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Lactococcosis is one of the main bacterial diseases affecting rainbow trout (Oncorhynchus mykiss), with significant economic and sanitary repercussion. Vaccination and antibiotic treatments are commonly used to prevent and control the infection outbreaks; however, these strategies have some drawbacks including limited coverage, handling costs, induction of antibiotic resistance and chemical residues in the environment. Selective breeding programs represent a promising complementary approach for increasing fish disease resistance in commercial farms and some immunological parameters may be tentatively used as indirect indicators for this purpose. The present study investigated for the first time some innate and adaptive immune responses in two groups of rainbow trout derived from selected lines (susceptible and resistant) showing a different “in field” phenotypical resistance to Yersinia ruckeri, Flavobacterium branchiophilum, F. psychrophilum, and Ichthyophthirius multifiliis, after an immersion-dilution based exposure to Lactococcus garvieae carried out in controlled experimental conditions. Twenty-six resistant and twenty-six susceptible female rainbow trout (mean body weight 80 g, 9 months aged, F5 generation) were obtained from an intensive farm considered L. garvieae free and were exposed to the pathogen. Moreover, 10 resistant and 10 susceptible fish were used as uninfected controls. After 5 days, blood and tissue samples were collected for immunological analyses. A significantly higher serum and mucus lysozyme activity was recorded in resistant rainbow trout compared to susceptible fish (P ≤ 0.05), both before and after exposure to L. garvieae. Similarly, respiratory burst activity of head kidney leukocytes resulted more intense in resistant fish (P ≤ 0.05), suggesting that phagocytes could more quickly activate their microbicidal mechanisms to counteract the bacterial spread. Resistant group displayed also an up-regulation of immunoglobulins M (IgM), major histocompatibility complex II (MHC-II) and interleukin 8 (IL-8) gene expression (P ≤ 0.05) and a significantly higher blood lymphocytes count (P ≤ 0.05), highlighting their potential better ability to trigger the recruitment of defensive cells and the initiation of specific immune processes such as antigen presentation to CD4+ T lymphocytes and IgM synthesis. The results herein presented might be useful for the identification of immunological markers to be used as indirect indicators in rainbow trout selective breeding programs.

Summary Lactococcus garvieae is a well-known fish pathogen, and in recent years, a human pathogen of increasing clinical significance. However, not much is known about the variances in characteristics of strains isolated locally and overseas. This study aims at conducting comparative genomic analysis on local and overseas L. garvieae isolates, to further understand the phylogenetic and virulence variances between the two groups. The genomic DNA of 11 local L. garvieae isolates (fish 6, human 5) were sequenced, annotated and typed using multi-locus sequence typing (MLST). A total of five novel sequence types (STs) were found in the local isolates. Genotypic overlapping of the STs was observed between local fish and human isolates with overseas fish, food and human clinical isolates. Thereby, suggesting a possible transmission between fish or food and humans. Virulence genes (putative internalin and putative mucus adhesin) were found to be specific to genomic clusters (GC), GC2 and GC3. A higher incidence of resistance genes was also observed in local isolates (n=8, 72.72%) when compared to the overseas isolates (n=7, 41.18%). This study represents the first evidence of genetic variances amongst local and overseas isolates, and virulence characteristics specific to the phylogeny of L. garvieae.

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G_DYNAMIN_2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish.

Nowadays, there is no suitable treatment for vibriosis in groupers. So an eco-efficient and environmentally friendly treatment is necessary for the grouper industry. Probiotic-feeding has been a promising strategy to control the bacterial pathogens in aquaculture. A new Bacillus velezensis strain named K2 was isolated from the intestinal tract of healthy grouper, and exhibited wide antimicrobial spectrum of against fish pathogens, including Vibrio harveyi, Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas veronii, Aeromonas caviae, Enterococcus casseliflavus and Lactococcus garvieae. Moreover, results of the safety of B. velezensis K2 showed that intraperitoneal injection of K2 in healthy grouper did not cause any pathological abnormality or death, indicating this bacteria could be considered as a candidate probiotic in aquaculture. Groupers were fed with the diets containing 1 × 107 cfu/g of B. velezensis K2 for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that diets supplemented with K2 significantly increased serum acid phosphatase (ACP) activity (P

The effectiveness of ionotropic gelation method (by combining alginate and chitosan) vaccine against Lactococcus garvieae and Streptococcus iniae was examined in rainbow trout. Fish were separated into four groups and fed the distinctive examined feeds. Our groups were included: A) fish immunized by chitosan-alginate coated vaccine, B) fish immunized by non-coated vaccine, C) fish feed by chitosan-alginate coated pellets without vaccine and D) fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days. Fish of group C, like groups A and B were fed 14 days with pellets covered with chitosan-alginate without vaccine and a short time later they were fed with control diet. On day 0, 20, 40 and 60 of the trial, serum samples were extracted. Fish were challenged with L. garvieae and S. iniae after 60 days of research. Innate immunity components containing complement activity, total protein and IgM appeared no significant changes nearly in all groups during the 60 days that the examination finished. Although, bactericidal activity and lysozyme activity demonstrated a significant increase on days 20, 40 and 60 in group A compared to control groups (C and D) (P

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses.

