Isolation of Lactic Acid Bacteria (LAB) from Salmonids for Potential Use as Probiotics: In Vitro Assays and Toxicity Assessment of Salmo trutta Embryonated Eggs.

This research investigates the potential of lactic acid bacteria (LAB) from freshwater salmonids as prospective probiotics for application in aquaculture. LAB and pathogenic bacteria were obtained from mucus and tissues of Oncorhynchus mykiss and Salmo trutta from fish farms in northeast Spain that had not used antibiotics for the six months preceding the study. Isolates were identified using Gram staining and sequencing of 16S rRNA and ITS-1. To assess the safety of the LAB, antibiotic susceptibility tests (ASTs) against 23 antimicrobials were performed. In vitro antagonism assays were conducted to evaluate the inhibitory effects of living LAB using the agar diffusion test method and their metabolites using the agar well diffusion method. The assays targeted six specific pathogens: Aeromonas salmonicida subsp. salmonicida, Carnobacterium maltaromaticum, Vagococcus salmoninarum, Yersinia ruckeri, Lactococcus garvieae, and the marine pathogen Vibrio jasicida. Additionally, a toxicity assay was conducted on embryonic eggs of S. trutta. The ASTs on probiotic LAB candidates revealed varied responses to antimicrobials, but no resistance to oxytetracycline or florfenicol, which are two antibiotics commonly used in aquaculture, was detected. The in vitro assays indicate that LAB exhibit antagonistic effects against pathogens, primarily when directly stimulated by their presence. In applications involving embryonic eggs or larvae, certain live strains of LAB were found to have adverse effects, with some isolates resulting in higher mortality rates compared to the control group or other isolates. Furthermore, the potential pathogenicity of certain LAB strains, typically considered safe in salmonids, warrants deeper investigation.

Limited Probiotic Effect of Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 to Protect Rainbow Trout against Saprolegniosis.

Previous studies have demonstrated that the strains Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 are probiotics against vibriosis or lactococosis in sea bass or rainbow trout. In this study, the utility of these bacterial strains in the control of saprolegniosis was evaluated. For this purpose, both in vitro inhibition studies and competition for binding sites against Saprolegnia parasitica and in vivo tests with experimentally infected rainbow trout were carried out. In the in vitro tests, the three isolates showed inhibitory activity upon mycelium growth and cyst germination and reduced the adhesion of cysts to cutaneous mucus; however, this effect depended on the number of bacteria used and the incubation time. In the in vivo test, the bacteria were administered orally at 108 CFU g-1 in the feed or at 106 CFU ml-1 in the tank water for 14 days. None of the three bacteria showed protection against S. parasitica infection either through water or feed, and the cumulative mortality reached 100% within 14 days post infection. The obtained results show that the use of an effective probiotic against a certain disease in a host may not be effective against another pathogen or in another host and that the results obtained in vitro may not always predict the effects when used in vivo.

Lactiplantibacillus plantarum DSM20174 Attenuates the Progression of Non-Alcoholic Fatty Liver Disease by Modulating Gut Microbiota, Improving Metabolic Risk Factors, and Attenuating Adipose Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, reaching epidemic proportions worldwide. Targeting the gut-adipose tissue-liver axis by modulating the gut microbiota can be a promising therapeutic approach in NAFLD. Lactiplantibacillus plantarum, a potent lactic-acid-producing bacterium, has been shown to attenuate NAFLD. However, to our knowledge, the possible effect of the Lactiplantibacillus plantarum strain DSM20174 (L.p. DSM20174) on the gut-adipose tissue axis, diminishing inflammatory mediators as fuel for NAFLD progression, is still unknown. Using a NAFLD mouse model fed a high-fat, high-fructose (HFHF) diet for 10 weeks, we show that L.p DSM20174 supplementation of HFHF mice prevented weight gain, improved glucose and lipid homeostasis, and reduced white adipose inflammation and NAFLD progression. Furthermore, 16S rRNA gene sequencing of the faecal microbiota suggested that treatment of HFHF-fed mice with L.p DSM20174 changed the diversity and altered specific bacterial taxa at the levels of family, genus, and species in the gut microbiota. In conclusion, the beneficial effects of L.p DSM20174 in preventing fatty liver progression may be related to modulations in the composition and potential function of gut microbiota associated with lower metabolic risk factors and a reduced M1-like/M2-like ratio of macrophages and proinflammatory cytokine expression in white adipose tissue and liver.

