Multiplex PCR assay for the accurate and rapid detection and differentiation of Lactococcus garvieae and L. petauri.

Lactococcosis is a common bacterial fish disease caused by Lactococcus garvieae, L. petauri and L. formosensis. Although there are different PCR-based techniques to identify the etiological agent, none of these can differentiate these two bacteria without sequencing PCR-amplified fragments. In the present study, we developed a multiplex PCR assay for simultaneous detection and differentiation of L. garvieae and L. petauri. The specificity of the primers was validated against the bacterial DNA of the targeted and non-targeted bacteria. The sizes of the PCR amplicons were obtained as 204 bp for the DUF1430 domain-containing protein gene of L. garvieae, 465 bp for the Lichenan permease IIC component gene of L. petauri, and 302 bp for the teichoic acid biosynthesis protein F gene of both L. garvieae and L. petauri. The PCR amplicons were clearly separated by agarose gel electrophoresis. The multiplex PCR assay did not produce any amplification products with the DNA of the non-targeted bacteria. The multiplex PCR detection limits for L. garvieae and L. petauri were 5 and 4 CFU in pure culture and 50 and 40 CFU/g in spiked tissue samples, respectively. It takes less than 2 h from plate-cultured bacteria and 3 h from tissue samples to get results. In conclusion, the developed multiplex PCR assay is a rapid, specific, accurate, and cost-effective method for the detection and differentiation of L. garvieae and L. petauri and is suitable to be used for routine laboratory diagnosis of L. garvieae and L. petauri.

Changes in blood serum parameters in farmed rainbow trout (Oncorhynchus mykiss) during a piscine lactococcosis outbreak.

The aquaculture sector plays a vital role in global food security, yet it grapples with significant challenges posed by infectious diseases. Piscine lactococcosis is one of the significant threats in rainbow trout aquaculture due to its potential to cause severe economic losses through mortalities, reduced growth rates, and increased susceptibility to other pathogens. It poses challenges in disease management strategies, impacting the sustainability and profitability of rainbow trout farming. The current study focuses on the variations in serum blood parameters of farmed rainbow trout Oncorhynchus mykiss during a lactococcosis outbreak caused by Lactococcus garvieae. Blood samples were collected for biochemical analysis, fish were examined for parasites and bacteria, and DNA from bacterial colonies was PCR-amplified and sequenced for identification. Overall, 13 biochemical parameters, including proteins, enzymes, lipids, chemicals, and minerals, were measured in serum blood samples from both diseased and healthy fish. The results indicate significant alterations in the levels of these parameters during the outbreak, highlighting the impact of infections on the blood profile of farmed rainbow trout. Urea levels were significantly higher in diseased fish compared to controls, and creatinine, phosphorus, and magnesium also showed similar trends. Alanine aminotransferase and total protein levels were higher in control fish. Chloride levels differed significantly between groups. Iron levels were higher in controls and lower in diseased fish. No significant differences were found in other parameters. This study reveals significant changes in serum blood parameters of rainbow trout during a lactococcosis outbreak caused by L. garvieae. These changes highlight the potential of these parameters as tools for monitoring health status, stress, and aquaculture management. Continuous monitoring can provide valuable insights into disease severity and overall fish health, aiding in the development of improved management practices. The presented data contribute to understanding the pathophysiology of piscine lactococcosis and developing effective mitigation strategies for farmed rainbow trout.

Lactococcus garvieae exerts a critical role in inducing inflammation in dairy mastitis by triggering NLRP3 inflammasome-mediated pyroptosis in MAC-T cells.

Lactococcus garvieae (L. garvieae) is a pathogenic bacterium that is Gram-positive and catalase-negative (GPCN), and it is capable of growing in a wide range of environmental conditions. This bacterium is associated with significant mortality and losses in fisheries, and there are concerns regarding its potential as a zoonotic pathogen, given its presence in cattle and dairy products. While we have identified and characterized virulent strains of L. garvieae through phenotyping and molecular typing studies, their impact on mammary tissue remains unknown. This study aims to investigate the pathogenicity of strong and weak virulent strains of L. garvieae using in vivo mouse models. We aim to establish MAC-T cell model to examine potential injury caused by the strong virulent strain LG41 through the TLR2/NLRP3/NF-kB pathway. Furthermore, we assess the involvement of NLRP3 inflammasome-mediated pyroptosis in dairy mastitis by silencing NLRP3. The outcomes of this study will yield crucial theoretical insights into the potential mechanisms involved in mastitis in cows caused by the L. garvieae-induced inflammatory response in MAC-T cells.

Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex).

Lactococcosis, caused by members of the genus Lactococcus, represents a devastating disease inducing mass mortalities and economic losses in many fish species worldwide. The present work aimed to compare the whole genome sequences of three different serotypes of Lactococcus garvieae isolated from diseased cultured striped jack (Pseudocaranx dentex) in Ehime prefecture, Japan. The three serotypes showed different virulence in the challenge test using Japanese amberjack (Seriola quinqueradiata). The genome sequencing revealed that two of the strains (serotype I and serotype III) were identified as L. garvieae, while the third strain (serotype II) was identified as L. formosensis. The chromosome sizes of the three serotypes ranged from 1.9 to 2.0 Mb; the GC content ranges were 38.2 to 38.9%; and the numbers of predicted protein-coding sequences (CDSs) were from 1922 to 1959. Only the serotype II harbours two plasmids, sizes of around 14 kb and 9 kb. The detected virulence factors varied among the different serotypes with some shared factors like adherence, anti-phagocytosis, secretion system, toxin (haemolysin), serum resistance, antimicrobial resistance and others. The genomes also contained factors responsible for resistance to toxic compounds. The genome of the serotype III tended to encode more prophage regions than the other serotypes.

First identification and histopathological analysis of Lactococcus garvieae infection in whiteleg shrimp, Penaeus vannamei cultured in low salinity water.

The isolation and characterization of bacterial species Lactococcus garvieae, previously unreported in whiteleg shrimp, Penaeus vannamei, has now been identified in the species. The pathogen was recovered from an affected shrimp farm in southern Taiwan. Bacterial characterization first identified the isolate as Gram-positive cocci, and biochemical profiles demonstrated that the causative agent of mortality was 97% L. garvieae. The bacterial cell DNA resulted in amplification of 1522 bp with 99.6% confirmation by PCR analysis. The phylogenetic tree revealed 100% evolutionary similarity among previously isolated strains. Experimental infection further confirmed higher susceptibility of whiteleg shrimp to L. garvieae in waters of lower salinity, particularly 5 ppt, than in higher salinity. Histopathological analysis showed severely damaged hepatopancreas with necrotized, elongated, collapsed tubules, dislodged membranes and granuloma formation in infected shrimp. Transmission electron microscopy observation indicated a hyaluronic acid capsular layer surrounding bacterial cell which is a virulence factor of L. garvieae and likely responsible for immunosuppression and higher mortality of shrimp cultured in lower salinity. Collectively, these findings report the first isolation of L. garvieae from whiteleg shrimp and shed new light on the disease that threatens the highly valuable species and accentuates the need for finding a solution.

First identification, molecular characterization, and pathogenicity assessment of Lactococcus garvieae isolated from cultured pompano in Taiwan.

Lactococcosis, caused by Lactococcus garvieae, is an acute hemorrhagic septicemia in fish recorded in marine and freshwater aquaculture during the summer months. In 2020-2021, several sea cage Pompano farms recorded sudden fish mortality events. Based on the results of phenotypic and biochemical tests, L. garvieae was predicted to be the cause. PCR with L. garvieae specific primers (pLG1 and pLG2) targeting the 16S rRNA region further confirmed the etiological agent as L. garvieae after amplifying an 1100 base pairs (bp) product. Furthermore, the 16S rRNA sequences of the two representative strains (AOD109-196-2B and AOD110-215-2B) shared 99.81% identity with L. garvieae (GenBank accession number: MT597707.1). The genetic profiles of the strains were classified using pulsed-field gel electrophoresis after digestion with SmaI and ApaI, which clustered our strains under the same pulsotype. Multiplex PCR targeting the capsule gene cluster and serotype-specific PCR collectively showed that the strains were non-capsulated; thus, they belonged to serotype I. An experimental infection was designed to fulfil Koch's postulates by infecting healthy Pompano with case-driven L. garvieae strains (AOD109-196-2B and AOD110-215-2B) with a cumulative mortality of 70%. Overall, L. garvieae infection in Pompano emphasizes the need for better monitoring and control procedures in aquaculture settings.

Resistance and genomic characterization of a plasmid pkh2101 harbouring erm(B) isolated from emerging fish pathogen Lactococcus garvieae serotype II in Japan.

The emergence of antibiotic-resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin-resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10-4 to 10-6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin-resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS-5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.

First preliminary study on identification of bacterial fish pathogens with Raman spectroscopy.