Lactococcus garvieae, Yersinia ruckeri and Flavobacterium psychrophilum are three of the most important pathogens in worldwide rainbow trout aquaculture (Oncorrhynchus mykiss Walbaum). In this work, a multiplex quantitative PCR (qPCR) method for the simultaneous detection of the three pathogens was developed and tested in a commercial trout farm. Fifty-four spleen and brain samples from a trout farm were analyzed using both multiplex qPCR and classic microbiological methods. When comparing qPCR results and classic plate diagnostic techniques no negative deviations were observed, which resulted in a 100% relative sensitivity of the multiplex qPCR method developed. In addition, efficiency of the qPCR method ranged between 97.5% and 108.8%. The specificity of the combination of primers and probes was successfully tested in 57 target and non-target bacterial strains Hence, the multiplex qPCR method developed in the present study could be used as a reliable diagnostic tool for the detection of L. garvieae, Y. ruckeri and F. psychrophilum in rainbow trout farms, enabling faster diagnostics (only a few hours) and contributing to a quick response and management of bacterial disease outbreaks.

The efficacy of a Eudragit L30D-55 encapsulated vaccine against Lactococcus garvieae and Streptococcus iniae was investigated in rainbow trout. Fish were divided into four groups and fed the different experimental feeds. Groups were: A) fish immunized by Eudragit-coated pellets containing vaccine, B) fish immunized by vaccine-coated pellets without Eudragit, C) fish fed Eudragit-coated pellets without vaccine and D) fish fed pellets without vaccine orEudragit (control group). In groups A and B, the vaccination was conducted for 14 days. Similar to groups A and B, fish of group C were fed 14 days with pellets coated with Eudragit and afterwards they were fed control diet. Serum samples were taken on day 0, 20, 40 and 60 of the experiment. After 60 days, fish were challenged with L. garvieae and S. iniae. In almost all groups, innate immunity components including alternative complement activity, lysozyme activity, bactericidal activity, IgM and total protein showed no significant changes during the 60 days that the experiment lasted. However, the blood respiratory burst activity and lysozyme activity showed a significant increase on day 20 of experiment in groups B and D respectively (P 0.05). The relative expression of immune-related genes including IL-6 and IgM genes was higher in vaccinated fish, with the highest expression in those immunized by Eudragit-coated pellets (Group A). In addition, the relative expression of IL-6 and IgM peaked on day 20 but decreased on day 60 in vaccinated groups. The ELISA antibody titer against L. garvieae increased from day 20 and peaked on day 60 of experiment (P 0.05). Also, the antibody titer against L. garvieae was higher in fish immunized by Eudragit-coated pellets (Group A) compared to fish of group C and control. After bacterial challenge, a survival percentages of % 85 ± 7.07% (challenged with S. iniae) and % 72.21 ± 7.8% (challenged with L. garvieae) were observed respectively in groups immunized with pellets coated with Eudragit L30D-55 (Group A), which were higher than survival percentages obtained in other experimental groups (P 0.05). The results of the present study demonstrate that the oral administration of Eudragit L30D-55-encapsulated vaccine appropriately protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

The purpose of this article is to study the effect of the probiotic on experimental infections of carp's fingerlings with Lactococcus garvieae. Lactic acid bacteria (LAB) (Lactobacillus fermentum 24с, Pediococcus pentosaceus 10/9к, Lactobacillus paracasei 9c) for the probiotic were previously isolated from the intestines of mature carps from Maybalyk commercial fisheries, which provided fingerlings for this experiment too as well. The feed-contained probiotic was given to fish in the experimental group for 14 days before challenge with pathogen L. garvieae. Throughout ten days after the infection, death of the fish was regularly recorded in the group, where the probiotic was not given with the feeding. Ten days after, all fish in this group died. In the probiotic group, the mortality on the tenth day after the challenge with pathogen was 10%. It was concluded the effect of the probiotic is not due to antibacterial action to the pathogen. The effectiveness of the probiotic can be associated with the displacement of the pathogen, due to the competitive adhesion and/or more likely, with the activation of the immune response from the fish organism due to the addition of the probiotic to the feed.

This study reports the characterization of a collection of 98 strains of L. garvieae using phenotypic, antigenic, genetic, proteomic, and chemotaxonomic methods, as well as the study of their virulence. Molecular (RAPD and REP-PCR) and proteomic (MALDI-TOF-MS) techniques allowed the separation of strains of L. garvieae isolated from human and bovine from those strains of L. garvieae isolated from the aquatic environment. On the other hand, PCR typing methods showed that the L. garvieae strains tested did not belong to serotype II and did not carry the capsule gene cluster described in the genome or plasmid of Japanese L. garvieae strains. The different clonal patterns observed in L. garvieae using RAPD and REP-PCR (7 profiles) and MALDI-TOF (six patterns) were closely related to the isolation source of L. garvieae strains but not with the virulence of the strains. In addition, two protein antigenic variants (W1 and W2), differing in reactivity in the range of 35 to 70 KDa, were detected using rabbit and rainbow trout antisera. Both antisera detected two highly immunogenic protein bands of 30 and 45 KDa. These results could contribute to a better understanding of the pathogenesis of lactococcosis and could help in the selection of targets for the development of effective vaccines.