Human Microbiota Network: Unveiling Potential Crosstalk between the Different Microbiota Ecosystems and Their Role in Health and Disease.

The human body is host to a large number of microorganisms which conform the human microbiota, that is known to play an important role in health and disease. Although most of the microorganisms that coexist with us are located in the gut, microbial cells present in other locations (like skin, respiratory tract, genitourinary tract, and the vaginal zone in women) also play a significant role regulating host health. The fact that there are different kinds of microbiota in different body areas does not mean they are independent. It is plausible that connection exist, and different studies have shown that the microbiota present in different zones of the human body has the capability of communicating through secondary metabolites. In this sense, dysbiosis in one body compartment may negatively affect distal areas and contribute to the development of diseases. Accordingly, it could be hypothesized that the whole set of microbial cells that inhabit the human body form a system, and the dialogue between the different host microbiotas may be a contributing factor for the susceptibility to developing diseased states. For this reason, the present review aims to integrate the available literature on the relationship between the different human microbiotas and understand how changes in the microbiota in one body region can influence other microbiota communities in a bidirectional process. The findings suggest that the different microbiotas may act in a coordinated way to decisively influence human well-being. This new integrative paradigm opens new insights in the microbiota field of research and its relationship with human health that should be taken into account in future studies.

Evaluation of three qPCR for the detection of pathogenic leptospires in domestic animals in Nicaragua / [Evaluación de tres PCR cuantitativas para la detección de leptospiras patógenas en animales domésticos en Nicaragua].

Introduction: Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals. Results: The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.8-86.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.

Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales. Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua. Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos. Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B. Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

Evaluación de tres PCR cuantitativas para la detección de leptospiras patógenas en animales domésticos en Nicaragua / Evaluation of three qPCR for the detection of pathogenic leptospires in domestic animals in Nicaragua

Resumen: Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales. Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua. Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos. Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B. Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

Abstract: Introduction: Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals. Results: The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.886.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.

Effect of a multi-citrus extract-based feed additive on the survival of rainbow trout (Oncorhynchus mykiss) following challenge with Lactococcus garvieae.

Growing global concerns about antibiotic resistance have generated a considerable interest in the search for alternative environmental-friendly approaches. This study was aimed to assess the antimicrobial activity of a multi-citrus extract-based feed additive (Biocitro®) against some fish pathogens, as well as evaluate its capacity to protect rainbow trout (Oncorhynchus mykiss) to lactococcosis. A broth dilution method was used to determine the minimum inhibitory concentration (MIC) of Biocitro®, and the results showed a strong antibacterial activity against Aeromonas salmonicida, Lactococcus garvieae and Yersinia ruckeri with MIC values of 2.0 µg/mL. Afterwards, rainbow trout juveniles were fed a Biocitro®-enriched diet (750 mg/kg feed) at a daily rate of 1.5% body weight for 4 weeks, then they were challenged with L. garvieae by the cohabitation method. At the end of the experimental period, fish treated with Biocitro® showed significantly (P < 0.001) improved protection against L. garvieae compared to control fish. Although further studies are needed to understand how Biocitro® increases rainbow trout resistance to L. garvieae, this feed additive could be considered as a useful alternative to chemotherapeutic treatment in aquaculture.

Effect of a novel postbiotic containing lactic acid bacteria on the intestinal microbiota and disease resistance of rainbow trout (Oncorhynchus mykiss).

