Search Results (31)

Streptococcus parauberis is a major pathogen responsible for streptococcosis in both marine and freshwater fish species, causing substantial economic losses in aquaculture. The increasing prevalence of multidrug resistance has highlighted the urgent need for alternative disease control strategies. Interference with bacterial quorum sensing (QS) systems represents a promising approach. This study aimed to identify and biochemically characterize an Rgg-family transcriptional regulator and evaluate its potential as a target for quorum sensing-related regulatory interference in vitro. We hypothesized that this Rgg regulator may function as a quorum sensing-associated transcription factor capable of promoter binding and modulation by small molecules. Bioinformatic analyses were used to identify the rgg gene encoding an Rgg-family transcriptional regulator and predict its structural features. The gene was cloned, heterologously expressed, and purified. Promoter binding activity was examined using electrophoretic mobility shift assay (EMSA), and key amino acid residues were identified through site-directed mutagenesis. The inhibitory effect of the cyclic peptide cyclosporin A (CsA) on Rgg-promoter binding was further assessed. The rgg gene (864 bp) encoding a 287-amino-acid protein (34.1 kDa) was successfully identified and expressed. Purified Rgg specifically bound to its own promoter region in a concentration-dependent manner. Mutations at conserved arginine residues R12 and R15 within the helix-turn-helix DNA-binding domain abolished promoter binding activity. Furthermore, CsA disturbed Rgg-promoter binding in a dose-dependent manner. This study provides the first in vitro characterization of an Rgg-family transcriptional regulator in fish-derived S. parauberis. The findings expand current understanding of Rgg-family regulators potentially associated with quorum sensing in aquatic streptococci and provide a preliminary basis for further investigation of quorum sensing-related regulatory interference strategies for controlling streptococcal diseases in aquaculture. Full article

This study evaluates the efficacy of a new florfenicol-based drug, both in vitro and in vivo, in Nile tilapia (Oreochromis niloticus) against pathogens commonly found in fish farming and its withdrawal period. The drug’s efficacy was tested using prophylactic, metaphylactic, and therapeutic approaches against Streptococcus agalactiae (serotypes Ib and III) and Francisella orientalis. The minimum inhibitory concentration of florfenicol was 4, 5, and 128 μg/mL for the different pathogens. Fish mortality was lower in the prophylactic treatment compared to the metaphylactic and therapeutic treatments for S. agalactiae. No difference in mortality was observed for F. orientalis across treatments. Mild to moderate lesions suggestive of intoxication were observed, mainly in the liver of fish treated with overdoses or exposed to low temperatures. Florfenicol reduced mortality rates, especially with early treatment (metaphylactic), in fish experimentally challenged with the pathogens. Moreover, prophylactic antimicrobial use is not recommended, as it promotes the selection of multidrug-resistant bacterial strains. Additionally, the residual concentration of the drug in muscle tissue lasted for a shorter period than that recommended by the manufacturer, and at lower concentrations than required by national and international legislation. Full article

Nile tilapia (Oreochromis niloticus) and tambaqui (Colossoma macropomum) are the two most produced freshwater fishes in Brazil. This study investigated the potential pathogenicity of Streptococcus agalactiae and Francisella orientalis, previously isolated from diseased Nile tilapia, to tambaqui. Experimental infection trials were conducted in juvenile tambaqui at a dose of approximately 10⁷ CFU fish⁻¹, assessing clinical signs, mortality, bacterial recovery, and histopathological changes. Results demonstrated that S. agalactiae exhibited high pathogenicity to tambaqui, causing rapid disease progression, high mortality (83.33%) within 48 h post-infection, and severe lesions in multiple organs, under the experimental conditions. In contrast, F. orientalis infection did not result in mortality or clinical signs, despite bacterial recovery and granulomatous inflammation observed in the tissues. This study highlights the need to consider the potential impact of these pathogens in tambaqui farming. Full article

Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. Streptococcus suis, a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into S. suis cells: HIV-1 TAT efficiently penetrated S. suis cells with low toxicity against S. suis; (RXR)₄XB had high penetration efficiency with inherent toxicity against S. suis; (KFF)₃K showed lower penetration efficiency than HIV-1 TAT and (RXR)₄XB; K8 failed to penetrate S. suis cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti-gyrA PNA) effectively inhibited the growth of S. suis. TAT-anti-gyrA PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of S. suis, which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating S. suis infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells. Full article

Streptococcus agalactiae is a highly invasive bacterium that causes significant economic losses in tilapia aquaculture around the world. Furthermore, it is a pathogen for mammals, including humans, emphasizing its importance in One Health. The aim of this work was to evaluate the evolution of clinical and histopathological lesions caused by acute infection with two serotypes of S. agalactiae. For this, two strains isolated from natural outbreaks in Brazilian aquaculture farms (S13, serotype Ib; S73, serotype III) were used to challenge juvenile Nile tilapia (Oreochromis niloticus) intraperitoneally. Target organ samples were collected ten times, between 1 and 96 h post-infection, for microbiological and histopathological analyses. Anorexia was the first clinical sign and the first death occurred at 24 and 30 h in the fish infected with strains S13 and S73, respectively. Serotype Ib initially caused more pronounced lesions in the nervous system; however, serotype III lesions progressed more aggressively, reaching the same severity as those of serotype Ib. This trend was repeated in the mortality curve after 32 h. These results elucidated the important stages in the pathogenesis of S. agalactiae serotypes Ib and III in tilapia and suggest “tips and tricks” to improve the positive culture rate in the clinical diagnosis of infections in some tissues. Full article

Streptococcosis caused by Streptococcus agalactiae (S. agalactiae) is a major bacterial disease affecting the production of Nile tilapia (Oreochromis niloticus L.), causing significant economic losses due to mortality in the growing phase. Vaccination is the most effective method for preventing streptococcosis on Nile tilapia farms. In Brazil, the major tilapia-producing regions have long production cycles (6–10 months) and harvest tilapias weighing over 900 g for fillet production. Thus, data on the duration of the humoral immune response and protection in farmed tilapia have not been reported or are poorly described. Furthermore, the efficiency of serological testing for the long-term monitoring of immune responses induced by vaccination against S. agalactiae has never been addressed. This study evaluated the duration of protection and humoral immune response induced in Nile tilapia vaccinated against S. agalactiae until 300 days post-vaccination (dpv). The immunization trial was composed of two groups: vaccinated (Vac), vaccinated intraperitoneally with a commercial vaccine, and unvaccinated (NonVac) group, injected fish with sterile saline solution. At 15, 30, 150, 180, 210, and 300 dpv, blood sampling was conducted to detect anti-S. agalactiae IgM antibodies using indirect Enzyme-Linked Immunosorbent Assay (ELISA), and the fish were challenged with pathogenic S. agalactiae to determine the duration of vaccine protection through relative percentage survival (RPS). Spearman’s rank correlation was performed between the ELISA optical density (OD) of vaccinated tilapia and the duration of vaccine protection (RPS). The mean cumulative mortality in NonVac and Vac groups ranged from 65 to 90% and less than 35%, respectively. The average RPS was 71, 93, 94, 70, 86, and 67% at 15, 30, 150, 180, 210, and 300 dpv, respectively. RPS revealed that the vaccine provided protection from 15 to 300 dpv. The specific anti-S. agalactiae IgM antibody levels were significantly higher in the Vac group than that non-Vac group up to 180 dpv. The vaccinated fish exhibited significant protection for up to 10 months after vaccination. There was a positive correlation between the antibody response and RPS. This study revealed that a single dose of commercial vaccine administered to Nile tilapia can confer long-term protection against S. agalactiae and that indirect ELISA can monitor the duration of the humoral immune response for up to six months following vaccination. Finally, vaccine protection over six months can be associated with other components of the fish immune system beyond the humoral immune response by IgM antibodies. Full article