The microbiota of the intestine produces a wide array of biologically active molecules and together act as a composite endocrine organ. Due to our limited understanding of bacterial communities in aquaculture ecosystems, it is necessary to evaluate the interactions between environmental and intestinal microbiota and the potential consequences of disease. This study taken the traditional P. clarkii culture in the Sichuan Basin as an example, and analyzed the relationships between the microbiota of the environment and host through microbial analysis and microbiological diagnosis. Our results showed that the bacterial abundance in sediment was greater than in water, followed by the intestine, and some of bacteria from the environment successfully selected to colonize the intestine. The bacterial composition in the intestines of diseased and healthy crayfish was significantly different. The bacteria that colonized and proliferated in the intestine had very low abundances in sediment and water. Two potential pathogens, Aeromonas veronii, and Citrobacter freundii, and two potential probiotics, Lactococcus garvieae and Exiguobacterium undae, were identified. Using multiple, real, and traditional P. clarkii aquaculture sites in the Sichuan Basin, this study revealed that the microbial communities of the environment and animal host did indeed interact. Furthermore, these results indicated that P. clarkii in a healthy status are capable of regulating which bacteria colonize their intestines.

The swimming crab Portunus trituberculatus is one of the most popular seafoods in South Korea. Despite yearly reports of foodborne illness caused by swimming crab consumption, the microbiota in the whole body of the crab has not been fully characterized. A total of 60 crabs were collected from different sites in the Yellow Sea in the spring and autumn, and the crab microbiota was analyzed by 16S rRNA pyrosequencing. Crab microbial diversity was higher in the autumn than in the spring. Psychrobacter, Vagococcus, Carnobacterium, Lactococcus, and Streptococcus were dominant genera in the spring, whereas Roseovarius and various other genera were dominant in the autumn. The composition of the microbiota differed significantly between spring and autumn (p Lactococcus garvieae, in crabs were higher in spring than in autumn. Thus, the potential risk of foodborne illness by crab intake could be higher in spring than autumn. This study extends our knowledge of potential foodborne illness according to microbiota and may improve the management of crab products.

The lactic acid bacteria (LAB) prevalence and dynamics of goatskin Tulum cheese produced in mountainous region of Karaman province of Turkey throughout 180 days of ripening were investigated using culture-dependent and -independent (PCR-DGGE) methods. This study used the V3-region of the 16S rRNA gene amplification and sequencing to characterise the LAB community of this cheese. Microbial counts were variable throughout the ripening. In the culture-dependent method, Enterococcus faecium shared dominance with Enterococcus hirae species at the end of the ripening, followed by Lactobacillus plantarum species. On the other hand, Streptococcus strains were predominant at the initial stage of the ripening, while E. faecium strains were identified, as well as Enterococcus faecalis and Lactococcus garvieae strains, using molecular techniques at the end of ripening. It was inferred that using these two methods together gave more information about the biodiversity and dynamics of LAB in artisanal goatskin Tulum cheeses during ripening.

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods.

We describe using a polyphasic approach that combines proteomic by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis, genomic data and phenotypic characterization the features of Lactococcus garvieae strain M14 newly isolated from the fermented milk (known as raib) of an Algerian cow. The 2188835 bp containing genome sequence displays a metabolic capacity to form acid fermentation that is very useful for industrial applications and encodes for two bacteriocins responsible for its eventual bioprotective properties.

This research was conducted to determine the effects of Lentinula edodes (L. edodes) medicinal mushroom extract as a trout feed supplement on the immune response and disease resistance against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss). For this purpose, the fish were fed with 1% and 2% L. edodes mushroom extract supplemented diet for six weeks. During the trial, blood samples were taken from 10 fish/group each week up to 6 weeks. Furthermore, at the end of the trial, the fish were challenged with L. garvieae pathogen by intraperitoneal injection. Upon the evaluation of the results, feeding the rainbow trout with mushroom extract supplemented diet showed a significant influence on immunological parameters. The parameters were found to be higher in fish fed with the experimental diet than the control group. Statistically significant levels of serum total immunoglobulin were detected only in the fish group fed with 2% L. edodes mushroom extract supplemented diet. The maximum influence on immune response occurred in rainbow trout fed with 2% L. edodes extract. As a result of the challenge test, the survival rate was found to be the highest in the 2% L. edodes supplemented feeding group. The results suggested that a diet supplemented with L. edodes mushroom extract enhanced the immune response and decreased the mortality rate of rainbow trout against L. garvieae. Statement of relevance This study determined the effects of an edible mushroom extract on the immune system and resistance to disease in rainbow trout. The use of mushroom extract in fish diet protected rainbow trout partially from lactococcosis disease, increased their survival rate, and also enhanced their immune response. This experiment was performed in a commercial trout farm at the region therefore the application was found to be suitable for aquacultural fields.

