No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Identification and Pathogenicity of Lactococcus Species in Nile Tilapia (Oreochromis niloticus) and Asian Sea Bass (Lates calcarifer)
Abstract
Lactococcosis, primarily caused by Lactococcus garvieae , is prevalent in various freshwater fish species globally. Yet, its significance in Thai fish aquaculture remains poorly understood. This study investigated Lactococcosis‐associated diseases across four red tilapia ( Oreochromis sp.) and one Asian sea bass ( Lates calcarifer ) farm in Thailand in 2022. Chronically diseased fish displaying clinical signs such as exophthalmia or corneal opacity, erratic swimming, scale loss and skin haemorrhage, as well as apparently healthy specimens, were studied. Bacterial isolation was performed from the fish internal organs. Gram‐positive bacterial isolates were selected and later identified as L. lactis ( n = 4), L. taiwanensis ( n = 1), L. garvieae ( n = 2) and L. petauri ( n = 2) based on bacterial 16S rDNA and gyrB partial sequences. Sub‐adults of Nile tilapia were intracoelomic (IC) injected with representatives of each of the three Lactococcus species ( L. garvieae , L. lactis and L. taiwanensis ) at varying doses (10 ³ –10 ⁷ CFU/fish), while juvenile Asian sea bass were IC injected with L. petauri at doses ranging from 10 ² to 10 ⁸ CFU/fish. Control groups received culture medium alone. By the end of the 21‐day test, mortality rates from all challenge groups ranged from 0% to 14.82%. Some clinical signs were observed in groups challenged with higher doses of the bacteria. Enzyme‐linked immunosorbent assay (ELISA) showed that survivor fish exposed to high bacterial doses elicited specific antibodies IgM. In summary, this study identifies the presence of Lactococcus species in farmed fishes, which exhibit low virulence in Nile tilapia and Asian sea bass under laboratory‐controlled challenges. These bacterial isolates appear to be opportunistic rather than pathogenic, potentially indicating underlying health challenges within the affected farms.
ResearchGate has not been able to resolve any citations for this publication.
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 .
The aquaculture sector is currently experiencing a global disease crisis. Emerging bacterial diseases—often opportunistic or syndromic—have collapsed production in nations across the world. Losses in the shrimp industry associated with opportunistic Vibrio spp. exceed 40% of global capacity. This paper reviews potential drivers of syndromic diseases involving opportunistic bacterial pathogens affecting global aquaculture. We provide key examples from major industries where such conditions have prompted greater antibiotic use and have resulted in significant mortality. We challenge the conventional definitions of opportunistic pathogens and propose a fluid categorisation that acknowledges the continuum of host adaptation and the complexity of microbial ecology. We discuss the implications of environmental and dietary stressors such as climate change, coastal eutrophication and pollution, and the transition to plant‐based feeds, which have been linked to impaired epithelial barrier function, gut health disorders and increased disease susceptibility. We critique the ‘one‐pathogen one‐disease’ paradigm, suggesting that Rothman's causal pie model is more useful for understanding opportunistic infections as it emphasises the multicausal nature of disease. We provide examples of bacterial and viral interactions in aquatic disease and occurrence of bacterial diseases resulting from host damage from eukaryotic parasites or increasing frequency and severity of interventions to control such parasites. We recognise the need for corroborative evidence to validate the rise of opportunistic bacterial pathogens as a global trend, and we advocate for the application of nuanced disease causation models to reduce the incidence of opportunistic infections and improve the sustainability of the aquaculture industry.
Lactococcus garvieae is a fish pathogen that can cause diseases in humans and cows. Two genetically related species, Lactococcus formosensis and Lactococcus petauri, may be misidentified as L. garvieae. It is unclear if these species differ in host specificity and virulence genes. This study analyzed the genomes of 120 L. petauri, 53 L. formosensis, and 39 L. garvieae isolates from various sources. The genetic diversity and virulence gene content of these isolates were compared. The results showed that 77 isolates previously reported as L. garvieae were actually L. formosensis or L. petauri. The distribution of the three species varied across different collection sources, with L. petauri being predominant in human infections, human fecal sources, and rainbow trout, while L. formosensis was more common in bovine isolates. The genetic diversity of isolates within each species was high and similar. Using a genomic clustering method, L. petauri, L. formosensis, and L. garvieae were divided into 45, 22, and 13 clusters, respectively. Most rainbow trout and human isolates of L. petauri belonged to different clusters, while L. formosensis isolates from bovine and human sources were also segregated into separate clusters. In L. garvieae, most human isolates were grouped into three clusters that also included isolates from food or other sources. Non-metric multidimensional scaling ordination revealed the differential association of 15 virulence genes, including 14 adherence genes and a bile salt hydrolase gene, with bacterial species and certain collection sources. In conclusion, this work provides evidence of host specificity among the three species.