Accurate and rapid determination of bacterial disease agents of fish is an important step for sustainable and efficient aquaculture production. In general, biochemical and molecular methods are used for pathogen detection but they are usually time-consuming and required qualified personnel. Recently spectroscopic methods are preferred in clinical and food microbiology and declared as a promising alternative method for pathogens diagnosis with many advantages. In this study, the significant spectra of three important bacterial fish pathogens (Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri) were determined by Raman spectroscopy. The first data of the pathogens were obtained and the distinctive differences in polysaccharides, nucleic acids, fatty acids and amino acids were identified. This preliminary study aimed to be pioneer for further studies in aquaculture and veterinary microbiology toward developing an alternative method for routine identification.

Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse-field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an evolving disease affecting many fish species and causing significant economic losses worldwide. Assessing pathogen relatedness and bacterial population structure is critical for determining the epidemiology of L. garvieae infections and in establishing effective pathogen management methods. The previously published morphological and genetic studies point to a clonal population structure, as seen in other fish bacteria. In the present study, the pulsed-field gel electrophoresis (PFGE) method was utilized to define a population of 41 Taiwanese isolates from outbreaks with comparisons to four well-characterized non-Taiwanese isolates previously published. Two restriction enzymes (ApaI and SmaI) were utilized individually for PFGE analysis (cut-off value = 90.0%), revealing genetic heterogeneity across L. garvieae isolates, with ApaI and SmaI yielding 12 and seven distinct PFGE band patterns, respectively. The phylogenic analysis using internal transcribed spacer region clustered all L. garvieae isolates in the same clad. Furthermore, the electron microscopic results confirmed the absence of capsular gene cluster (CGC) in previously characterized Taiwanese vaccine strain (S3) from grey mullet. Overall, our findings emphasize the importance of analysing the morphological and genetic diversity in L. garvieae being correlated for proper taxonomic classification in vaccine strain selection and epidemiological studies.

NGS analysis revealed that Lactococcus garvieae Lg-Per was Lactococcus petauri in Türkiye.

Lactococcus garvieae Lg-per was originally isolated from rainbow trout cultured in cages located on the Turkish coast of the Black Sea in 2011. A whole genome sequence of Lg-per was performed in the present study. The complete genome of Lg-per mapped to the reference genomes of L. garvieae (GCF_000269925.1) and Lactococcus petauri (GCF_014830225.1) had a total of 1,694,407 and 1,945,297 base pairs, respectively. Lg-per had 1955 protein-coding genes and 4 rRNA, 46 tRNA and 1 tmRNA operons. The orthoANI value was 98.30% between Lg-per and L. petauri (GCF_014830225.1) and 93.1% between Lg-per and L. garvieae (GCF_000269925.1). A phylogenetic tree generated from the whole genome sequences (WGS) of several Lactococcus species found that L. petauri (GCA 002154895) was closely related to the Lg-per strain with 98% similarity. Although L. garvieae Lg-per was confirmed as L. garvieae based on phenotypical, biochemical and 16S rRNA sequence, WGS of the Lg-per strain revealed that Lg-per was L. petauri. Using a 16S rRNA-based PCR detection approach, Lg-per was misdiagnosed as L. garvieae since its 16S rRNA gene was 99.9% similar to that of L. garvieae strains. Consequently, the 16S rRNA-based PCR detection approach may not be adequate for the identification of the Lactococcus genus. This is the first study to document the presence of L. petauri in Türkiye. L. garvieae isolates should be analysed using WGS since the same issue might occur in other countries.

Garvicin KS, a Broad-Spectrum Bacteriocin Protects Zebrafish Larvae against Lactococcus garvieae Infection.

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda. In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri. There was no inhibition of three foodborne bacterial species, namely Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration of 33 µg/mL exhibited low cytotoxicity, while concentrations ≤3.3 µg/mL had no cytotoxicity on CHSE-214 and RTG-2 cells. In vivo tests showed that zebrafish larvae treated with 33 µg/mL and 3.3 µg/mL GarKS prior to challenge had 53% and 48% survival, respectively, while concentrations ≤0.33 µg/mL were nonprotective. Altogether, these data show that GarKS has a broad inhibitory spectrum against Gram positive and negative bacteria and that it has potential applications as a therapeutic agent for a wide range of bacterial pathogens. Thus, future studies should include clinical trials to test the efficacy of GarKS against various bacterial pathogens in farmed fish.

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9 kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35 °C and pH 5. rLgGad showed a melting temperature of 43.84 °C. At 30 °C, more than 80% of the activity was maintained even after 7 h; however, it rapidly decreased at 50 °C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3 min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Efficiency of a probiotic in carp lactococcosis in an in vitro experiment.