Lactococcus garvieae is one of the emerging zoonotic bacterial pathogen, causes fatal hemorrhagic septicemia in cultured fish species, animals and humans, worldwide. Here, we report the genomic features of whole-genome sequence (WGS) of L. garvieae strain RTCLI04, recovered from lower intestine of farmed rainbow trout, Oncorhynchus mykiss in the northwest Himalayan region India. The genome of L. garvieae RTCLI04 is a single circular chromosome of 2,054,885 base pairs (bp), which encodes 1993 proteins and has G + C content of 39%. The bioinformatics analysis of WGS of RTCLI04, confirmed the presence of 51 tRNAs genes (including two pseudogenes), six rRNAs genes (four genes for 5S rRNA; one gene for 16S rRNA and one gene for 23S rRNA), five virulent domains, and twenty eight different genetic pathways. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) finder tool indicates that three different CRISPR and one cas system with common spacer was present in the genome of L. garvieae RTCLI04. Pan-genome analysis of RTCLI04 and all the other reference L. garvieae strains shows that pan-genome of this bacterium consisted of 2239 putative protein-coding genes in which 1850 genes are core gene, 389 genes are dispensable gene, and 221 genes are unique to RTCLI04. L. garvieae RTCLI04 lacks genomic island of 16.5 Kb capsule gene cluster. In addition, 39 virulence-associated genes (VAGs) including hly1,-2,-3; PavA, PsaA; eno; LPxTG containing surface proteins 1, 2, 3 and 4; pgm, sod and 29 antimicrobial resistant genes (ARGs) including mefE (clindamycin), srmB (lincomycin), dfrA26 (trimethoprim), gyrB (nalidixic acid), arr-3 (rifampin), otrB (tetracycline), aac(6)-Ic (tobramycin), IrgB (penicillin), mecA (oxacillin), vanRB (vancomycin) and mfpA (fluoroquinolone) were also predicted in the genome of L. garvieae RTCLI04. Our study provides new insight into understanding the virulence mechanism, antimicrobial resistance, and development of effective therapeutic measures against L. garvieae during a disease outbreak in aquaculture.

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.

Infective endocarditis (IE) is a known complication of heart valve disorders. There is no consensus on whether bioprosthetic valves are associated with more episodes of IE in comparison to mechanical valves. Here, we present IE with a rare organism in a patient with bioprosthetic mitral valve. A 71

To date, a number of bacteriophages that infect Lactococcus garvieae isolated from marine fish have been identified. However, the evolutionary insight between L. garvieae phages and other viral community have not yet been immersedly investigated. In this study, completed genomic sequence of phage PLgY-30 was obtained, a comparative analysis of three lytic phages, which have been using for phage typing and treatment of L. garvieae infecting marine fish, is conducted. The results revealed that the genomes of lytic phages specific for L. garvieae isolated from diseased marine fish share a high level of homology and almost all proteins are conserved. At genome level, no similarity was detected for either PLgY-30 or PLgY-16, while PLgW-1 shares only very limited homology (1%) with other sequences in Genbank database. In addition, the function of only 35% of ORFs in the PLgY-30 phage genomes could be predicted, demonstrating that it is novel phage. At protein level, lytic phage proteins shared a significant similarity to various proteins of global phage species isolated from dairy fermentation facilities that utilize L. lactis as a primary starter culture, called the 936 phage group. Genome organization and architecture of three lytic phages are also similar to that of the 936 phage group. To our knowledge, this is the first time lytic bacteriophages infecting L. garvieae from marine fish were characterized to genome level.

The present study was undertaken to investigate the microbial quality of raw milk from dairy farms in Kosovo with emphasis on the potential pathogen Lactococcus garvieae . Based on selective media and growth profiles approximately 770 isolates were obtained from raw milk collected from 221 farms and 119 of these isolates were shown to belong to L . garviae based on 16S rRNA gene sequencing. The 119 L . garvieae isolates were derived from 43 farms. It also appeared that L . garviae was established as a persistent bacterium in these farms since it was found in most samples collected over a period of 6 months. The L . garvieae -containing farms were not geographically related as isolates of this bacterium were found across the country. Genetic diversity assessed by rep-PCR fingerprinting revealed 8 major groups and the largest group contained isolates from 13 farms. Isolates within the individual groups 1, 2, 3, 4, and 5 showed strong genetic relatedness while the isolates from the remaining groups 6, 7 and 8, did not give a clear picture of relatedness. Twenty one isolates showed weak alpha haemolytic activity and antibiotic resistance was demonstrated in 39 of the 43 isolates. Diversity in the fermentation profiles was also observed among the 43 L . garvieae isolates that could be divided into 5 separate fermentation groups.

