A rare case of primary cutaneous Nocardia vinacea in an immunocompetent patient: A case report and a review of the literature.

The nocardiae are a complex group of bacteria belonging to the aerobic saprophytes actinomycetes. Although nocardiosis typically occurs in immunocompromised patients, infection may occasionally develop in immunocompetent patients as well. Here we describe a rare case of primary cutaneous nocardiosis due to Nocardia vinacea in an immunocompetent 79-year-old patient. Since cutaneous nocardiosis presents variably and mimics other cutaneous infections, acid-fast and Gram stainings on clinical samples are significant to obtain a rapid and presumptive diagnosis.

Characterization of the equine placental microbial population during nocardioform placentitis.

Nocardioform placentitis is a poorly understood disease of equine late gestation. The presence of nocardioform, filamentous branching gram-positive bacteria, has been linked to the disease, with Crossiella equi, Amycolatopsis spp., and Streptomyces spp. being the most frequently identified bacteria. However, these bacteria are not found in all clinical cases in addition to being isolated from healthy, normal postpartum placentas. To better understand this form of placentitis, we analyzed the microbial composition in the equine placenta (chorioallantois) of both healthy postpartum (control; n = 11) and nocardioform-affected samples (n = 22) using 16S rDNA sequencing. We found a lower Shannon index in nocardioform samples, a higher Chao1 index in nocardioform samples, and a difference in beta diversity between control and nocardioform samples (p < 0.05), suggesting the presence of dysbiosis during the disease. In the majority of the NP samples (77 %), one of the following genera-Amycolatopsis, Crossiella, Lentzea, an unidentified member of the Pseudonocardiaceae family, Mycobacterium, or Enterococcus -represented over 70 % of the relative abundance. Overall, the data suggest that a broader spectrum of potential opportunistic pathogens could be involved in nocardioform placentitis, extending beyond the traditionally recognized bacteria, resulting in a similar histomorphological profile.

Complement C1q is involved in the activation of membrane attack complexes, regulation of bacterial infectious inflammation, and apoptosis through overexpression in primary cells of silver pomfret (Pampus argenteus) in vitro.

The complement system is pivotal in innate immune defense, with Complement 1qb (C1qb) playing a key role in recognizing immune complexes and initiating the classical pathway. In this research, we cloned the full-length cDNA of silver pomfret (Pampus argenteus) c1qb and demonstrated its role in mediating defense responses against Nocardia seriolae (N. seriolae) infection, which notably causes significant economic losses in the aquaculture industry. Our investigation revealed that N. seriolae infection led to tissue damage in fish bodies, as observed in tissue sections. Subsequent analysis of differential genes (DEGs) in the transcriptome highlighted genes linked to apoptosis and inflammation. Through experiments involving overexpression and interference of c1qb in vitro, we confirmed that c1qb could suppress N. seriolae-induced apoptosis and inflammation. Moreover, overexpression of c1qb hindered N. seriolae invasion, and the purified and replicated C1qb protein displayed antimicrobial properties. Additionally, our study unveiled that overexpression of c1qb might stimulate the expression of membrane attack complexes (MAC), potentially enhancing opsonization and antibacterial effects. In conclusion, our findings offer valuable insights into the immune antibacterial mechanisms of c1qb and contribute to the development of strategies for controlling N. seriolae.

In vitro activity of omadacycline against clinical isolates of Nocardia.