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and septicemia in swine and humans. Among numerous pathogenic serotypes, S. suis serotype 8 has distinctive characteristics such as a high detection rate and causing multi-host infection. There is no complete genome of serotype 8 strains so far. In this study, the complete genome of two S. suis serotype 8 strains, virulent strain 2018WUSS151 and non-virulent strain WUSS030, were sequenced. Comparative genomic analysis showed that the homology of the two genomes reaches 99.68%, and the main difference is the distinctive prophages. There are 83 genes unique to virulent strain 2018WUSS151, including three putative virulence-associated genes (PVGs). Two PVGs, padR and marR, are passenger genes in ISSsu2 family transposons that are able to form circular DNA intermediates during transposition, indicating the possibility of horizontal transmission among S. suis strains. The deletion mutant of PVGs marR or atpase attenuated the virulence of serotype 2 virulent SC070731 in a mouse infection model, confirming their role in S. suis virulence. These findings contribute to clarifying the genomic characterization of S. suis serotype 8 and S. suis pathogenesis. Full article

Streptococcus suis (S. suis) is a zoonotic pathogen with multiple serotypes, and thus, multivalent vaccines generating cross-protection against S. suis infections are urgently needed to improve animal welfare and reduce antibiotic abuse. In this study, we established a systematic and comprehensive epitope prediction pipeline based on immunoinformatics. Ten candidate epitopes were ultimately selected for building the multi-epitope vaccine (MVSS) against S. suis infections. The ten epitopes of MVSS were all derived from highly conserved, immunogenic, and virulence-associated surface proteins in S. suis. In silico analyses revealed that MVSS was structurally stable and affixed with immune receptors, indicating that it would likely trigger strong immunological reactions in the host. Furthermore, mice models demonstrated that MVSS elicited high titer antibodies and diminished damages in S. suis serotype 2 and Chz infection, significantly reduced sequelae, induced cytokine transcription, and decreased organ bacterial burdens after triple vaccination. Meanwhile, anti-rMVSS serum inhibited five important S. suis serotypes in vitro, exerted beneficial protective effects against S. suis infections and significantly reduced histopathological damage in mice. Given the above, it is possible to develop MVSS as a universal subunit vaccine against multiple serotypes of S. suis infections. Full article

In the present study, chitosan-based bivalent nanovaccines of S. iniae and F. covae were administered by immersion vaccination at 30 and 40 days after hatching (DAH), and the third vaccination was orally administered by feeding at 50 DAH. ELISA revealed that the levels of total IgM and specific IgM to S. iniae and F. covae were significantly elevated in all vaccinated groups at 10, 20, and 30 days after vaccination (DAV). A qRT-PCR analysis of immune-related genes revealed significantly higher IgT expression in the vaccinated groups compared to the control group, as revealed by 44–100-fold changes in the vaccinated groups compared to the control (p < 0.001) at every tested time point after vaccination. All vaccinated groups expressed IgM, MHCIIα, and TCRα at significantly higher levels than the control group at 10 and/or 20 DAV (p < 0.05). In the S. iniae challenge tests, the survival of vaccinated groups ranged from 62.15 ± 2.11 to 75.70 ± 3.36%, which significantly differed from that of the control group (44.44 ± 1.92%). Similarly, all vaccinated groups showed higher survival rates of 68.89 ± 3.85 to 77.78 ± 5.09% during F. covae challenge than the control groups (50.00 ± 3.33%) (p < 0.05). Full article