Lactococcosis caused by Lactococcus garvieae is known as one of the serious economical bacterial diseases in aquaculture species of both freshwater and marine fish at a wide range of water temperature mainly from 13 °C to 37 °C. The PavA gene and Hly gene, the genes responsible for encoding fibronectin and hemolysin proteins, respectively are the well known virulent factors of L. garvieae . The effect of essential oil of Zataria multiflora was evaluated on expression of these genes in two virulent isolates of L. garvieae isolates recovered from diseased rainbow trout. Fresh cultures of the bacterial isolates were exposed to 1/2–1/16 (0.12–0.015 μl/ml) of the minimum inhibitory concentrations (MIC) of Z. multiflora essential oil and the expression of PavA and Hly genes was assessed by real time PCR works. The results showed that under MIC concentration (0.12 μl/ml) of the essential oil, the expression of both Hly and PavA genes was significantly suppressed in both bacterial isolates (P Z. multiflora in forms of oral and/or bath administration to reduce morbidity and mortality by Lactococcus garvieae in fish via the suppression of expression of these virulent genes.

In the present study, Macrobrachium rosenbergii were fed with diets containing extracts of banana, Musa acuminate , fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg −1 . The non-specific immune parameters, disease resistance and anti-hypothermal stress were evaluated at 2, 4, 8, 16 and 32 days of post feeding. Also, we demonstrated the percent weight gain (PWG), percent length gain (PLG), feeding efficiency (FE), and survival rate of giant freshwater prawn at 30, 60, 90, and 120 days of post feeding. The PWG, PLG, FE and survival rate of prawns fed at 0, 1.0, 3.0 and 6.0 g kg −1 BPE-containing diets after 120 days were 69.5%, 75.4%, 77.8% and 83.3%; 21.8%, 23.6%, 27.8% and 33.9%; 0.60, 0.72, 0.75 and 0.90; and 55.4%, 62.2%, 62.3% and 75.3%, respectively. After 32 days of post feeding, a significant increase in total haemocyte count (THC), different haemocyte count (DHC), respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, phenoloxidase (PO) activity and transglutaminase (TG) activity, and meanwhile, a decreased haemolymph coagulation time was observed. Furthermore, phagocytic activity and clearance efficiency of prawns against Lactococcus garvieae infection were significantly increased. Prawns challenged with L. garvieae after 32 days of feeding at 1.0, 3.0 and 6.0 g kg −1 had a significantly higher survival rate (33.3%, 40.0% and 56.7%) than those fed with the control diet. Subsequently, hypothermal (14 °C) stress was 43.4%, 50.0% and 50.0%, respectively. Altogether, we therefore recommend the dietary BPE administration at 6.0 g kg −1 promotes growth, anti-hypothermal stress, and enhance immunity and resistance against L. garvieae in M. rosenbergii .

Bacteriocin-producing Lactococcus garvieae LG34 was isolated from traditional Chinese fermented cucumber. Garviecin LG34 was a Class IIa bacteriocin consisting of 46 amino acid residues. No identical sequence with garviecin LG3 was obtained from the Database of NCBI. Garviecin LG34 exhibited inhibitory activity against not only Gram-positive bacteria but also Gram-negative bacteria. Garviecin LG34 showed resistance to heat and low pH, and was sensitive to proteolytic enzymes but not to lipase and amylase. These results indicated that garviecin LG34 was a novel Class IIa bacteriocin with a broad inhibitory spectrum produced by Lactococcus garvieae , and had the potential to be used as food biopreservative.

Insertion sequences are the simplest intracellular Mobile Genetic Elements which can occur in very high numbers in prokaryotic genomes, where they play an important evolutionary role by promoting genome plasticity. As such, the studies on the diversity and distribution of insertion sequences in genomes not yet investigated can contribute to improve the knowledge on a bacterial species and to identify new transposable elements. The present work describes the occurrence of insertion sequences in Lactococcus garvieae, an opportunistic emerging zoonotic and human pathogen, also associated with different food matrices. To date, no insertion elements have been described for L. garvieae in the IS element database. The analysis of the twelve published L. garvieae genomes identified 15 distinct insertion sequences that are members of the IS3, IS982, IS6, IS21 and IS256 families, including five new elements. Most of the insertion sequences in L. garvieae show substantial homology to the Lactococcus lactis elements, suggesting the movement of IS between these two species phylogenetically closely related. ISLL6 elements belonging to IS3 family were most abundant, with several copies distributed in 9 of the 12 genomes analyzed. An alignment analysis of two complete genomes carrying multi-copies of this insertion sequence indicates a possible involvement of ISLL6 in chromosomal rearrangement.