IMPORTANCE
Lactococcus formosensis and Lactococcus petauri are two newly discovered bacteria, which are closely related to Lactococcus garvieae, a pathogen that affects farmed rainbow trout, as well as causes cow mastitis and human infections. It is unclear whether the three bacteria differ in their host preference and the presence of genes that contribute to the development of disease. This study shows that L. formosensis and L. petauri were commonly misidentified as L. garvieae. The three bacteria showed different distribution patterns across various sources. L. petauri was predominantly found in human infections and rainbow trout, while L. formosensis was more commonly detected in cow mastitis. Fifteen genes displayed a differential distribution among the three bacteria from certain sources, indicating a genetic basis for the observed host preference. This work indicates the importance of differentiating the three bacteria in diagnostic laboratories for surveillance and outbreak investigation purposes.
Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)—L. garvieae, L. petauri, and L. formosensis—which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention.
IMPORTANCE
Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.
In aquaculture, Lactococcus garvieae is a common fish pathogen that can cause significant economic losses in several fresh and saltwater species. Despite the extensive range of hosts, L. garvieae infection in sea bass (Dicentrarchus labrax) has rarely been reported. During the summer of 2023, an outbreak occurred in an inland farm in the Gulf of Follonica (Tuscany, Italy). Fish of various sizes were affected, showing apathy, inappetence, erratic swimming and eye lesions, while the mortality was low (2–3% per month). Anatomopathological examinations suggested a septicaemic infection characterised by melanosis, diffuse redness (skin and fins), paleness (gills and internal organs), haemorrhages and splenomegaly. Seventy swabs from the viscera of 14 subjects were collected and colonies similar to Streptococcus spp. grew from all the samples. Lactococcus garvieae was identified via the biochemical tests, API20STREP, MALDI-TOF, 16S rDNA and whole genome sequencing. Genetical characterisation revealed remarkable differences between this isolate and the strains previously isolated in Italian fish farms. Feed treatments with flumequine and erythromycin were ineffective. Considering the limited effects of antimicrobials, preventive measures, such as vaccination and biosecurity, should be implemented.
Lactococcus lactis (L. lactis) is the primary organism for lactic acid bacteria (LAB) and is a globally recognized safe microorganism for the regulation of the intestinal micro-ecological balance of animals and improving the immune performance of the host. L. lactis is known to play a commercially important role in feed fortification, milk fermentation, and vaccine production, but pathogenic L. lactis has been isolated from many clinical cases in recent years, such as the brain of silver carp with Lactococcosis, the liver and spleen of diseased waterfowl, milk samples and padding materials with cow mastitis, and blood and urine from human patients with endocarditis. In dairy farming, where L. lactis has been used as a probiotic in the past, however, some studies have found that L. lactis can cause mastitis in cows, but the lack of understanding of the pathogenesis of mastitis in cows caused by L. lactis has become a new problem. The main objective of this review is to analyze the increasingly serious clinical mastitis caused by L. lactis and combined with the wide application of L. lactis as probiotics, to comprehensively discuss the characteristics and diversity of L. lactis.
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 a 1,100 base pair (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, the L. garvieae infection in Pompano emphasizes the need for better monitoring and control procedures in aquaculture settings.
Background
Tilapia tilapinevirus , also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems.
Methods
Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV.
Results
The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms ( i.e ., river water samples from affected cages (8.50 × 10 ³ to 2.79 × 10 ⁵ copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 10 ³ to 3.53 × 10 ⁴ copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.
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.
Simple Summary
Lactococcus garvieae is an emerging pathogen that causes lactococcosis, which is a septicemia disease that, as per reports, primarily affects rainbow trout and tilapia in various parts of the world. In rainbow trout, the clinical disease is very similar to other diseases caused by Gram-positive bacteria, including weissellosis, which has been previously reported in trout in Mexico. Following septicemia outbreaks that tested negative for weissellosis, a study was conducted to determine the etiology. This study confirms, for the first time, the presence of L. garvieae in processes of a septicemia disease affecting five rainbow trout farms in Mexico. Fourteen isolates were obtained from different geographical regions, but presented homogenous biochemical and protein profiles, as well as weak genetic heterogeneity. This study evidences the wide distribution and incidence of lactococcosis cases at rainbow trout farms in Mexico, demonstrating the need to develop vaccination programs against this disease.
