Comparative pathogenicity of Nocardia seriolae in Nile tilapia (Oreochromis niloticus), milkfish (Chanos chanos) and Asian seabass (Lates calcarifer).

Nocardiosis, caused by Nocardia seriolae, has been a prominent disease in Southeast Asian aquaculture in the last three decades. This granulomatous disease reported in various fish species is responsible for significant economic losses. This study investigated the pathogenicity of N. seriolae in three cultured species in Taiwan: Nile tilapia (omnivore), milkfish (herbivore) and Asian seabass (carnivore). Administration of an infective dose of 1 × 106 CFU/ fish in tilapia, seabass and milkfish demonstrated mortalities of 100%, 90% and 75%, respectively. Additionally, clinical signs namely, granuloma and lesions displayed varying intensities between the groups and pathological scores. Polymerase chain reaction (PCR) amplification specific for N. seriolae was confirmed to be positive (432 bp) using NS1/NG1 primers. Post-mortem lesions revealed the absence of granulomas in tilapia and milkfish and their presence in the seabass. Interestingly, the gut in tilapia showed an influx of eosinophils suggesting its role during the acute stages of infection. However, post-challenge, surviving milkfish exhibited granulomatous formations, while surviving seabass progressed toward healing and tissue repair within sampled tissues. Overall, in conclusion, these results demonstrate the versatility in the immunological ability of individual Perciformes to contain this pathogen as a crucial factor that influences its degree of susceptibility.

Nocardiosis and other common diseases of cultured golden pompano (Trachinotus blochii) in Taiwan.

Golden pompano (Trachinotus blochii) is a carnivorous teleost cultured in the Asia-Pacific region. Fish culture in high densities and numbers results in disease outbreaks, causing huge economic losses. Here, we collected cultured golden pompanos from 2021 to 2022 and identified the pathogens isolated from the diseased fish. Out of a total of 64 clinical cases observed in both sea cages and fish ponds, it was found that Nocardia seriolae was the predominant pathogen (26%), followed by Lactococcus garvieae (13%). Trichodina spp. was the most prevalent parasite in sea cages and earthen ponds (21%), while Neobenedenia spp. was the primary parasitic pathogen (16%) in sea cages. Given these findings, further investigations were conducted, including antibiotic susceptibility and pathogenicity tests specific to N. seriolae in golden pompanos. Antibiotic susceptibility tests of N. seriolae revealed that all strains were susceptible to doxycycline, oxytetracycline, florfenicol and erythromycin but resistant to amoxicillin and ampicillin. Additionally, a pathogenicity assessment was carried out by administering an intraperitoneal injection of 0.1 mL containing 107 CFU of N. seriolae per fish. The mortality rates observed varied between 40% and 90%, with the P2 strain exhibiting the highest level of virulence, resulting in a cumulative mortality of 90%. Therefore, disease outbreaks in fish can be minimized by developing effective treatments and prevention methods.

First report of a Kudoa lutjanus outbreak in farmed Chicken Grunts Parapristipoma trilineatum.

OBJECTIVE: As part of the National Disease Surveillance Program for Taiwanese Aquaculture, we investigated the causative agent of disease outbreaks in farmed Chicken Grunts Parapristipoma trilineatum. METHODS: In this study, outbreak cases on two separate farms were noticed in coastal Pingtung County, Taiwan. In total, 50 juvenile fish showing clinical signs (such as emaciation and erratic swimming behavior) and broodstock (two females and two males) from both farms were collected to perform gross lesion assessment, histopathological examination, and molecular identification of the pathogen. RESULT: Clinical symptoms were infected fish exhibited erratic swimming behavior, such as whirling and floating on the surface of the water. In the following months, cumulative mortality had reached 19% and 24%, respectively. The gross lesions in the infected fish included white oval cysts in the muscle, serosa of the internal organs, sclera of the eyes, and cerebral meninges. After conducting a wet mount examination of cysts using a light microscope, we observed a significant quantity of spores with morphological characteristics, suggesting their affiliation with the Myxosporea group. The spores were semiquadrate, with four tiny suture notches at the periphery; the mean spore length was 7.3 µm (SD = 0.5), and the mean spore width was 8.2 µm (SD = 0.6). The mean length and width of the pyriform polar capsules (nematocysts) were 3.6 µm (SD = 0.5) and 2.2 µm (SD = 0.5), respectively. The 18S and 28S ribosomal RNA sequences of these specimens were identical to those of Kudoa lutjanus. CONCLUSION: As this was the first time an outbreak of K. lutjanus in Chicken Grunts was confirmed, its reappearance with substantial mortality should serve as a warning to the aquaculture industry.