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.

Proposal of Lactococcus garvieae subsp. bovis Varsha and Nampoothiri 2016 as a later heterotypic synonym of Lactococcus formosensis Chen et al. 2014 and Lactococcus formosensis subsp. bovis comb. nov.

Currently, Lactococcus garvieae contains two subspecies: L. garvieae subsp. bovis and L. garvieae subsp. garvieae. In a study by Varsha and Nampoothiri, high pheS (99.7 %) and rpoA (99.6 %) sequence similarities indicated that L. garvieae subsp. bovis and Lactococcus formosensis probably have a close taxonomic relationship; low pheS (92.2 %) and rpoA (97.8 %) sequence similarities and relatively low DNA-DNA hybridization value (75.8 %) indicated that L. garvieae subsp. bovis and L. garvieae subsp. garvieae probably represent two different species. In the present study, the taxonomic relationships between L. garvieae subsp. bovis, L. garvieae subsp. garvieae and L. formosensis were re-examined based on sequence analyses of 16S rRNA, pheS, recA, rpoA and rpoB genes, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) values, average amino acid identity (AAI), fatty acid methyl ester analysis and phenotypic characterization. L. garvieae subsp. bovis LMG 30663T exhibited 97.3 % ANI, 78.3 % dDDH and 96.4 % AAI values to L. formosensis NBRC 109475T, higher than the threshold for species demarcation (95-96, 70 and 95-96 %, respectively), indicating that L. garvieae subsp. bovis LMG 30663T and L. formosensis NBRC 109475T belong to the same species. L. garvieae subsp. bovis LMG 30663T had 91.2 % ANI, 43.3 % dDDH and 92.9-93.0% AAI values with the type strain of L. garvieae subsp. garvieae, indicating that they represent two different species. Because L. formosensis has been proposed and validated before L. garvieae subsp. bovis, L. garvieae subsp. bovis is transferred to L. formosensis as L. formosensis subsp. bovis comb. nov. The type strain of L. formosensis subsp. bovis is BSN307T (=DSM 100577T=MCC 2824T=KCTC 21083T=LMG 30663T). The type strain of L. formosensis subsp. formosensis is 516T (=NBRC 109475T=BCRC 80576T).

Hepatic antioxidant activity, immunomodulation, and pro-anti-inflammatory cytokines manipulation of κ-carrageenan (κ-CGN) in cobia, Rachycentron canadum against Lactococcus garvieae.

The effects of dietary k-Carrageenan (k-CGN) at 10, 20, and 30 g kg-1 on growth rate, hemato-biochemical indices, innate-adaptive parameters and modification of pro- and/or anti-inflammatory cytokines and chemokines pathway in cobia, Rachycentron canadum against Lactococcus garvieae is reported. The weight gain (WG) increased substantially (P < 0.05) in all k-CGN treated groups; the specific growth rate (SGR) was significant in healthy uninfected normal (HuN) and L. garvieae challenged (LaC) groups fed with 20 g kg-1k-CGN diet on 45 and 60 days. The white blood cell (WBC) counts, total protein (TP) level, total anti-oxidant (T-AOC), catalase (CAT), and glutathione (GSH) activities increased significantly when fed with 20 g and 30 g kg-1k-CG diets on 45th and 60th day. The immunological parameters such as phagocytic (PC) index and the activity of phagocytic (PC), respiratory burst (RB), superoxide dismutase (SOD), alternate complement pathway (ACH50), and lysozyme (LZM) were significantly enhanced with all k-CG diets in 45 and 60 days of treatment. No cumulative mortality (CM) in HuN group fed by control or any k-CGN diets. CM was 5% in LaC group fed with 20 g kg-1k-CGN diet whereas in LaC groups fed with 10 g and 30 g kg-1k-CGN diets the CM was 10%. The interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα) pro-inflammatory cytokines mRNA transcripts were one-fold high (P < 0.05) in both HuN and LaC group fed all k-CGN enriched diets on 45 and 60 days. Similarly, IL-18 and TLR2 mRNA was one-fold high expression in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 or 60 days. Interferon gamma (IFNγ) and interferon regulatory factor 3/7 (IRF3/IRF7) mRNA transcripts did not change with any diet. IL-6, IL-10, and IL-11 mRNA were one-fold high expressions in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 and 60 days. However, the expression of CC1, CC3, and CCR9 pro-inflammatory chemokines mRNA did not vary with any control or k-CGN enriched diets. The results indicate that diet enriched with k-CGN at 20 g kg-1 significantly influences the growth, antioxidant and innate-adaptive immune performance, and pro-anti-inflammatory cytokines and chemokines regulation in cobia against L. garvieae.