Abstract
Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a Mexican surveillance program for the detection of fish diseases. Fourteen L. garvieae isolates were recovered from sampled fish, as confirmed by biochemical tests, 16S rRNA gene sequencing, and clinical and histological insights. The biochemical and protein profiles of the isolates obtained were homogeneous. Repetitive extragenic palindromic—(REP)—and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) analyses established weak genetic heterogeneity. Rainbow trout challenged with two of the isolates used at different bacterial concentrations (10⁻² and 10⁻⁴ CFU/mL) showed melanosis, and hemorrhages were observed in the fins, liver, kidney, and spleen. Isolates were obtained from all of the organs sampled, including from surviving fish, as either pure or mixed cultures. The present study is the first to confirm the presence of L. garvieae as the agent of severe lactococcosis outbreaks in the two primary Mexican states for trout farming.
Tilapia (Oreochromis niloticus) farming on Lake Kariba, Zambia has been practiced since early 2000s. In recent years, the industry has experienced disease outbreaks with clinical signs consistent with streptococcus-like infections. Affected fish were growers weighing approximately 200 g or above from farms designated A and B, reared in cages on the lake. The objective of this study was to identify and characterize the causative bacteria of these outbreaks. A total of eighty-six diseased fish were sampled between 2014 and 2016. Bacteria were cultured from internal organs and were characterized by biochemical tests, PCR and sequencing of the 16S rRNA gene or 16S23S intergenic spacer region. The results showed the prevalence of Lactococcus garvieae at farm A in 2015 and 2016 outbreaks (15/20 and 23/40, respectively) and at farm B (6/20) in 2016. Infection of naïve fish with one isolate of L. garvieae recovered from the outbreaks resulted in clinical signs and mortalities akin of what had been observed during the outbreaks, confirming Koch's postulates. In the 2014 outbreaks, the low prevalence of Streptococcus agalactiae (4/15 and 2/11 respectively at farms A and B) and S. iniae (2/15) at farm A suggests that other pathogens were likely involved in the disease outbreak observed.
Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.
Aims:
The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods.
Methods and results:
The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92·5 % at a threshold Cq value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a Cq value of 33·7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the Cq threshold of 35 (34·5 and 34·4 respectively), while only S. agalactiae were detected with a Cq value of 30. The limit of detection of the assay was 1·7 copies µl-1 at Cq 35. Discrepancies between molecular and culture-based methods of enumeration were noted.
Conclusions:
The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a Cq 30 would be a very meaningful cut-off, allowing for detection of infected fish whilst ruling out all false positives.
Significance and impact of the study:
This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.
In this study, the diseased fish samples were collected from fish farms in the nine different dam lakes during the summer
months between July 2014 and September 2018. It is isolated twenty-two bacteria from diseased rainbow trout (Oncorhynchus mykiss) eyes
showing exophthalmos symptoms. All isolates identification was carried out by using conventional biochemical tests, API 20 Strep test kit and
PCR test. ATCC 43921 reference strain of the Lactococcus garvieae was used as a positive control. The PCR test was confirmed by using the
pLG-1 and pLG-2 reference genes (16S rRNA) specific for the L. garvieae species. As a result of this study, all of the twenty-two isolates
isolated from the eyes of diseased rainbow trout were confirmed as L. garvieae by PCR test. According to the results of antibiotic susceptibility
test, all of the L. garvieae isolates were resistant to streptomycin, sulfamethoxazole, and sulfamethoxazole/trimethoprim. The same isolates have
been determined to be resistant to ampicillin (94.2%), oxytetracycline (72.5%), erythromycin (55.07%), oxolinic acid (43.5%), enrofloxacin
(26.09%), doxycycline (20.3%), amoxicillin and florfenicol (8,7%) as well. Thus, florfenicol, amoxicillin, doxycycline, and enrofloxacin were
found as the most effective antibiotics.