Nocardiosis typically requires a prolonged treatment duration of ≥6 months and initial combination therapy with 2-3 antibiotics. First-line regimens for nocardiosis are associated with considerable toxicity; therefore, alternative therapies are needed. Omadacycline is an aminomethylcycline with broad antimicrobial activity whose in vitro activity against Nocardia species has not been formally assessed. The in vitro potency of omadacycline was evaluated against 300 Nocardia clinical isolates by broth microdilution. The most common Nocardia species tested were N. cyriacigeorgica (21%), N. nova (20%), and N. farcinica (12%). The most common specimens were respiratory (178 isolates, 59%) and wound (57 isolates, 19%). Omadacycline minimum inhibitory concentrations (MICs) across all Nocardia species ranged from 0.06 µg/mL to 8 µg/mL, with an MIC50 of 2 µg/mL and MIC90 of 4 µg/mL. The lowest MICs were found among N. paucivorans (MIC50 = 0.25 µg/mL, MIC90 = 0.25 µg/mL), N. asiatica (MIC50 = 0.25 µg/mL, MIC90 = 1 µg/mL), N. abscessus complex (MIC50 = 0.5 µg/mL, MIC90 = 1 µg/mL), N. beijingensis (MIC50 = 0.5 µg/mL, MIC90 = 2 µg/mL), and N. otitidiscaviarum (MIC50 = 1 µg/mL, MIC90 = 2 µg/mL). The highest MICs were found among N. farcinica (MIC50 = 4 µg/mL, MIC90 = 8 µg/mL). In vitro potency differed by species among Nocardia clinical isolates. Further studies are warranted to evaluate the potential clinical utility of omadacycline for nocardiosis.

Aggregation-induced emission of TTCPy-3: A novel approach for eradicating Nocardia seriolae infections in aquatic fishes.

Aquatic fishes are threatened by the strong pathogenic bacterium Nocardia seriolae, which challenges the current prevention and treatment approaches. This study introduces luminogens with aggregation-induced emission (AIE) as an innovative and non-antibiotic therapy for N. seriolae. Specifically, the AIE photosensitizer, TTCPy-3 is employed against N. seriolae. We evaluated the antibacterial activity of TTCPy-3 and investigated the killing mechanism against N. seriolae, emphasizing its ability to aggregate within the bacterium and produce reactive oxygen species (ROS). TTCPy-3 could effectively aggregate in N. seriolae, generate ROS, and perform real-time imaging of the bacteria. A bactericidal efficiency of 100% was observed while concentrations exceeding 4 µM in the presence of white light irradiation for 10 min. In vivo, evaluation on zebrafish (Danio rerio) confirmed the superior therapeutic efficacy induced by TTCPy-3 to fight against N. seriolae infections. TTCPy-3 offers a promising strategy for treating nocardiosis of fish, paving the way for alternative treatments beyond traditional antibiotics and potentially addressing antibiotic resistance.

Disseminated nocardiosis and anti-GM-CSF antibodies.

Infections that are unusually severe or caused by opportunistic pathogens are a hallmark of primary immunodeficiency (PID). Anti-cytokine autoantibodies (ACA) are an emerging cause of acquired immunodeficiency mimicking PID. Nocardia spp. are Gram-positive bacteria generally inducing disseminated infections in immunocompromised patients, but seldom also occurring in apparently immunocompetent hosts. Anti-GM-CSF autoantibodies are associated with autoimmune pulmonary alveolar proteinosis (PAP). In those patients, an increased incidence of disseminated nocardiosis and cryptococcosis has been observed. It is unclear whether the PAP or the autoantibodies predispose to the infection. We report an apparently immunocompetent woman presenting with disseminated nocardiosis without any evidence of PAP. Clinical data and radiological images were retrospectively collected. Lymphocyte populations were analyzed by flow cytometry. Anti-GM-CSF autoantibodies were measured by ELISA. A 55-year-old otherwise healthy woman presented with cerebral and pulmonary abscesses. Personal and familial history of infections or autoimmunity were negative. After extensive examinations, a final diagnosis of disseminated nocardiosis was made. Immunologic investigations including neutrophilic function and IFN-γ/IL-12 circuitry failed to identify a PID. Whole-exome sequencing did not find pathogenic variants associated with immunodeficiency. Serum anti-GM-CSF autoantibodies were positive. There were no clinical or instrumental signs of PAP. Trimethoprim-sulfamethoxazole and imipenem were administered, with progressive improvement and recovery of the infectious complication. We identified anti-GM-CSF autoantibodies as the cause of disseminated nocardiosis in a previously healthy and apparently immunocompetent adult. This case emphasizes the importance of including ACA in the differential diagnosis of PID, especially in previously healthy adults. Importantly, anti-GM-CSF autoantibodies can present with disseminated nocardiosis without PAP.