Streptococcus agalactiae is a major health concern in tilapia farming worldwide. In contrast to the availability of susceptibility profile results, interpretative criteria for disk diffusion assays and the influence of serotypes on resistance profiles are not available. To address this, sixty isolates (thirty of each serotype, Ib and III) were evaluated using the disk diffusion assay against six antibiotics, and the epidemiological cut-off value (ECV) was calculated. All the isolates were classified as non-wild type (NWT) for sulfamethoxazole (SUT) and norfloxacin (NOR). The inhibition zones for oxytetracycline (OXY) and doxycycline (DOX) were largely distinct; all serotype Ib and III isolates were classified as wild-type (WT) and NWT, respectively. The results for serotype III of fish group B Streptococcus (GBS) were comparable to the NWT tetracycline profile of human GBS available in EUCAST, suggesting the presence of resistance mechanisms in these fish isolates. The calculation of the cut-off wild type (CO_WT) values for OXY and DOX was appropriate for both serotypes. Differences between the distribution of florfenicol (FLO) and amoxicillin (AMO) were found, and we attribute this to the faster growth rate of serotype III, which promotes smaller inhibition zones. Therefore, using separate CO_WT for each serotype is necessary. In conclusion, the serotype of fish GBS affects its susceptibility profile, and it is recommended to use serotype-specific CO_WT values as interpretative criteria for disk diffusion assays against FLO and AMO. Full article

Necropsies can reveal herd problems or comorbidities that can lead to management corrections, improvements in animal performance, and better decision making. Furthermore, the pattern and causes of mortality might differ when different systems are evaluated. The present study was conducted to establish the main causes of death in nursery pigs from different systems in Brazil, as well as the clinical, microbiological, and pathological aspects of these mortalities. Eighteen nurseries were analyzed (a total of 120,243 housed piglets), and 557 necropsies were performed. Streptococcus suis infection was the most prevalent cause of death (21.2%), followed by bacterial polyserositis (16.7%), chronic atrophic enteritis (13.5%), salmonellosis (8.8%), pneumonia (8.6%), and colibacillosis (6.1%). The increase in mortality rate in individual nurseries and, consequently, in the diagnoses was commonly associated with disease outbreaks. Infectious diseases constituted the largest portion of the diagnoses, making a great opportunity for improving production rates in herds. Moreover, the extensive range of observed diagnoses highlights the importance of conducting preliminary diagnostic investigations based on necropsy to determine the causes of death. This approach allows for the direction of complementary tests, which can diagnose agents with greater specificity. As a result, this allows for the implementation of more effective prevention and control strategies. Full article

Streptococcus pasteurianus is a zoonotic pathogen causing meningitis and bacteremia in animals and humans. A lack of accurate and convenient detection methods hinders preventing and controlling diseases caused by S. pasteurianus. Additionally, there is limited knowledge about its pathogenicity and antimicrobial resistance characteristics, as there are only three complete genome sequences available. In this study, we established a multiplex PCR assay for the detection of S. pasteurianus, which was applied to six fecal samples from cattle with diarrhea and 285 samples from healthy pigs. Out of the samples tested, 24 were positive, including 5 from pig tonsils, 18 from pig hilar lymph nodes, and 1 from cattle feces. Two strains were isolated from positive samples, and their complete genomes were sequenced. The two strains were non-virulent in mice and multidrug-resistant by the antimicrobial susceptibility test. We first found the presence of genes tet(O/W/32/O) and lsa(E) in S. pasteurianus, leading to resistance to lincosamides and tetracyclines. The convenient and specific multiplex PCR assay provides essential technical support for epidemiological research, and the complete genome sequence of two non-virulent strains contributes to understanding this zoonotic bacterium’s genomic characteristics and pathogenesis. Full article