Co-infection of Lactococcus garvieae and Tilapia lake virus (TiLV) in Nile tilapia Oreochromis niloticus cultured in India.

Tilapia lake virus (TiLV) and Lactococcus garvieae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus. In June-July 2018, a disease outbreak was reported in Nile tilapia cultured in brackish water floating cages in Kerala, India. Affected fish died gradually, and cumulative mortality reached ~75% within 1 mo. In the present study, TiLV and L. garvieae were isolated from the infected fish and confirmed. Nucleotide analysis of the partial sequence of segment 3 revealed that the present TiLV isolate showed 100% similarity with TiLV MF574205 and 97.65% similarity with TiLV KU552135 isolated in Israel. The partial 16S rDNA nucleotide sequence of L. garvieae shared 99% similarity with the 16S rDNA nucleotide sequence of L. garvieae isolated from Nile tilapia in Brazil. Eight virulence genes (hly1, hly2, hly3, NADH oxidase, adhPav, LPxTG-1, LPxTG-4, adhC1) were amplified in the present isolate. In the experimental challenge study, the onset of mortality started earlier in fish co-infected with TiLV and L. garvieae (3 d post-infection [dpi]) compared to other groups. Cumulative mortality (90% at 12 dpi) was significantly higher in the co-infected group than in fish infected with TiLV (60% at 12 dpi) and L. garvieae (40% at 12 dpi) alone. This study reveals that synergistic co-infection with TiLV and other bacteria may increase mortality in disease outbreaks. To the best of our knowledge, this is the first reported co-infection of L. garvieae with TiLV associated with mass mortality in Nile tilapia in India.

Lactococcus garvieae isolated from Lake Kariba (Zambia) has low invasive potential in Nile tilapia (Oreochromis niloticus).

The pathogenesis of Lactococcus garvieae (L. garvieae) was assessed in Nile tilapia (Oreochromis niloticus) following administration by two different routes of infection (intraperitoneal versus immersion), using 180 fish divided into three groups. The first group of fish was injected intraperitoneally (IP) with 3 × 105 colony-forming units (cfu) of L. garvieae; the second group was infected by immersion (IMM) into water containing 9.6 × 105  cfu/ml L. garvieae, and in group 3 (Control), the fish were injected IP with sterile normal saline. Mortalities were recorded daily, and on 3, 5, 7, and 13 days post-infection (dpi), liver, kidney, spleen, brain and eyes were sampled. The level of infection between groups was assessed by number of mortalities that occurred, pathology/histopathology of internal organs, bacterial re-isolation and presence of bacteria in situ determined using immunohistochemistry. A significant difference (p < .0001) was observed between L. garvieae re-isolation from tilapia following administration by IP injection and IMM. Similarly, more clinical signs and mortalities (p < .001) were observed in the IP group compared to the IMM group where no mortalities were observed. These findings suggest that L. garvieae has a low invasive potential in Nile tilapia with intact skin/external barriers and highlights the importance of maintaining fish without cuts or abrasions under field conditions.

Changes in Intestinal Microbiota Due to the Expanded Polystyrene Diet of Mealworms (Tenebrio molitor).

Expanded polystyrene (EPS), which is difficult to decompose, is usually buried or incinerated, causing the natural environment to be contaminated with microplastics and environmental hormones. Digestion of EPS by mealworms has been identified as a possible biological solution to the problem of pollution, but the complete degradation mechanism of EPS is not yet known. Intestinal microorganisms play a significant role in the degradation of EPS by mealworms, and relatively few other EPS degradation microorganisms are currently known. This study observed significant differences in the intestinal microbiota of mealworms according to the dietary results of metagenomics analysis and biodiversity indices. We have proposed two new candidates of EPS-degrading bacteria, Cronobacter sakazakii and Lactococcus garvieae, which increased significantly in the EPS feeding group population. The population change and the new two bacteria will help us understand the biological mechanism of EPS degradation and develop practical EPS degradation methods.

Emerging bacterial fish pathogen Lactococcus garvieae RTCLI04, isolated from rainbow trout (Oncorhynchus mykiss): Genomic features and comparative genomics.

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.

Resistant and susceptible rainbow trout (Oncorhynchus mykiss) lines show distinctive immune response to Lactococcus garvieae.

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.