Co-infection of Nocardia and Aspergillus fumigatus in a immunosuppressed patient: Case report.

BACKGROUND: Nocardia and Aspergillus fumigatus are opportunistic pathogenic fungus that has a major impact on the mortality of rheumatoid arthritis patients. Opportunistic infections in immunocompromised patients present diagnostic challenges. Nocardia and A fumigatus are both easily overlooked because of their rarity, leading to delayed diagnosis and treatment. CASE PRESENTATION: We report an infection caused by steroid use in a patient with rheumatoid arthritis. A 76-year-old man with a history of rheumatoid arthritis was admitted to our hospital because of cough, expectoration and fever for 10 days. The patient had low immune function, granulocytopenia, diffuse infiltration could be seen on chest computed tomography, and BAL fluid galactomannan level of 1.3 S/CO. The microbiological findings reflect a possible co-infection with Nocardia and A fumigatus. Voriconazole was used to treat pulmonary aspergillosis, ceftriaxone and Trimethoprim-Sulfamethoxazole were used to treat Nocardia. After timely targeted medication administration, the patient was discharged with a good prognosis. CONCLUSION: Co-infection is more common in immunosuppressed patients and warrants attention in clinical practice. Early diagnosis and treatment can help patients with Co-infection of Nocardia and A fumigatus achieve better prognosis.

Disruption of a kasB homolog gene (kasB) causes attenuation of cell invasion and virulence of Nocardia seriolae.

Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. ß-ketoacyl-ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene-deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC-Ns. Compared to the wild-type (WT) strain, the ΔkasB showed a measurably growth defect in vitro but retained the acid-fastness in acid-fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron-transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head-kidney of the large yellow croaker (LYC-hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell-wall lipids in N. seriolae and play a crucial role in its pathogenicity.

Gastrointestinal dysbiosis induced by Nocardia sp. infection in tilapia.

Nocardiosis has caused high mortalities among fish cultures; however, the effects of Nocardia infections in the fish gastrointestinal microbiota are unknown. In this research, tilapia was infected with Nocardia sp., to analyze the effect of infection on the gastrointestinal microbiota. Tilapia infected with Nocardia sp. reported a 46 % survival (100 % in non-infected). Moreover, the infection caused severe damage to the stomach microbiota, with a loss of diversity and a significant increase of Proteobacteria (94.8 %), resulting in a negative correlation network between Proteobacteria and other important phyla. Nocardia sp. is an emerging pathogen capable of inducing dysbiosis and causing significant mortalities.

Trimethoprim-sulfamethoxazole significantly reduces the risk of nocardiosis in solid organ transplant recipients: systematic review and individual patient data meta-analysis.

