Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Effects of dietary myo-inositol on growth, antioxidative capacity, and nonspecific immunity in skin mucus of taimen Hucho taimen fry.

In this study, the effects of dietary myo-inositol on the skin mucosal immunity and growth of taimen (Hucho taimen) fry were determined. Triplicate groups of 500 fish (initial weight 5.58 ± 0.15 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, and 738.15 mg kg-1) until satiation for 56 days. Thereafter, the nonspecific skin mucus immune parameters, antioxidative capacity, and growth performance were measured. The skin mucus protein and the activities of alkaline phosphatase were significantly higher than those in the control group (P < 0.05). However, there were no significant differences in lysozyme activity among the treatments (P > 0.05). The antimicrobial activity and minimum inhibitory concentration of the skin mucus were increased significantly by myo-inositol supplementation (P < 0.05). The superoxide dismutase, catalase, and glutathione peroxidase activities were significantly elevated in the treatment groups (P < 0.05), whereas the malondialdehyde contents were significantly decreased (P < 0.05). Low-level myo-inositol (28.75 mg kg-1) led to a significantly lower weight gain, feed efficiency, condition factor, and survival rate compared with the other treatments (P < 0.05). In conclusion, dietary myo-inositol deficiency (28.75 mg kg-1) adversely affects the skin mucus immune parameters, antioxidative capacity, and growth performance of Hucho taimen fry.

The Infection Process of Yersinia ruckeri: Reviewing the Pieces of the Jigsaw Puzzle.

Finding the keys to understanding the infectious process of Yersinia ruckeri was not a priority for many years due to the prompt development of an effective biotype 1 vaccine which was used mainly in Europe and USA. However, the gradual emergence of outbreaks in vaccinated fish, which have been reported since 2003, has awakened interest in the mechanism of virulence in this pathogen. Thus, during the last two decades, a large number of studies have considerably enriched our knowledge of many aspects of the pathogen and its interaction with the host. By means of both conventional and a variety of novel strategies, such as cell GFP tagging, bioluminescence imaging and optical projection tomography, it has been possible to determine three putative Y. ruckeri infection routes, the main point of entry for the bacterium being the gill lamellae. Moreover, a wide range of potential virulence factors have been highlighted by specific gene mutagenesis strategies or genome-wide transposon/plasmid insertion-based screening approaches, such us in vivo expression technology (IVET) and signature tagged mutagenesis (STM). Finally, recent proteomic and whole genomic analyses have allowed many of the genes and systems that are potentially implicated in the organism's pathogenicity and its adaptation to the host environmental conditions to be elucidated. Altogether, these studies contribute to a better understanding of the infectious process of Y. ruckeri in fish, which is crucial for the development of more effective strategies for preventing or treating enteric redmouth disease (ERM).

Effect of quorum quenching bacteria on growth, virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout.

Five N-acyl homoserine lactone-degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo-C8-HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo-C8-HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 108  CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.

Modulation of rainbow trout (Oncorhynchus mykiss) cutaneous mucosal immune responses following anesthesia: A comparative study on different anesthetic agents.

The present study investigated the possible effects of different anesthetic agents including MS222 (50 ppm), 2-Phenoxyethanol (2-PE) (0.2 mL L-1) and clove oil (25 ppm), on cutaneous mucosal immune parameters in rainbow trout (Oncorhynchus mykiss). The induction and recovery times for each anesthetic agent were assessed. Also, the immune parameters were measured in skin mucus, 1 and 24 h post anesthesia. No significant difference was observed among treatments at 1 h post-anesthesia except for bactericidal and alkaline phosphatase (ALP) activities which was significantly enhanced in fish exposed to 2-PE compared to other anesthetics. At 24 h post-anesthesia, most of the skin mucosal immune parameters were increased upon exposure to clove oil but decreased following exposure to 2-PE. However, no significant change was noticed after MS222 exposure. These results demonstrated that the anesthetics type should be considered to avoid possible immunosuppression in farmed fish. Furthermore, the present results could be useful for better understanding of alterations in cutaneous mucosal immunity in response to chemical stressors such as anesthetic agents.

Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs.

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.

The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence.

BACKGROUND: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. RESULTS: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. CONCLUSIONS: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.

In vivo monitoring of Yersinia ruckeri in fish tissues: progression and virulence gene expression.

In this study, the utilization of bioluminescence imaging (BLI) allowed us to define the progression of Yersinia ruckeri during the infection of rainbow trout. A luminescent Y. ruckeri 150 strain was engineered using the pCS26-Pac plasmid containing the lux operon from Photorhabdus luminescens. Two different models of infection of rainbow trout were defined depending on the route in which bacteria were administered, being the gut the major organ affected following bath immersion. This indicates that this organ is important for bacterial dissemination inside the fish and the establishment of the infection. Moreover, the expression of three previously selected operons by in vivo expression technology (IVET) was analysed, the yhlBA involved in the production of a haemolysin, the cdsAB related to the uptake of cysteine and the yctCBA implicated in citrate uptake. Apart from these factors, the expression of yrp1 encoding a serralysin metalloprotease involved in pathogenesis was also analysed. The results indicated that all of the assayed promoters were expressed during infection of rainbow trout. In addition to these findings, the methodology described in this work constitutes a useful model for studying the infection process in other fish pathogenic bacteria.

The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence.

A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.

The ZnuABC operon is important for Yersinia ruckeri infections of rainbow trout, Oncorhynchus mykiss (Walbaum).

Signature-tagged mutagenesis was used to identify genes essential for survival of Yersinia ruckeri in its natural host, rainbow trout, Oncorhynchus mykiss. A mini-Tn5-Km2 signature-tagged mutant, C6-1, was missing from rainbow trout kidney at 7 days after an immersion challenge. The transposon insertion in C6-1 was in a homologue of the znuA gene of Escherichia coli that encodes ZnuA, a zinc-binding periplasmic protein of the high-affinity zinc transporter ZnuABC. Further sequencing of the C6-1 locus in Y. ruckeri identified homologues of two other genes: znuB, encoding a putative inner membrane permease, and znuC, encoding a putative ATPase. When present on a low-copy plasmid, the znuABC locus of Y. ruckeri fully restored growth of a zinc transport-deficient DeltaznuABC mutant of E. coli. Unlike DeltaznuABC mutants of E. coli and Salmonella typhimurium, the DeltaznuABC mutant of Y. ruckeri did not demonstrate significantly slower growth in zinc-deficient M9 minimal medium or in Luria-Bertani (LB) medium supplemented with the metal chelators EDTA and tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). In LB medium, the znuA::lacZ and znuCB::lacZ transcriptional fusions of Y. ruckeri were derepressed by addition of EDTA and TPEN and were repressed by addition of zinc and manganese. In a competitive challenge by immersion, the DeltaznuABC mutant was unable to compete with the parental strain and survived poorly in rainbow trout kidney, indicating that the ZnuABC transporter has a role in establishing and maintaining a rainbow trout infection by Y. ruckeri.