The virulent factor of Lactococcus garvieae contains a specific capsule gene cluster including epsD. Some herbal essential oils have antibacterial activities and could negatively affect the virulence of pathogenic bacteria. In this study, 15 strains of L. garvieae were recovered from diseased rainbow trout, and were identified by molecular works. The encapsulated strains were then detected using both staining and molecular studies. The effect of Shirazi thyme ( Zataria multiflora ) essential oil was then assessed on the expression of the encapsulated positive epsD gene strains with a higher genetic diversity. The phylogenic features of the five identified encapsulated positive epsD gene categorized them into a group, two clusters and three genogroups. Also, the obtained results showed that from the five encapsulated positive epsD strains, only two strains showed a higher genetic diversity. The antibacterial activity of essential oils of Shirazi thyme ( Z. multiflora ), Cinnamon ( Cinnamomum zeylanicum ) and Garlic ( Allium sativum ) to these two encapsulated positive epsD gene strains revealed the highest inhibition effect by Z. multiflora with a minimum inhibitory concentration (MIC) of 0.12 μl/ml. Subinhibitory concentrations of 1/2, 1/4, 1/8 and 1/16 MICs of Z. multiflora on the epsD gene production showed a suppression effect in reducing this gene production. Therefore, this essential oil could be advantageous for the control of lactococcosis in aquaculture.

This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

Lactococcosis is prevalent on grey mullet (Mugil cephalus) in Hong Kong aquaculture resulting in serious economic loss. A compound formulation of Traditional Chinese Medicines (TCM) (modified Huanglian Jiedu decoction (HLJDD)) comprising Rhizoma coptidis, Radix scutellaria, Cortex phellodendri, Fructus gardeniae, Fructus forsythiae and Flos lonicerae japonicae (in a ratio of 3:2:2:3:3:5) were applied as feed supplements to deal with the disease. The Nitroblue tetrazolium activity in blood, bactericidal activity and total immunoglobulin in plasma were significantly enhanced after feeding 1% of this TCM for 28 days. The disease resistances to Lactococcus garvieae in 1% and 2% TCM feeding groups were significantly enhanced. In the in vitro study, the modified HLJDD also activated the plasma bactericidal activities (p

Environmental change is contributing to change the pattern of diseases in aquatic settings. In this study we have analyzed the disease outbreaks that occurred in North Spanish Salmonid hatcheries during the last two decades. Six pathogens were considered: Aeromonas hydrophila, Aeromonas salmonicida, Flavobacterium psychrophilum, Lactococcus garvieae, infectious pancreatic necrosis virus and Yersinia ruckeri. Vaccines against Lactococcus and Yersinia are currently applied in the region. A shift in the dominant pathogen from Y. ruckeri to F. psychrophilum occurred after the year 2000, coincident with application of vaccines and climate warming in the region. Positive association of vaccines against some pathogens and prevalence of other pathogens was found, as well as a significant change in the temperature range at which outbreaks occurred. Both environmental perturbations and opportunistic development of pathogens when others are controlled by vaccines contribute to explain the stability of disease outbreaks in the region despite the introduction of vaccines.

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Five diets were formulated with 0% (control), 4% (D4), 8% (D8), 12% (D12), and 16% (D16) Tenebrio molitor protein in prepared aquafeed to investigate the effects of T. molitor protein on growth performance, immunological parameters, and resistance against Lactococcus garvieae and Aeromonas hydrophila in Macrobrachium rosenbergii for 10 weeks. The weight gain (WG), percentage of weight gain (PWG), daily growth rate (DGR), specific growth rate (SGR), and protein efficiency ratio (PER) of D12 were significantly higher than those of the control, D4, and D8, but not significantly different from those of D16. The condition factor (K), feed conversion ratio (FCR), and survival rate (SR) did not differ significantly among groups. Lipid content decreased and protein content increased in the carcass and muscle of prawns as T. molitor protein contents increased. The total haemocyte count (THC) and numbers of semigranular cells (SCs), granular cells (GCs), and hyaline cells (HCs) significantly increased from the control to D16. Increased activities of superoxide dismutase (SOD), phenoloxidase (PO), lysozyme (LZ), and alkaline phosphatase (ALP) were detected as T. molitor protein contents increased. mRNA expression of prophenoloxidase (proPO), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), and α2-macroglobulin (α2-M) increased in haemocytes of M. rosenbergii from the control to D16. Phagocytosis of L. garvieae and the relative survival rate (RPS) against A. hydrophila were positively correlated with activities of protective enzymes and expression of proPO system immune-related genes in prawns fed diets with T. molitor protein. These results demonstrated that dietary inclusion of 12% T. molitor protein had the best effects on growth performance, immunological parameters, and resistance against L. garvieae and A. hydrophila, when balanced against the additional feed cost. This study supports the inclusion of T. molitor protein in functional aquafeed.

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α(+) T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α(+) T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1-2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α(+) T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α(+) T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4(+) T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM(+) cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α(+), CD4(+) T-cells and surface IgM(+) cells in fish.