BACKGROUND: Whether trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis prevents nocardiosis in solid organ transplant (SOT) recipients is controversial. OBJECTIVES: To assess the effect of TMP-SMX in the prevention of nocardiosis after SOT, its dose-response relationship, its effect on preventing disseminated nocardiosis, and the risk of TMP-SMX resistance in case of breakthrough infection. METHODS: A systematic review and individual patient data meta-analysis. DATA SOURCES: MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, Web of Science Core Collection, and Scopus up to 19 September 2023. STUDY ELIGIBILITY CRITERIA: (a) Risk of nocardiosis between SOT recipients with and without TMP-SMX prophylaxis, or (b) sufficient details to determine the rate of TMP-SMX resistance in breakthrough nocardiosis. PARTICIPANTS: SOT recipients. INTERVENTION: TMP-SMX prophylaxis versus no prophylaxis. ASSESSMENT OF RISK OF BIAS: Risk Of Bias In Non-randomized Studies-of Exposure (ROBINS-E) for comparative studies; dedicated tool for non-comparative studies. METHODS OF DATA SYNTHESIS: For our primary outcome (i.e. to determine the effect of TMP-SMX on the risk of nocardiosis), a one-step mixed-effects regression model was used to estimate the association between the outcome and the exposure. Univariate and multivariable unconditional regression models were used to adjust for the potential confounding effects. Certainty of evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. RESULTS: Individual data from three case-control studies were obtained (260 SOT recipients with nocardiosis and 519 uninfected controls). TMP-SMX prophylaxis was independently associated with a significantly decreased risk of nocardiosis (adjusted OR = 0.3, 95% CI 0.18-0.52, moderate certainty of evidence). Variables independently associated with an increased risk of nocardiosis were older age, current use of corticosteroids, high calcineurin inhibitor concentration, recent acute rejection, lower lymphocyte count, and heart transplant. Breakthrough infections (66/260, 25%) were generally susceptible to TMP-SMX (pooled proportion 98%, 95% CI 92-100). CONCLUSIONS: In SOT recipients, TMP-SMX prophylaxis likely reduces the risk of nocardiosis. Resistance appears uncommon in case of breakthrough infection.

Central nervous system nocardiosis diagnosed by metagenomic next-generation sequencing: A case series and literature review.

BACKGROUND: Central nervous system (CNS) nocardiosis is a rare suppurative disease caused by the genus Nocardia. It is found most frequently in immunocompromised individuals. OBJECTIVES: In this study, we retrospectively reviewed the clinical presentations, laboratory examination, therapy and outcomes of 9 patients with CNS nocardiosis diagnosed using metagenomic next-generation sequencing (mNGS) in our hospital. MATERIAL AND METHODS: We reviewed 9 patients with confirmed diagnosis of CNS Nocardia infection from January 2017 to December 2021 in the Department of Neurology at The Third Affiliated Hospital, Sun Yat-sen University (Guangzhou, China). In addition, we searched literature related to CNS Nocardia infection on PubMed and included all case reports with proven CNS nocardiosis since 2016. RESULTS: The metagenomic next-generation sequencing (mNGS) of CSF can be used for the rapid diagnosis of nocardiosis in CNS and N. farcinica are the most commonly isolated species. Underlying autoimmune diseases, immunosuppressive agents including corticosteroids and organ transplantation are predisposing factors of developing CNS nocardiosis. Single or multiple hyper-enhanced ring lesions indicative of cerebral abscesses are commonly presented in brain imaging. Trimethoprim-sulfamethoxazole (TMP-SMX) is used as the primary agent for the antibacterial therapy and in combination with other antibacterial agents. CONCLUSION: Our study demonstrated that mNGS of CSF can be conducted for definitive and rapid diagnosis for CNS nocardiosis.

Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

Clinical and microbiological characteristics of nocardiosis: A 5-year single-center study in Crete, Greece.

Nocardiosis is a rare disease affecting both immunocompromised and immunocompetent hosts, presented in various clinical forms ranging from localized to disseminated infection. Aim of the present study was to investigate the clinical and microbiological characteristics of nocardiosis, antimicrobial resistance profiles, treatment, and outcomes of Nocardia infection over the last 5 years at our institution. The medical records and microbiological data of patients affected by nocardiosis and treated at the university hospital of Heraklion, Crete, Greece, between 2018 and 2022, were retrospectively analyzed. The isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and through sequencing of 16S rRNA. Antimicrobial susceptibility for 17 agents was determined by E-test and results were interpreted according to CLSI guidelines. Among the 28 Nocardia isolates, eight species were identified, with Nocardia brasiliensis being the most prevalent (32.1%), followed by Nocardia otitidiscaviarum (25%), and Nocardia farcinica (14.3%). Skin and soft tissue infections were the most common presentations, noted in 13 (50%) patients, followed by pulmonary infection presented in 10 (38.5%) patients. Fifteen patients (57.7%) had at least one underlying disease, and 11 (42.3%) were on immunosuppressive or long-term corticosteroid treatment. Susceptibility rates of linezolid, tigecycline, amikacin, trimethoprim-sulfamethoxazole, moxifloxacin, and imipenem were 100, 100, 96.4, 92.9, 82.1, and 42.9%, respectively. The 26 patients in this study were treated with various antibiotics. Mortality rate was 3.8%, and the patient who died had disseminated infection. Since epidemiology and antimicrobial susceptibility are evolving, continuous surveillance is mandatory in order to initiate appropriate treatment in a timely manner.