In 2016, Barramundi, Lates calcaifer juveniles imported from Thailand to the aquaculture station at Sultan Qaboos University exhibited remarkable external clinical signs of losing scales, severe muscle necrosis, and ascites. Fin rot was also observed in the diseased fish. The mortality reached more than 90 % and it was assumed that certain bacterial species were associated with the diseased fish. The objective of the present study was to investigate the infection route of disease, revealing the causative agent and finding the most effective antibiotic treatment. The suspected pathogen vehicle was mosquito larvae in the outlet of the culture tank as bacterium was isolated inside the mosquito larvae. Primary, traditional phenotypic tests and the vitic test confirmed that the bacteria were pathogenic Aeromonas sobria and Lactococcus garvieae. Eight of the most commonly used antibiotics in the aquaculture industry was used for antibiotic susceptibility test. It showed that that Gentamycin was the most effective antibiotic while the most effective environmentally friendly source was henna, Lawsonia inermis, at a concentration of 10%.
A strain of lactic acid bacteria, designated 159469 T , isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469 T was closely related to Lactococcus garvieae ATCC 43921 T , showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA–DNA hybridization value of 50.7 % were determined for the genome of strain 159469 T , when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469 T (=LMG 30040 T =DSM 104842 T).
Lactococcus garvieae is the causative agent of lactococcosis, a hyperacute, haemorrhagic septicaemia of fish. This bacterium is also considered an emerging zoonotic pathogen, as reports of human infection are increasing. Significant economic loss in aquaculture is suffered as a result of lactococcosis, as numerous freshwater and marine species of commercial interest are affected. Development of antibiotic resistance in L. garvieae to several chemotherapeutic agents complicates and restricts treatment options. Effective, sustainable treatment and prevention options are thus needed, but progress is impeded by the lack of knowledge concerning several aspects of the disease and the pathogen. This review aims to present the latest research on L. garvieae, with specific focus on pathogenesis, virulence factors, risks associated with chemotherapeutic administration and possible control options.
This manuscript describes a series of Lactococcus garvieae outbreaks in rainbow trout farms in N. and N.W. Greece. The disease, termed "Lactococcosis", is associated with water temperatures usually above 14°C; in the first year of disease appearance under such circumstances, mortalities often reached 40% of the total predicted production. The outbreaks occurred during the warm seasons of 2003-2006, and affected mainly the fish over 20 g. The disease was diagnosed in farms located at the two main trout culturing areas of Greece (Epirus and Macedonia) and the suspected initial source of the disease was the import of infected rainbow trout from Spain. The phenotypic characteristics of the isolates indicated that they belong to Lactococcus garvieae; final identification was confirmed by PCR amplification.
The disease outbreak occurred in rainbow trout (15-107g) at an average water temperature of 13.5°C during september-december 2012. The cumulative mortality rate was approximately 15 % within 4 months. The most typical symptoms in infected fish were marked mono and bilateral exophthalmos, opacity and thickening, along with pseudomembrane formation in swim-bladder and bulbus arteriosus walls described for the first time during an epizootic outbreak of lactococcosis, petechial and focal haemorrhages in viscera, enlarged liver and spleen. 25 bacterial isolates from sick fish were identified as Lactococcus garvieae by morphological, physiological, and biochemical features and confirmed by PCR using species-specific primers. Further more, 16S rRNA gene of one isolate (KC4) was partially sequenced and showed 100 % identity with the Genbank sequences of L. garvieae. According to the histopathological examination, severe panophthalmitis, peri and myocarditis and gastritis were described. Pathological findings were also observed in kidney and liver. Subcapsular and sinuzoidal mononuclear cell infiltration and necrosis of the hepatocytes has not been described previously for lactococcosis. Moreover, this is the first report of a focal mononuclear cell infiltration in the lamina propria of stomach in some fish. In conclusion, this study presents interesting and original data on the caracterization of L. garvieae isolated from rainbow trout, particularly new histopathological observations.
Five isolates of Gram positive, cocci-shaped, non-hemolytic bacteria were isolated from dis-eased rainbow trout, Oncorhynchus mykiss in Taiwan. The bacteria were identified as Lactococcus garvieae by physiological, biochemical and 16S rRNA gene sequence analysis. The diseased fish showed erratic swimming, dark discoloration, and exophthalmia; the mortality varied from 10 to 20% in the farms. Histological examination revealed lesions in the brain, heart, spleen, kidney, small intestine and eye. These lesions were characterized by fibroplasia at the serosa of organs. This is the first report of L. garvieae associated with rainbow trout disease in Taiwan.
