Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous ...vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.
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•Serial passaging in increasing concentrations of rifampin attenuated Streptococcus iniae.•Passaged strains produced specific and cross-reactive antibody responses in tilapia.•A strain offering high homologous protection has potential as a live vaccine.
The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive ...aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram‐positive cocci and cream‐coloured, round and convex colonies of rod‐shaped gram‐negative bacteria. Additional biochemical and species‐specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole‐trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole‐trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.
Outbreaks of an infectious disease affecting cultured white sturgeon (Acipenser transmontanus) were investigated. Clinical signs included erratic swimming, arching of the back and mortality. Necropsy ...findings included poorly demarcated yellow to dark‐red and friable lesions in the epaxial muscle, ulcerative skin lesions and haemorrhages in the swim bladder and coelomic wall. Histological evaluation revealed areas of necrotizing and heterophilic myositis with aggregates of bacterial cocci. The lumen of blood vessels in the dermis, under ulcerated areas, and in the posterior kidney, was occluded by fibrin thrombi. Aggregates of Gram‐positive cocci were observed in the muscle lesions and within the fibrin thrombi in the dermis and kidney. Genetically homogeneous Streptococcus iniae strains were recovered from affected fish from different outbreaks. The isolates shared high degree of similarity at gene locus (gyrB) with previously characterized S. iniae from cultured fish in California, confirming the emergence of this particular strain of S. iniae in US aquaculture.
Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative ...agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S–23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non‐target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non‐target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.
Lactococcus petauri
is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, ...laboratory-controlled challenges were used as models of infection. Rainbow trout
Oncorhynchus mykiss
maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 10
1
, 10
3
or 10
5
colony-forming units per fish (CFU fish
-1
) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon
Oncorhynchus tshawytscha
, white sturgeon
Acipenser transmontanus
, Nile tilapia
Oreochromis niloticus
, and koi
Cyprinus carpio
to infection by
L. petauri
was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of
L. petauri
that can be used in the development of better management practices to protect against piscine lactococcosis.
Streptococcus iniae
is a Gram-positive, opportunistically zoonotic bacterium infective to a wide variety of farmed and wild fish species worldwide. Outbreaks in wild fish can have detrimental ...environmental and cultural impacts, and mortality events in aquaculture can result in significant economic losses. As an emerging or re-emerging pathogen of global significance, understanding the coalescing factors contributing to piscine streptococcosis is crucial for developing strategies to control infections. Intraspecific antigenic and genetic variability of
S. iniae
has made development of autogenous vaccines a challenge, particularly where the diversity of locally endemic
S. iniae
strains is unknown. This study genetically and phenotypically characterized 11
S. iniae
isolates from diseased wild and farmed fish from North America, Central America, and the Caribbean. A multilocus sequence analysis (MLSA) scheme was developed to phylogenetically compare these isolates to 84 other strains of
Streptococcus
spp. relevant to aquaculture. MLSA generated phylogenies comparable to established genotyping methods, and isolates formed distinct clades related to phenotype and host species. The endothelial
Oreochromis mossambicus
bulbus arteriosus cell line and whole blood from rainbow trout
Oncorhynchus mykiss
, Nile tilapia
Oreochromis niloticus
, and white sturgeon
Acipenser transmontanus
were used to investigate the persistence and virulence of the 11 isolates using
in vitro
assays.
In vivo
challenges using an
O. niloticus
model were used to evaluate virulence by the intragastric route of infection. Isolates showed significant differences (p < 0.05) in virulence and persistence, with some correlation to genogroup, establishing a basis for further work uncovering genetic factors leading to increased pathogenicity.
Complex and interacting selective pressures can produce bacterial communities with a range of phenotypes. One measure of bacterial success is the ability of cells or populations to proliferate while ...avoiding lytic phage infection. Resistance against bacteriophage infection can occur in the form of a metabolically expensive exopolysaccharide capsule. Here, we show that in Caulobacter crescentus, presence of an exopolysaccharide capsule provides measurable protection against infection from a lytic paracrystalline S-layer bacteriophage (CR30), but at a metabolic cost that reduces success in a phage-free environment. Carbon flux through GDP-mannose 4,6 dehydratase in different catabolic and anabolic pathways appears to mediate this trade-off. Together, our data support a model in which diversity in bacterial communities may be maintained through variable selection on phenotypes utilizing the same metabolic pathway.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lactococcus garvieae is an emergent bacterial pathogen of salmonid fish in North America that causes acute infections particularly at water temperatures above 15°C. During 2020, L. garvieae was ...detected in rainbow trout, Onchorhyncus mykiss, cultured in Southern California and the Eastern Sierras. Infected fish exhibited high mortalities and nonspecific clinical signs of lethargy, erratic swimming, dark skin pigmentation, and exophthalmia. Macroscopic changes included external and internal hemorrhages, mainly in the eyes, liver, coelomic fat, intestine, and brain. Histological examination revealed splenitis, branchitis, panophthalmitis, hepatitis, enteritis, and coelomitis, with variable degrees of tissue damage among evaluated fish. Pure colonies of L. garvieae were isolated from infected trout and specific PCR primers for L. garvieae confirmed the preliminary diagnosis. Multilocus sequence analysis showed that the strains recovered from diseased trout represent a novel genetic group. Isolates were able to form biofilms within 24 h that increased their resistance to disinfection by hydrogen peroxide. Laboratory challenge methods for inducing lactococcosis in steelhead trout, O. mykiss, were evaluated by intracoelomic injection with serial dilutions of L. garvieae. The median lethal dose 21 days post challenge was ∼20 colony‐forming units/fish. Experimentally infected trout presented similar clinical signs, gross changes, and microscopic lesions as those with natural disease, fulfilling Koch's postulates and demonstrating the high virulence of the recovered strains.
Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups ...(GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.
Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera
(Family
) and
(
) encompass ...the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive
and
isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the
gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as
species and 131 as
. The majority of
isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and
into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two
clades (F & G), and four
clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established
breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies.
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