Molecular detection of Nocardia: development and application of a real-time PCR assay in sputum and bronchoalveolar lavage fluid samples.

The diagnosis of pulmonary nocardiosis remains challenging. Rapid detection of Nocardia is of primary importance for early diagnosis and precise treatment of nocardiosis. In this study, our objective was to develop and validate a new TaqMan real-time PCR (qPCR) assay for rapidly detecting Nocardia spp. in respiratory samples. Based on published sequence data, primers in a conserved region of the 16S rRNA gene and a probe within that region that was specific for Nocardia were designed. The distinction effect of the qPCR assay was assessed between Nocardia and other respiratory-associated bacteria. Furthermore, the specificity and sensitivity of the assay were evaluated in respiratory clinical samples (n = 205), compared to the results of 16S rRNA gene amplicon sequencing and clinical diagnosis. The qPCR assay exhibited high specificity, sensitivity, repeatability, and reproducibility. The limit of detection of standard plasmid DNA was 3 × 102 copies/mL. Additionally, the qPCR assay was applied to the direct detection of 205 clinical respiratory samples. The specificity and sensitivity of the qPCR were all 100% compared to 16S rRNA gene amplicon sequencing, as well as 98.4% and 100% compared to clinical diagnosis respectively. The qPCR yielded results within 3 h of sample processing, compared to several days for culture, significantly reducing turnaround time. The results suggest that the new qPCR assay developed in this study provides reliable and rapid detection of Nocardia spp. in the respiratory tracts and is expected to reduce the time required for diagnosing and treating nocardiosis.

Molecular identification and antimicrobial resistance pattern of Nocardia isolated from 14 diseased dogs and cats.

Nocardia are ubiquitous, saprophytic and opportunistic bacteria. They cause a set of pyogenic clinical infections in animals and humans, particularly immunocompromised patients, mostly affecting the skin and respiratory tract, with refractoriness to conventional therapy. The most descriptions of nocardial infections in companion animals involve case reports, and there are scarce case series studies focused on canine and feline nocardiosis in which diagnosis has been based on molecular techniques. We investigated epidemiological aspects, clinical findings, in vitro susceptibility profile, and molecular identification of Nocardia using PCR-based method targeted 16S rRNA gene in twelve dogs and two cats. Among dogs were observed cutaneous lesions (8/12 = 67%), pneumonia (3/12 = 25%), and encephalitis (2/12 = 17%), whereas cats developed cutaneous lesions and osteomyelitis. Nocardia and canine morbillivirus coinfection was described in six dogs (6/12 = 50%). A high mortality rate (6/8 = 75%) was seen among dogs. Three dogs (3/4 = 75%) and one cat (1/2 = 50%) with systemic signs (pneumonia, encephalitis, osteomyelitis), and 83% (5/6) of dogs with a history of concomitant morbillivirus infection died. N. nova (5/12 = 42%), N. cyriacigeorgica (3/12 = 25%), N. farcinica (2/12 = 17%), N. veterana (1/12 = 8%), and N. asteroides (1/12 = 8%) species were identified in dogs, whereas N. africana and N. veterana in cats. Among the isolates from dogs, cefuroxime (12/12 = 100%), amikacin (10/12 = 83%), gentamycin (10/12 = 83%), and imipenem (10/12 = 83%) were the most effective antimicrobials, whereas cefuroxime, cephalexin, amoxicillin/clavulanic acid, imipenem, and gentamycin were efficient against isolates from cats. Multidrug resistance was observed in 36% (5/14) of isolates. We describe a variety of Nocardia species infecting dogs and cats, multidrug-resistant ones, and a high mortality rate, highlighting a poor prognosis of nocardiosis in companion animals, particularly among animals systemically compromised or coinfected by canine morbillivirus. Our study contributes to species identification, in vitro antimicrobial susceptibility profile, clinical-epidemiological aspects, and outcome of natural Nocardia-acquired infections in dogs and cats.

