the previous assay. The lower protection across different treatments could be due to the higher weight of fish (30-40 g versus 15-20 g), suggesting that the probiotic presents better properties in early stages of rainbow trout. When assessing weight or length of the surviving fish at the end of the experiment no significant differences were found (FIG.14), suggestion that the probiotic does not alter the physical state of fish.

The potential use of the probiotic inSalmo salarchallenged withPisciricketssia salmoniswas studied, fish fed withL. lactis-IFNg for 7 days (Treatment a: 28% survival) present a slight increase in protection compared to fish treated with the probiotic for 14 days (treatment b: 25% survival) suggesting that the protective effect againstP. salmonisis given when feeding fish for 7 days and then it is maintained constant. On the other hand, fish fed withL. lactiswith an empty vector (treatment c: 0% survival) presented total mortality, however, fish fed withL. lactiswith the plasmid without insert/commercial feed showed significant disparity in their mortality (Treatment d: 12% and 50% each replica). Therefore,L. lactis-IFNγ can contribute to the survival of fish infected withP. salmonis(FIG.15).

To determine ifL. lactispNZ8149 without insert has an effect on survival of fish challenged withP. salmonis, the experiment was repeated withSalmo salarspecimens (average weight: 50 g) infected and fed with probiotic (FIG.16), observing that their registered mortality was the same than for infected fish fed with commercial feed, which indicates that the bacteria per se does not confer protection.

Sequences<110>Consorcio Tecnológico de Sociedad Acuícola S.A. y Universidad de Santiago<120>Lactococcus Lactisbacteria transformed, RGM2416, producing Salmo salarinterferon gamma (IFNg), feed an composition that comprises it, to immunostimulateaquatic species and prevent infection ofF.psychrophilium,P.salmonisor both andmethos to obtain it.<160>5<210>1<211>779<212>ADN<400>1ccatggtata gatctaatta atctataaac catatccctc tttggaatca aaatttatta60tctactcctt tgtagatatg ttataataca agtatcaatg atctgggaga ccacaacggt120ttcccactag aaataatttt gtttaacttt agaaaggaga tatacgcatg aaaaaaaaga180ttatctcagc tattttaatg tctacagtga tactttctgc tgcagccccg ttgtcaggtg240tttacgctgc tcaatataca tcaattaata tgaaatcaaa tattgataaa cttaaagtac300attataaaat tagtaaagat caattgttta atggaaaacc agtttttcct aaagatacat360ttgaagattc agaacgtaga gtttggatgt ctgttgtatt agatgtatat cgttcaattt420ttaatcaaat gcttaatcaa acaggtgatc aagaagtacg tgaaagatta gatcaagtta480aaggaaaagt acaagaaact caaaaacatt attttcttaa acgaattcca gaattgagaa540cacatttgca aaatctttgg gctattgaaa ctagtaatac aactgttcaa ggaaaagcat600tgtcagaatt tattactatt tatgaaaaag cttctaaatt agcacttaaa attcatttaa660agaaagataa tcgacgtaaa agacgacaag cacaacgatt gaaaagtagt attatgggag720gtggacatca tcatcatcat cattaaaaaa aagtcttaaa ataataaaaa tagtctaga<210>2<211>161<212>ADN<400>2tatagatcta attaatctat aaaccatatc cctctttgga atcaaaattt attatctact60cctttgtaga tatgttataa tacaagtatc aatgatctgg gagaccacaa cggtttccca120ctagaaataa ttttgtttaa ctttagaaag gagatatacg c<210>3<211>81<212>ADN<400>3atgaaaaaaa agattatctc agctatttta atgtctacag tgatactttc tgctgcagcc60ccgttgtcag gtgtttacgc t<210>4<211>468<212>ADN<400>4gctcaatata catcaattaa tatgaaatca aatattgata aacttaaagt acattataaa60attagtaaag atcaattgtt taatggaaaa ccagtttttc ctaaagatac atttgaagat120tcagaacgta gagtttggat gtctgttgta ttagatgtat atcgttcaat ttttaatcaa180atgcttaatc aaacaggtga tcaagaagta cgtgaaagat tagatcaagt taaaggaaaa240gtacaagaaa ctcaaaaaca ttattttctt aaacgaattc cagaattgag aacacatttg300caaaatcttt gggctattga aactagtaat acaactgttc aaggaaaagc attgtcagaa360tttattacta tttatgaaaa agcttctaaa ttagcactta aaattcattt aaagaaagat420aatcgacgta aaagacgaca agcacaacga ttgaaaagta gtattatg<210>5<211>24<212>ADN<400>5ggaggtggac atcatcatca tcat