OBJECTIVE: This study was aimed to assess the effect of a novel postbiotic on bacterial community composition and structure within the intestinal ecosystem of rainbow trout (Oncorhynchus mykiss), as well as evaluate its capacity to protect rainbow trout from Lactococcus garvieae infection. RESULTS: After 30 days of dietary postbiotic supplementation, high-throughput 16S rRNA gene sequencing revealed that bacterial community composition, diversity and richness were significantly higher in treated fish than in control fish. The proportion of sequences affiliated to the phylum Tenericutes, and to a lesser extent, the phyla Spirochaetes and Bacteroidetes was increased in fish fed a postbiotic-enriched diet compared to control fish, whereas the abundance of Fusobacteria was higher in control fish. Moreover, the treated fish showed significantly (p < 0.05) improved protection against L. garvieae compared to control fish. CONCLUSIONS: These findings suggest that dietary postbiotic supplementation may represent an environmentally friendly strategy for preventing and controlling diseases in aquaculture.

Phylogenetic analysis of intestinal microbiota reveals novel Mycoplasma phylotypes in salmonid species.

This study describes the bacterial community composition within the intestinal ecosystem of rainbow trout (Oncorhynchus mykiss) using high-throughput 16S rRNA gene sequence analysis. Sequences from intestinal samples from Chinook salmon (Oncorhynchus tshawytscha) farmed in New Zealand and rainbow trout farmed in Turkey were also included for comparative purposes. The results revealed that the most abundant operational taxonomic units (OTUs) were affiliated to the genus Mycoplasma, but were not specifically associated with any known species. Comparative analysis of 16S rRNA gene sequences indicated that these OTUs represent potentially novel species within the genus Mycoplasma.

Changes in intestinal microbiota and disease resistance following dietary postbiotic supplementation in rainbow trout (Oncorhynchus mykiss).

This experimental study was aimed to investigate whether the dietary supplementation of a postbiotic obtained as a food product fermented with two lactic acid bacteria could induce changes in the intestinal microbiota and prevent the development of Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss). After 30 days of dietary postbiotic supplementation, bacterial community composition and structure was significantly different between the treated and control groups. A higher bacterial diversity and richness in the intestinal samples was found in treated fish, as compared to those samples from untreated fish. Dietary postbiotic supplementation also conferred increased protection against L. garvieae infection. These findings suggest that the establishment of a beneficial microbiota is essential to prevent diseases or protect the host from foreign agents.

Leptospira spp in Rodents from Peridomestic Sites in Endemic Regions of Nicaragua.

Leptospirosis is one of the most important zoonoses in tropical countries, including Nicaragua, where it is considered endemic. The aim of this study was to determine the frequency of Leptospira spp in rodents captured from peridomestic sites in leptospirosis endemic regions of Nicaragua. Using live traps, 191 rodents were captured in 2012 and 2013 between April and December. Kidney samples were collected and processed for Leptospira detection from 166 animals by direct culture and isolation. The isolates were tested by PCR for LipL32 and lfb1-F genes specific to pathogenic Leptospira species. The trapping success over all sites was 20.2%, with higher rates of success in rainy season (p < 0.05). Leptospira spp were detected in 22.3% of rodents by direct culture methods. Significant differences (p < 0.01) were found in the frequencies of Leptospira positive rodents per month as well as per region. Of the isolated Leptospira spp, 37.5% were positive for pathogenic species by PCR. The frequency of Leptospira positive rodents by isolation could be used as a predictive indicator for the risk of human leptospirosis in Nicaragua.

Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges.

Although aquaculture activity has experienced a great development over the past three decades, infectious diseases have become a limiting factor for further intensification. Because the use of antibiotics has led to the widespread emergence of antibiotic resistance, the search for alternative environmentally friendly approaches is urgently needed. This Opinion paper offers an update on the successes and challenges of biological approaches for bacterial disease prevention and control in aquaculture. Although most of these approaches are still in research and development stages, some of them have shown promising results in field trials. Therefore, a better understanding of the mechanisms of action of these approaches will help to maximise their beneficial properties.

A cross-sectional epidemiological study of domestic animals related to human leptospirosis cases in Nicaragua.