Eight hundred lactic acid bacteria (LAB) strains isolated from golka, one of the oldest and most popular regional cheese produced in Poland, were screened for anti- Listeria activity. Nearly one third of isolates showed antagonistic activity against Listeria monocytogenes. Seven strains produced other substances active against L. monocytogenes. These substances were detected in neutralized cell-free supernatant fluids and were classified as bacteriocins. 16S rDNA identification of the selected bacteriocin-producing bacteria was performed. By using primers specific to a fragment of class IIa bacteriocin-coding genes, developed by the authors, presence of the bacteriocin structural gene was confirmed for all Lactococcus garvieae strains, Leuconostoc mesenteroides 453Lab and Lactobacillus plantarum Lab572. Presence of the class IIa bacteriocin-coding genes within genomes of non-bacteriocin-producing strains Lactococcus lactis subsp. lactis was also detected. Bacteriocins with the highest activity against Listeria, i.e . bacteriocins of L. garvieae Lab428, Lc. mesenteroides Lab25 and Lb. plantarum Lab572 strains, were partially characterized. The bacteriocins did not adhere to surface of the producer cells and exhibit a bactericidal mode of action. Bacteriocin of L. garvieae 428Lab was produced at much higher levels (12.800 AU/ml), as compared to the other bacteriocins (3.200 AU/ml). Our results indicate that golka cheese is a rich source of LAB active against L. monocytogenes . The bacteriocinogenic isolates from golka described in this work may potentially find application as starter or protective cultures and could constitute a superb genetic pool for production of new bacteriocins.

Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.

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-1β, 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-1β, 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.

In this study, we determined the effects of norepinephrine (NE) on immunity and the pathway of its function in the freshwater giant prawn, Macrobrachium rosenbergii. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Lactococcus garvieae, were measured when the freshwater giant prawn, M. rosenbergii (16.2±2.1 g) was individually injected with saline or NE at 0.5, 5.0, and 50.0 pmol prawn(-1). Results showed that semi-granular cells, respiratory bursts and phagocytic activity at 2 h, PO activity and clearance efficiency from 2 to 4 h, THC at 8 h, and SOD activity from 4 to 8 h significantly decreased, but hyaline cells at 2 h, and respiratory bursts at 8 h had significantly increased after injection of NE at 50.0 pmol prawn(-1). In prawns that had received 5.0 pmol NE prawn(-1), the PO activity had decreased at 2 h, SOD activity at 8 h, and the clearance efficiency at 2 h. PO activity had decreased at 2 h after prawns had received 0.5 pmol NE prawn(-1). All of the immune parameters had returned to control values by 16 h after receiving NE. However, no significant differences were observed in the granular cells during the experimental period. An injection of NE also significantly increased the mortality of prawns challenged with L. garvieae, which appeared to be dose dependent. In another experiment, NE co-injected with prazosin, metoprolol, or propranolol significantly decreased the mortality of challenged prawns, especially when co-injected with prazosin and metoprolol. These results suggest that stress-inducing NE suppresses the immune system, which in turn promotes the susceptibility of M. rosenbergii to L. garvieae via both α1- and β1-adrenergic receptors.

Streptococcus iniae is a hemolytic, Gram-positive bacterium that causes significant disease problems in freshwater and marine fish species throughout the world. Since first diagnosed in March 1992, S. iniae has become one of the most common bacterial diseases and causes significant economic losses in cultured hybrid tilapia ( Oreochromis niloticus × Oreochromis aureus ) in Saudi Arabia. The specificity of the two sets of oligonucleotide primers used for PCR detection was conformed to generate specific amplicons to S. iniae . Polymerase chain reaction amplification was specific, giving rise to products of expected size for S. iniae . With the 16S rRNA gene primers (Sin-1 and Sin-2) the PCR assay confirmed and resulted in the amplification of 300 bp specific band detected among the local strain tested (AH1) and the reference strain S. iniae ATCC 29178. The primers combination of the lactate oxidase gene ( lctO ) (Lox-1 and Lox-2) gave the expected amplification product of 870 bp with local S. iniae (AH1) only which was identical to that given by the type strain S. iniae ATCC 29178. No specific band was amplified in other type strains, such as S. agalactiae , and Lactococcus garvieae ATCC 43921. The sequence analysis of the 16S rDNA product clearly revealed that the fish isolate was S. iniae identical and showed 99.8% similarity to the ATCC 29178 S. iniae type strain.

Microbial diversity and dynamic changes of sliced vacuum-packed cooked ham during refrigerated storage (0–90 days) after high pressure processing (400 MPa at 22 °C for 10 min) was investigated by using culture-dependent and culture-independent approaches. Isolation of genome DNA and total RNA directly from meat samples, followed by PCR–denaturing gradient gel electrophoresis (DGGE) and RT-PCR–DGGE on 16S rDNA V3 region, was performed to describe the structure of the bacterial community and active species in pressurized sliced cooked ham. The DGGE profile showed that most spoilage bacteria including Lactococcus garvieae , Weissella cibaria , Lactobacillus sakei , Lactobacillus curvatus , Weissella paramesenteroides , Leuconostoc carnosum and Lactococcus lactis subsp. lactis were completely inactivated after high pressure processing (HPP), whereas Weissella viridescens and Weissella minor survived HPP and induced the final spoilage. The microbial diversity of HPP samples during the whole refrigerated storage period was extremely simple. Our results clearly indicated that HPP was an efficient method for avoiding the growth of the major spoilage bacteria and could be used to prolong the shelf-life of sliced vacuum-packed cooked ham.