Lactococcus garveiae outbreaks in farmed rainbow trout are described from different parts of Iran. The disease outbreaks were associated with water quality parameters particularly high water temperature (>14°C) and poor health management. The peak of outbreaks occurred during late spring till late summer with mortalities varied from 10-50%. The disease was diagnosed in fish of mainly above 50 g in seven provinces of Iran located at the northwest, northeast and southwest. The phenotypic and molecular studies of the recovered isolates of bacteria showed that they belonged to both L. garvieae and Streptococcus iniae but isolation of L. garvieae was more frequent than S. iniae.
The population structure and diversity of Lactococcus garvieae, an emerging pathogen of increasing clinical significance, was determined at both gene and genome level. Selected lactococcal isolates of various origins were analyzed by a multi locus sequence typing (MLST). This gene-based analysis was compared to genomic characteristics, estimated through the complete genome sequences available in database. The MLST identified two branches containing the majority of the strains and two branches bearing one strain each. One strain was particularly differentiated from the other L. garvieae strains, showing a significant genetic distance. The genomic characteristics, correlated to the MLST-based phylogeny, indicated that this "separated strain" appeared first and could be considered the evolutionary intermediate between Lactococcus lactis and L. garvieae main clusters. A preliminary genome analysis of L. garvieae indicated a pan-genome constituted of about 4100 genes, which included 1341 core genes and 2760 genes belonging to the dispensable genome. A total of 1491 Clusters of Orthologous Genes (COGs) were found to be specific to the 11 L. garvieae genomes, with the genome of the "separated strain" showing the highest presence of unique genes.
An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at >= 107.0, which corresponded with an increase in mortality. The kinetics of IFNalpha and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.
Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 1×10(8) and 1×10(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon.
Gram-positive cocci recovered from diseased rainbow trout from a farm in England were characterized by different methods, including pulsed field gel electrophoresis, as virulent Lactococcus garvieae serogroup 2 (pulsotype A1). Groups of rainbow trout were kept at a range of temperatures and injected intraperitoneally (i.p.) with one of the UK isolates, L. garvieae 00021. The 18 degrees C and 16 degrees C groups showed 67% and 28% mortality, respectively, by day 27 post-injection. Fish kept at 14 degrees C or lower were less susceptible (< or =3% mortality). Raising the temperature of all groups to 18 degrees C at day 27 post-injection did not result in recurrence of the disease, even though viable bacteria were recovered from all groups 42 days later. Grayling were highly susceptible, with 65% mortalities when challenged with 200 colony forming unit fish(-1) by i.p. injection and 37% mortalities when exposed to effluent water from tanks containing affected rainbow trout. Other fish species tested, Atlantic salmon, brown trout and seven cyprinid species, were less susceptible. Viable L. garvieae was isolated from the internal organs of all species tested at the end of the trials, suggesting that they may pose a threat as possible carriers to susceptible farmed and wild fish.
Black rockfish (Sebastes schlegeli) is an important mariculture species in Korea. The production of this fish is drastically declined due to bacterial diseases, particularly streptococcosis caused by Lactococcus garvieae. The bacterial surface characteristics of SJ7 and TY6 were found to have capsule but not NB13 and YS18. The experiential evaluation of L. garvieae pathogenicity, the capsular isolates showed high cumulative mortality i.e. SJ7 (100%) and TY6 (60%) compared to non-capsular isolates. Based on this result the capsular isolates L. garvieae were highly suspected as the causative agent of streptococcosis in rockfish.
Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch’s postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.
Objective: Acute mortality with clinical symptoms of streptococcal-like infections was observed in red tilapia Oreochromis sp. cultured in floating cages in Prachin Buri Province, Thailand, during May 2023. Herein, we identified an emerging pathogen, Lactococcus garvieae, as the etiological agent.
Methods: After bacterial isolation from the brain and kidney of diseased fish, identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK 2 system. Sequencing of the 16S ribosomal RNA (rRNA) gene and phylogenetic analysis were applied to confirm bacterial species. Antimicrobial susceptibility testing was conducted. Histopathological findings in the brain, kidney, spleen, liver, and heart were evaluated.
Result: From 20 fish samples, L.garvieae (n=18 isolates) and Streptococcus agalactiae (n=2 isolates) were identified. A phylogenetic tree of the 16S rRNA gene revealed that Thai isolates of either L. garvieae or S. agalactiae clustered with reference piscine isolates from intercontinental locations. Our isolates showed resistance against quinolones while being susceptible to other antimicrobials. Histopathological changes demonstrated severe septicemic conditions, with more invasive lesions—especially in the heart and liver—being apparent in L. garvieae-infected fish compared to S. agalactiae-infected fish.