Genetic variability of the 16S rRNA gene of Nocardia brasiliensis, the most common causative agent of actinomycetoma in Latin America and the Caribbean.

Mycetoma is a neglected tropical disease (NTD) declared by the World Health Organization (WHO) in 2016. It is characterized by the progressive growth of nodules and granulomatous lesions on the legs, arms, and trunk. It is potentially disfiguring and causes disability or amputations in working-age people from marginalized areas. The causative agents can be fungi (eumycetoma) or actinobacteria (actinomycetoma), the latter being the most common in America and Asia. Nocardia brasiliensis is the most important causal agent of actinomycetoma in the Americas. Taxonomic problems have been reported when identifying this species, so this study aimed to detect the 16S rRNA gene variations in N. brasiliensis strains using an in silico enzymatic restriction technique. The study included strains from clinical cases of actinomycetoma in Mexico, isolated from humans and previously identified as N. brasiliensis by traditional methods. The strains were characterized microscopically and macroscopically, then subjected to DNA extraction and amplification of the 16S rRNA gene by PCR. The amplification products were sequenced, and consensus sequences were constructed and used for genetic identification and in silico restriction enzyme analysis with the New England BioLabs® NEBcutter program. All study strains were molecularly identified as N. brasiliensis; however, in silico restriction analysis detected a diversity in the restriction patterns that were finally grouped and subclassified into 7 ribotypes. This finding confirms the existence of subgroups within N. brasiliensis. The results support the need to consider N. brasiliensis as a complex species.

Intracellular behavior of Nocardia seriolae and its apoptotic effect on RAW264.7 macrophages.

Nocardia seriolae, an intracellular gram-positive pathogen, is prone to infecting immunocompromised and surface-damaged fish, causing serious losses to the aquaculture industry. Although a previous study has demonstrated that N. seriolae infects macrophages, the persistence of this bacterium in macrophages has not been well characterized. To address this gap, we used the macrophage cell line RAW264.7, to investigate the interactions between N. seriolae and macrophages and deciphered the intracellular survival mechanism of N. seriolae. Confocal and light microscopy revealed that N. seriolae entered macrophages 2 hours post-inoculation (hpi), were phagocytosed by macrophages at 4-8 hpi, and induced the formation of multinucleated macrophages by severe fusion at 12 hpi. Flow cytometry, evaluation of mitochondrial membrane potential, release of lactate dehydrogenase, and observation of the ultrastructure of macrophages revealed that apoptosis was induced in the early infection stage and inhibited in the middle and later periods of infection. Additionally, the expression of Bcl-2, Bax, Cyto-C, Caspase-3, Capase-8, and Caspase-9 was induced at 4 hpi, and then decreased at 6-8 hpi, illustrating that N. seriolae infection induces the activation of extrinsic and intrinsic apoptotic pathways in macrophages, followed by the inhibition of apoptosis to survive inside the cells. Furthermore, N. seriolae inhibits the production of reactive oxygen species and releases large amounts of nitric oxide, which persists in macrophages during infection. The present study provides the first comprehensive insight into the intracellular behavior of N. seriolae and its apoptotic effect on macrophages and may be important for understanding the pathogenicity of fish nocardiosis.