Leptospirosis is one of the most extended zoonosis worldwide and humans become infected most commonly through contact with the urine of carrier animals, either directly or via contaminated water or soil. The aim in this study was to analyse the epidemiological behaviour of Leptospira spp., from domestic animals around the sites of human leptospirosis cases in Nicaragua, from 2007 through 2013. We report the results of a cross-sectional epidemiological study with a non-probability sampling of blood (n=3050) and urine (n=299) from Domestic Animals (DA) around the sites of human leptospirosis cases in Nicaragua. We analysed data obtained through Microscopic Agglutination Test (MAT), in-vitro culture, real time PCR and sequencing of lfb1 locus. Frequencies of 30.31% (95% CI: 28.66-31.95) and 15.38% (95% CI: 11.12-19.64) were obtained from serological test and from in-vitro culture, respectively. Although similar frequencies from serology test (P≥0.05) were found in DA species, in-vitro culture frequencies were significantly higher from bovine, equine and sheep (P<0.05) in comparison with swine and canine species. Ten serogroups of pathogenic Leptospira spp. were encountered, with the highest presence of Icterohaemorrhagiae serogroup 34.65% (95% CI: 29.35-39.94). We identified 7 samples homologous to L. interrogans species Pyrogenes serovar and 3 samples as L. noguchii Louisiana or Panama serovars by analysis of lfb1 sequences. We were able to establish a temporal and spatial correlation from DA and cumulative incidence of human cases. Therefore an effective epidemiological surveillance should be implemented with a specific control program toward DA in order to reduce human leptospirosis incidence.

Isolation, characterization and evaluation of probiotic lactic acid bacteria for potential use in animal production.

In livestock production, lactic acid bacteria (LAB) are the most common microorganisms used as probiotics. For such use, these bacteria must be correctly identified and characterized to ensure their safety and efficiency. In the present study, LAB were isolated from broiler excreta, where a fermentation process was used. Nine among sixteen isolates were identified by biochemical and molecular (sequencing of the 16S rRNA gene) methods as Lactobacillus crispatus (n=1), Lactobacillus pentosus (n=1), Weissella cibaria (n=1), Pediococcus pentosaceus (n=2) and Enterococcus hirae (n=4). Subsequently, these bacteria were characterized for their growth capabilities, lactic acid production, acidic pH and bile salts tolerance, cell surface hydrophobicity, antimicrobial susceptibility and antagonistic activity. Lactobacillus pentosus strain LB-31, which showed the best characteristics, was selected for further analysis. This strain was administered to broilers and showed the ability of modulating the immune response and producing beneficial effects on morpho-physiological, productive and health indicators of the animals.

Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae.

Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p < 0.01) the mortality (20 %) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50 %) and the control (72.5 %). We demonstrated the effectiveness of L. cremoris WA2-67 to protect rainbow trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.

Identification and in vitro screening of avian yeasts for use as probiotic.

UNLABELLED: The objective of this study was to isolate and identify yeast strains from broilers excreta and to evaluate in vitro their potential for probiotic use in animal production. METHODS AND RESULTS: Nine yeast strains were isolated and presumptively pre-identified by biochemical assays. These isolates were grouped in six different molecular profiles using PCR-fingerprinting technique. Each profile was identified by sequencing of the D1/D2 domains of the large subunit of the 26S rRNA gene. These yeasts were identified as: Trichosporon sp. (LV-2), Wickerhamomyces anomalus (LV-6), Pichia kudriavzevii (LV-8), Kodamaea ohmeri (LV-9) and Trichosporon asahii (LV-10). A pre-screening of the strains for probiotic use was based on their ability to agglutinate pathogenic micro-organisms. These yeast strains were characterized for specific growth rate, duplication time, their cell surface hydrophobicity, medium acidification, resistance to low pH (2.0, 2.5 and 3.0) and concentrations of bile salts (0.3% and 0.6%). The isolate of W. anomalus (LV-6) had the highest agglutinating and adherence capacity, a growth rate of 2.07×10(8) cfu/mL in 24 h at 30 °C, decreasing the medium pH from 6.5 to 5.23, a 25% hydrophobicity and an elevated capacity to grow under stress conditions. CONCLUSIONS: W. anomalus strain LV-6 showed the best characteristics for use as a probiotic candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study helped in choosing a probiotic candidate from yeast to use in broiler production.

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection.

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 106 CFU g⁻¹ feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1ß, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1ß, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.