Conclusion: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.
Streptococcus iniae is a bacterial pathogen that causes streptococcosis, leading to significant losses in fish aquaculture globally. This study reported a newly developed probe‐based quantitative polymerase chain reaction (qPCR) method for the detection of S. iniae . The primers and probes were designed to target the lactate oxidase gene. The optimized method demonstrated a detection limit of 20 copies per reaction and was specific to S. iniae , as evidenced by no cross‐reactivity when assayed against genetic materials extracted from 23 known aquatic animal pathogens, and fish samples infected with Streptococcus agalactiae or Streptococcus dysgalactiae . To validate the newly developed qPCR protocol with field samples, fish specimens were systematically investigated following the Food and Agriculture Organization of the United Nations & Network of Aquaculture Centres in Asia‐Pacific three diagnostic levels approach, which integrated basic and advanced techniques for disease diagnosis, including observation of gross signs (level I), bacterial isolation (level II), qPCR and 16S rDNA sequencing (level III). The result showed that 7/7 affected farms (three Asian seabass farms and four tilapia farms) experiencing clinical signs of streptococcosis were diagnosed positive for S. iniae . qPCR assays using DNA extracted directly from fish tissue detected S. iniae in 11 out of 36 fish samples (30.6%), while 24 out of 36 samples (66.7%) tested positive after an enrichment step, including apparently healthy fish from affected farms. Bacterial isolation of S. iniae was only successful in a proportion of clinically diseased fish but not in healthy‐looking fish from the same farm. Overall, the newly developed qPCR protocol combined with enrichment would be a useful tool for the diagnosis and surveillance of S. iniae infections in fish populations, thereby aiding in the disease control and prevention.
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.
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.
Lactococcosis in fish has been associated with Lactococcus garvieae and the recently described L. petauri. However, the relevance of these emerging fish pathogens to Nile tilapia still requires thorough understanding. This study investigated lactococcosis outbreaks in Nile tilapia on Brazilian farms and characterized the isolates through multilocus sequence typing (MLST), virulence to Nile tilapia, and antimicrobial susceptibility. Lactococcosis outbreaks were monitored from 2019 to 2022 throughout Brazil. The outbreaks occurred mainly during warmer months, and co-infections were observed in four farms, whereas concurrent bacterial infections were identified in all farms. Since the sequence of the 16S rRNA was not capable of differentiating between L. petauri and L. garvieae, Lactococcus spp. isolates were identified at the species level using the gyrB gene sequence. In total, 30 isolates were classified as L. petauri and two as L. garvieae. According to the MLST, all L. petauri isolates were grouped in the sequence type (ST) 24, except for one isolate which belonged to the newly described ST47. A new ST was also described for the L. garvieae isolates identified, ST46. The L. petauri ST24 and ST47 were characterized as singletons, whereas L. garvieae ST46 was grouped with ST16 and ST17 and formed CC17. For the challenge trial, an L. petauri ST24 isolate was chosen considering that this MLST lineage was the most frequently observed. Fish were challenged by intraperitoneal injection and different bacterial doses were used (10 6 to 10 2 CFU per fish). The infection in the challenged fish progressed very rapidly, and within 48 h post-challenge clinical signs and the first mortalities were observed. The pathogenicity of L. petauri to Nile tilapia was confirmed. The estimated LD50 was 5.74 × 10 3 CFU 15 days post-challenge. Provisional epidemiological cutoff values were determined for L. petauri for six antimicrobial agents from different drug classes. All isolates were characterized as wild type (WT) for neomycin and J o u r n a l P r e-p r o o f Journal Pre-proof oxytetracycline, whereas 96.67 % of the isolates were characterized as WT for amoxicillin, erythromycin, and florfenicol, and 83.33 % were WT for norfloxacin. The L. petauri ST24 was observed in different regions of the country, illustrating a rapid expansion of this bacterial lineage.
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.
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.