Streptococcosis and motile Aeromonad septicemia (MAS) are the main bacterial diseases in tilapia culture worldwide, causing significant economic losses. Vaccination is an effective method of preventing diseases and contributes to economic sustainability. This study investigated the immuno-protective efficacy of a newly developed feed-based bivalent vaccine against streptococcosis and MAS in red hybrid tilapia. The feed-based bivalent vaccine pellet was developed by incorporating the formalin-killed S. agalactiae and A. hydrophila antigens into a commercial feed pellet with palm oil as the adjuvant. The bivalent vaccine was subjected to feed quality analyses. For immunological analyses, 900 fish (12.94 ± 0.46 g) were divided into two treatment groups in triplicate. Fish in Group 1 were unvaccinated (control), while those in Group 2 were vaccinated with the bivalent vaccine. The bivalent vaccine was delivered orally at 5% of the fish’s body weight for three consecutive days on week 0, followed by boosters on weeks 2 and 6. Lysozyme and enzyme-linked immunosorbent assays (ELISAs) on serum, gut lavage, and skin mucus were performed every week for 16 weeks. Lysozyme activity in vaccinated fish was significantly (p ≤ 0.05) higher than in unvaccinated fish following vaccination. Similarly, the IgM antibody levels of vaccinated fish were significantly (p ≤ 0.05) higher after vaccination. The bivalent vaccine provided high protective efficacy against S. agalactiae (80.00 ± 10.00%) and A. hydrophila (90.00 ± 10.00%) and partial cross-protective efficacy against S. iniae (63.33 ± 5.77%) and A. veronii (60.00 ± 10.00%). During the challenge test, fewer clinical and gross lesions were observed in vaccinated fish compared with unvaccinated fish. Histopathological assessment showed less severe pathological changes in selected organs than the unvaccinated fish. This study showed that vaccination with a feed-based bivalent vaccine improves immunological responses in red hybrid tilapia, and thus protects against streptococcosis and MAS. Full article

Streptococcus suis serotype 2 (SS2) is a noteworthy zoonotic pathogen that has been responsible for large economic losses in pig production and a great threat to human health. Pentraxin 3 (PTX3) is an essential regulator of the innate immune response to bacterial pathogens; however, its role during SS2 infection is not fully understood. In this study, we found that the SS2 strain HA9801 induced a significant inflammatory response in the mouse air pouch model; this response was amplified by the treatment of exogenous PTX3 simultaneously in terms of the results of inflammatory cell recruitment and proinflammatory cytokine IL-6 production. In addition, PTX3 facilitated the phagocytosis of macrophage Ana-1 against SS2 strain HA9801. The supplementation of exogenous PTX3 significantly reduced the bacterial loads in a dose-dependent manner in lungs, livers and bloods of SS2-infected mice compared to the samples with HA9801 infection alone; this finding indicated that PTX3 may facilitate the bacterial clearance through enhancing the host inflammatory response during SS2 infection. Both PTX3 and SS2 capsular polysaccharide (CPS2) were required for the robust inflammatory response, implying that the host PTX3 protein and SS2 surface CPS2 modulate the host innate immune response in concert. All of these results suggested that PTX3 is a potential novel biological agent for the SS2 infection; however, the recommended dose of PTX3 must be evaluated strictly to avoid inducing an excessive inflammatory response that can cause serious tissue injury and animal death. Full article

Juvenile Asian seabass (Lates calcarifer) (body weight 10 ± 0.7 g) were intraperitoneally injected with 10¹² CFU fish⁻¹ of formalin-killed Streptococcus iniae. The protective efficacy of the vaccine on survival and infection rate was assessed upon challenge at 4, 8, 12, 20, and 28 weeks post-vaccination. The results revealed that the challenged vaccinated fish showed no mortality at all time points, and the control fish presented 10–43.33% mortality. The infection rate at 2 weeks post-challenge was 0–13.33% in the vaccinated fish and 30–82.35% in the control group. At 8 weeks post-vaccination, the vaccinated fish showed comparable ELISA antibody levels with the control; however, the antibody levels of the vaccinated fish increased significantly after the challenge (p < 0.05), suggesting the presence of an adaptive response. Innate immune genes, including MHC I, MHC II, IL-1β, IL-4/13B, and IL-10, were significantly upregulated at 12 h post-challenge in the vaccinated fish but not in the control. In summary, vaccination with S. iniae bacterin provided substantial protection by stimulating the innate and specific immune responses of Asian seabass against S. iniae infection. Full article