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.

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.
RESULTS: 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.
CONCLUSIONS: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.

Lactococcus garvieae is the causative agent of lactococcosis in fish and an emerging zoonotic pathogen with high levels of antimicrobial resistance. We report a case of L. garvieae-associated septicemia in a central bearded dragon (Pogona vitticeps) confirmed via whole-blood PCR and direct sequencing. Following a 30-d course of ceftazidime (20 mg/kg IM q72h), the animal\'s clinical condition had not resolved; leukopenia persisted, with heterophil toxic change. Coelomic ultrasound findings were consistent with preovulatory follicular stasis, folliculitis, and coelomitis. Following surgical ovariectomy and an additional 30-d course of ceftazidime, the animal\'s behavior and appetite returned to normal, the animal tested negative via whole-blood PCR assay, and the CBC was unremarkable. To our knowledge, L. garvieae with L. garvieae-associated clinical disease has not been reported previously in a bearded dragon. We conclude that L. garvieae should be considered as a possible etiologic agent in cases of septicemia in bearded dragons, with the potential for zoonotic transmission warranting further investigation.

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 petauri is a recently described species of the genus Lactococcus. It was reported as an etiological agent of piscine lactococcosis together with Lactococcus garvieae. L. garvieae was already described as an opportunistic pathogen in human infections, with a potential zoonotic role. This paper represents the first report of a human urinary tract infection caused by L. petauri. A 91-year-old man was admitted to the emergency department for a femur fracture consequent to a domestic accident. The fracture was reduced by surgery and a catheterized specimen urine culture revealed a high bacterial load sustained by Gram-positive cocci, identified by Vitek 2 compact as L. garvieae, and subsequently as L. petauri through Internal Transcribed spacer 16S-23S r-RNA amplification. The number of L. petauri infections in humans is expected to rise in the near future mainly due to diagnostic improvement. A dedicated survey on L. garvieae and L. petauri infections in humans should be performed to better understand their role as pathogens and as zoonotic agents.

The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides.
Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc).
Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments.
The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

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 101, 103 or 105 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.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

Lactococcus petauri is an important emergent bacterial pathogen of salmonids in the USA. The purpose of this study was to evaluate the protection conferred to rainbow trout (Oncorhynchus mykiss) against L. petauri by formalin-killed vaccines in immersion and injectable forms, as well as the enhanced protection afforded by booster vaccination. In the first challenge, fish were immunized via intracoelomic injection (IC) or immersion (Imm) routes alone. Approximately 418 degree days (Temperature in degree Celsius × days post-immunization) (dd) Imm, or 622 dd IC post-vaccination, fish were challenged via IC with wild-type L. petauri. In the second experiment, initial Imm vaccination was followed by booster vaccination via Imm or IC routes 273 dd post-immunization along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. petauri by cohabitation with diseased fish 399 dd post-booster administration. A relative percent survival (RPS) of 89.5% and 28% was recorded in the IC and Imm single immunization treatments, respectively. In the second study, an RPS of 97.5%, 10.2%, 2.6% and -10.1% plus approximately 0%, 50%, 20%, and 30% bacterial persistence was recorded in the Imm immunized + IC boosted, Imm immunized + mock IC boosted, Imm immunized + Imm boosted, and Imm immunized + mock Imm boosted treatments, respectively. Only the Imm immunized + IC injection boosted treatments provided significant protection when compared to unvaccinated and challenged treatments (p < 0.05). In conclusion, although both Imm and IC vaccines appear safe for trout, the inactivated Imm vaccines seem to provide only mild and temporary protection against lactococcosis; whereas IC immunized trout develop a significantly stronger protective response in both challenges.