Tilapia is one of the most extensively farmed fish on a global scale. Lately, many studies have been carried out to select and produce probiotics for cultured fish. Bacteria from the genera Bacillus, Lactiplantibacillus (synonym: Lactobacillus), and Lactococcus are the most widely studied with respect to their probiotic potential. Among these microorganisms, Lactococcus lactis has outstanding prospects as a probiotic because it is generally recognized as safe (GRAS) and has previously been shown to exert its probiotic potential in aquaculture through different mechanisms, such as competitively excluding pathogenic bacteria, increasing food nutritional value, and enhancing the host immune response against pathogenic microorganisms. However, it is not sufficient to simply select a microorganism with significant probiotic potential for commercial probiotic development. There are additional challenges related to strategies involving the mass production of bacterial cultures, including the selection of production variables that positively influence microorganism metabolism. Over the last ten years, L. lactis production in batch and fed-batch processes has been studied to evaluate the effects of culture temperature and pH on bacterial growth. However, to gain a deeper understanding of the production processes, the effect of hydrodynamic stress on cells in bioreactor production and its influence on the probiotic potential post-manufacturing also need to be determined. This review explores the trends in tilapia culture, the probiotic mechanisms employed by L. lactis in aquaculture, and the essential parameters for the optimal scale-up of this probiotic.
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 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 non-specific 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 24h 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 LD50 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.
This article is protected by copyright. All rights reserved
Ozone nanobubble (NB-O3) is a promising technology for improving dissolved oxygen and reducing bacterial concentration in aquaculture systems. Here, we investigated the effects of NB-O3 on the innate immunity of fish by monitoring the expression levels of nonspecific immune-related genes (IL-1β, IL-2β, TNF-α), heat-shock protein genes (HSP70, HSP90-α), and a bacteriolytic enzyme, C-type lysozyme, gene (LYZ) post-treatment with this technology. Following exposure to NB-O3, the different tissues of Nile tilapia (Oreochromis niloticus) were collected over time for quantitative real-time PCR (qPCR) analysis. The expression of all the genes evaluated in the gills, the head kidney, and the spleen of the NB-O3 treated group was significantly up-regulated compared to that in the untreated control group. The expression levels were the highest (approx. 2 to 4-fold) at 15 min and 3 h post-exposure and then decreased from 6 to 24 h. These findings suggested that NB-O3 could switch on the innate immunity genes of Nile tilapia. Thus, we hypothesized that the NB-O3-immune-activated fish would respond more effectively to subsequent bacterial infections, thereby improving survivability compared to that of untreated fish. To test this hypothesis, 3 h post NB-O3 exposed fish and unexposed fish were challenged with a lethal dose of Streptococcus agalactiae. Interestingly, the survival rate of the NB-O3 group was significantly higher than that of the non-treated controls, with a relative percent survival (RPS) of 60-70%. Together, these findings indicate, for the first time, that NB-O3 may trigger the nonspecific defense system of the fish, thereby improving fish survivability during subsequent bacterial infections. This research identified another potential benefit of NB-O3 in aquaculture for preventing infectious bacterial diseases.
Scale drop disease virus (SDDV) is a Megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, a sensitive and specific SYBR Green qPCR assay is reported. The qPCR primers target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected as few as 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens found commonly in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 SDD-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously using a semi-nested conventional PCR. The higher sensitivity of the SDDV qPCR assay will thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed SDDV DNA loads to vary unpredictably (0.08-6.80 × 104 viral DNA copies per 200 ng DNA template) among and between 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of >1 tissue type could thus be beneficial.
A total of 200 moribund rainbow trout with clinical signs of a hyperacute haemorrhagic septicemia were collected from rainbow trout farms in Fars, Kohkiloyeh-Boyer Ahmad and Charmohal-Bakhtiari provinces in the south and southwest of Iran during summer 2002 to winter 2008 for detection of Lactococcus garvieae, the causative agent of lactococcosis. Fish kidney samples were cultured aseptically onto brain heart infusion agar plates and incubated at 25°C for 48 h. Using conventional biochemical tests, L. garvieae was detected from 32 fish (16% of total fish samples). Additionally, isolates were confirmed as L. garvieae using a specific PCR assay based on 16S rDNA gene by producing a single band of 1107 bp. Partial analysis of 16S rDNA revealed 100% sequence similarity for all Iranian isolates and there was a close genetic relationship among these isolates and those previously reported from mullet in Taiwan (AF352166) and yellowtail in Japan (AB267897) based on GenBank data. Results of antibiogram tests on L. garvieae isolates showed a high susceptibility to erythromycin, enrofloxacin, chloramphenicol and clarithromycin. In pathogenicity tests, immersion of fish in a bacterial suspension of 6 × 10 5 colony forming unit/ml of L. garvieae in challenge experiments showed 60% mortality during 14 days post-infection. Experimentally, infected fish showed typical blackening of skin haemorrhages, exophthalmia and wide haemorrhages on viscera. The present study provides useful molecular and biochemical information for L. garvieae isolates in Iran compared with those from different hosts and geographic locations.