Dairy and fish isolates of Lactococcus garvieae were tested for their ability to utilize lactose and to grow in milk. Fish isolates were unable to assimilate lactose, but unexpectedly, they possessed the ability to grow in milk. Genetic studies, carried out constructing different vectorette libraries, provided evidence that in fish isolates, no genes involved in lactose utilization were present. For L. garvieae dairy isolates, a single system for the catabolism of lactose was found. It consists of a lactose transport and hydrolysis depending on a phosphoenolpyruvate-dependent phosphotransferase system combined with a phospho-beta-galactosidase. The genes involved were highly similar at the nucleotide sequence level to their counterparts in Lactococcus lactis; however, while in many L. lactis strains these genes are plasmid encoded, in L. garvieae they are chromosomally located. Thus, in the species L. garvieae, the phospho-beta-galactosidase gene, detectable in all strains of dairy origin but lacking in fish isolates, can be considered a reliable genetic marker for distinguishing biotypes in the two diverse ecological niches. Moreover, we obtained information regarding the complete nucleotide sequence of the gal operon in L. garvieae, consisting of a galactose permease and the Leloir pathway enzymes. This is one of the first reports concerning the determination of the nucleotide sequences of genes (other than the 16S rDNA gene) in L. garvieae and should be considered a step in a continuous effort to explore the genome of this species, with the aim of determining the real relationship between the presence of L. garvieae in dairy products and food safety.

We report a case of definite Lactococcus garvieae native-valve endocarditis. The diagnosis was suspected in a patient presenting with congestive heart failure and found to have Enterococcus hirae bacteremia, with a history of L. garvieae bacteremia 1 month prior. Diagnosis was confirmed by 16S rRNA gene sequencing of the 2 isolates and the demonstration of aortic valve vegetations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 °C in summer months. Normally, it causes a hyperacute and haemorrhagic septicemia. This paper presents a state of the art review of fish Lactococcosis including aspects such as pathogen characterization, pathogenesis, epidemiology, diagnosis and control measures of the disease in farmed fish.

This paper reports the development of genetic tools in Lactococcus garvieae, an important Gram-positive bacterial pathogen affecting both fish and mammals. The vector pGKV210, a broad host range vector, was introduced by electroporation into L. garvieae UNIUD074. The maximal frequency obtained was 3.2 x 10(5) transformants/mug of DNA. Moreover, this effect is highly reproducible and appears to be constant, since all L. garvieae strains tested were transformed. Once the optimal transformation procedure was established, it was used to generate isogenic and transposition mutants. Insertional mutagenesis of the L. garvieae SA9H10L gene, similar to a Streptococcus pyogenes gene encoding the M protein (emm64), was carried out using the conditional replication plasmid pORI19. Transposition mutagenesis using the streptococcal temperature-sensitive suicide vector pTV408 to deliver Tn917 into the chromosome of L. garvieae was also achieved at a frequency of ca. 10(-4). Transposon flanking DNA sequences were obtained by plasmid rescue in Escherichia coli and their sequencing analysis demonstrated that the transposon was inserted at different chromosomal loci. Tn917 also made it possible to select a mutant in the operon involved in mannitol fermentation in this microorganism. The results obtained in the present study lay the foundation for future research on the virulence mechanisms of L. garvieae.

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.

Ninety-seven lactic acid bacteria (LAB) were isolated from slaughterhouse porcine blood in order to select autochthonous LAB strains for use as biopreservatives of this by-product. They were identified by 16S rDNA sequencing; and their inhibition capacity was determined against four bacterial species frequently found in contaminated blood, i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens and Bacillus spp. The taxonomic study showed an unexpected low diversity of LAB in blood, i.e. only 8 different species were found, from which just 4, i.e. Enterococcus raffinosus, Lactobacillus murinus, Lactobacillus reuteri and Lactococcus garvieae, amounted to more than 90% of all isolates. Inhibition tests in solid culture media proved that S. aureus and Bacillus spp. were inhibited by most LAB strains obtained from porcine blood. E. coli was the indicator less affected by the isolated LAB species. Several isolates efficiently inhibited the growth of all tested indicators.

In this work, we evaluated the effect of the inclusion of different adjuvants in the vaccine formulation against Lactococcus garvieae in comparison with an aqueous bacterin. The aqueous and non mineral oil adjuvanted vaccines (Montanide-ISA-763-A and Aquamun) yielded a good protection 4 weeks after the immunization (RPS of 82.6, 84.8 and 86.9, respectively). The protective rates obtained for the adjuvants Montanide-IMS-2212 (water based nanoparticles with immunostimulant) and Carbomer (high molecular weight polymer organic of polyacrylic acid) were 45.7% and 56.5%, respectively. We also evaluated the duration of protection confered by Aquamun adjuvanted-vaccine in comparison with the aqueous bacterin. Three months after vaccination, the RPS values obtained with the aqueous vaccine was 40% whereas with the Aquamun adjuvanted-vaccine the protection increased to values of 92%. In addition, a long lasting protection was observed with this adjuvanted vaccine with RPS values of 90% and 83.3% in the challenges performed at 6 and 8 months post-vaccination, respectively.