Vaccination is one of the greatest triumphs of modern medicine, yet we remain largely ignorant of the mechanisms by which successful vaccines stimulate protective immunity. Two recent advances are beginning to illuminate such mechanisms: realization of the pivotal role of the innate immune system in sensing microbes and stimulating adaptive immunity, and advances in systems biology. Recent studies have used systems biology approaches to obtain a global picture of the immune responses to vaccination in humans. This has enabled the identification of early innate signatures that predict the immunogenicity of vaccines, and identification of potentially novel mechanisms of immune regulation. Here, we review these advances and critically examine the potential opportunities and challenges posed by systems biology in vaccine development.
Journal HP: http://www.int-res.com/journals/dao/ Enterococcus seriolicida is the causative agent of an enterococcal infection in yellowtail Seriola quinqueradiata and is antigenically classified into 2 phenotypes, KG+ and KG-. Phenotypic variation from KG- to KG+ occurs readily on an artificial medium. The surface morphologies of the KG- and KG+ phenotypes were differentiated by scanning electron microscopy. KG- cells were more hydrophilic than KG+ cells and were resistant to phagocytosis by yellowtail head kidney phagocytes. The chemiluminescent response of these phagocytic cells was lower with the KG- phenotype than with the KG+ phenotype. The immune responses of yellowtail following injection of the 2 phenotypes differed: higher agglutinating titers were obtained with the KG+ phenotype compared to the KG- phenotype. Thus, the cell surface may function as an anti-phagocytic factor in E. seriolicida and apparently affects its immunogenicity.
A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare.
Genetic variability among Fl. columnare isolates was characterized using restriction fragment length polymorphism analysis of the 16S rDNA gene, intergenic spacer region (ISR) sequencing, and amplified fragment length polymorphism (AFLP) fingerprinting. Thirty Fl. columnare cultures isolated from different fish species and geographical origins as well as reference strains were included in the study. Fifteen isolates belonged to genomovar I while eleven were ascribed to genomovar II. Analysis of the ISR sequence confirmed the genetic differences between both genomovars but revealed a higher diversity among genomovar I isolates. The maximum resolution was provided by AFLP fingerprinting, as up to 22 AFLP profiles could be defined within the species.
We confirmed the division of Fl. columnare isolates from cultured fish into different genogroups. We showed that both genomovars I and II are present in channel catfish from the US. We described a unique genetic group represented by four Fl. columnare isolates from tilapia in Brazil which appears to be related to both genomovars. We were able to further subdivide the species by analysing the ISR. Finally, the use of AFLP allowed us to fingerprint the species at clone level without losing the higher genetic hierarchy of genomovar division.
This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the fish pathogen Fl. columnare.
The antimicrobial susceptibility of 151 clinical Streptococcus suis strains isolated from diseased pigs in Spain was determined by a microdilution method. Isolates were mostly susceptible to beta-lactam antimicrobials, aminoglycosides, enrofloxacin, novobiocin and spectinomycin. More than 87% of the S. suis isolates were resistant to tetracyclines, sulphonamides, macrolides and clindamycin. Strains of serotype 9 were significantly more resistant than strains of serotype 2 (P<0.05) to tylosin (94% versus 77%) and clindamycin (94% versus 64%). Eighty-seven percent of the S. suis isolates were resistant to at least four antimicrobials and nine isolates (6%) were resistant to at least six antimicrobials. The most frequently identified multidrug pattern involved resistance against tetracyclines, sulphonamides, macrolides and lincosamides, with 69% of the isolates exhibiting this resistotype. Fifteen out of the 22 strains of serotype 2 (68.2%), and 84 out of the 98 of the strains of serotype 9 (85.7%) exhibited this resistotype, indicating its widespread distribution among the strains of the two most frequently isolated serotypes.
To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan.
Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3.
The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related.
This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.
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 degrees 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.
The State of World Fisheries and Aquaculture 2022
- Fao
FAO. 2022. The State of World Fisheries and Aquaculture 2022. Towards
Blue Transformation. FAO. https:// doi. org/ 10. 4060/ cc0461en.