Pathogenicity characterization of nocardiosis in orange-spotted grouper (Epinephelus coioides) via exposure to nine distinct isolates of Nocardia seriolae and four administrative routes.

Nocardia seriolae causes chronic nocardiosis in various marine and freshwater aquatic animals; however, grouper species have rarely been investigated. This study evaluated the pathogenicity of nocardiosis following N. seriolae infection in the orange-spotted grouper Epinephelus coioides. Nine identified genetic isolates of N. seriolae were tested in vivo using the intraperitoneal method and observed daily for 35 days. The most virulent isolate was then used to evaluate transmission through different routes (intraperitoneal IP, intramuscular IM, oral OR, and immersion IS) in the same fish model and was observed daily for 42 days. The results showed mild variation in virulence among N. seriolae isolates. AOD107132-2 K and OT103003-N11 strains displayed the highest and lowest risk virulence, respectively, based on the accumulation and kinetics of mortality. IM and IP administrations showed an early phase response with early mortality by 5 dpc (30%-100%), while slower kinetics of nocardiosis occurred in the OR and IS routes with slow mortality at 35 dpc (4%-8%). Histopathology revealed typical granulomas, confirming the progression of nocardiosis in the diseased fish. These results provide the basis for further studies on the virulence profile of N. seriolae in Taiwan and a well-suited route of administration in orange-spotted groupers for further prevention development.

Pathogenicity and Genomic Characterization of a Novel Genospecies, Bacillus shihchuchen, of the Bacillus cereus Group Isolated from Chinese Softshell Turtle (Pelodiscus sinensis).

The Chinese softshell turtle (CST; Pelodiscus sinensis) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen. Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin.

Nocardia seriolae cell wall lipids: An effective protective mechanism in resistance and virulence.

Piscine nocardiosis, caused by Nocardia seriolae, is a refractory granulomatous disease in South-East Asian aquaculture. This study investigates the virulence of nocardial lipids essential for pathogenesis among Actinomycetes. Petroleum ether (PE) was used to selectively delipidate two groups of N. seriolae, namely, live cell (LC) and killed cell (KC); resulting in delipidated live cell (DLC) and delipidated killed cell (DKC), respectively. Changes post-delipidation on genus characteristics, such as loss in acid-fast nature and resistance to lysozyme were observed. Transmission electron microscopy revealed notable changes in the lipid layer. Additionally, Lates calcarifer, Asian seabass intraperitoneally injected with LC and DLC had mortality rates of 90% and 50%, respectively, with the latter exhibiting a delay in mortality. Reverse-transcription quantitative PCR (RT-qPCR) analysis of host cytokines from the spleen and head kidney showed delipidation contributed to the induction of an immune response with increased transcriptional levels of interferon-γ (ifn-γ). Histopathological samples collected on day 7 post-inoculation displayed a varied granulomatous response between the treatment groups and scored for pathological changes. These findings affirm that the virulence of the lipids remains independent of the living state of the cell, significantly altering the immune and granulomatous responses in L. calcarifer to N. seriolae.

First report of acanthocephalan parasite, Longicollum pagrosomi Yamaguti, 1935 in cultured red snapper (Lutjanus erythropterus) in Taiwan.

For the first time, Longicollum pagrosomi Yamaguti, 1935, an acanthocephalan parasite, has been characterized with morphological, histopathological and molecular detail in farmed red snapper (Lutjanus erythropterus) in Taiwan. The diseased fish showed clinical signs of anorexia, emaciation and were gasping for air at the water's surface. Gross examination revealed extensive necrosis in the intestine and even penetration at the site of parasitic attachment, resulting in a large number of parasites being lodged in the peritoneal cavity, surrounded by black hyperplastic connective tissue. The parasites collected from the intestine were studied using optical microscopy, histopathology, scanning electron microscopy (SEM) and molecular phylogenetic analysis. They were creamy white in colour, and were separated into their proboscis, neck and metasoma (trunk) under optical microscopy. Histopathological examination revealed ovarian balls (floating ovaries) inside the ligament sac and eggs within the egg sorting apparatus. The SEM images revealed that the proboscis had 11-14 longitudinal rows with 9-12 recurrent, backward-facing, organized hooks. In the 18S and 28S phylogenetic tree, sequences of the specimens were identical to those of other Longicollum pagrosomi, in addition to the morphological features. Thus, we confirmed that the parasites belonged to Longicollum pagrosomi in this study.

First report on genetic characterization, cell-surface properties and pathogenicity of Lactococcus garvieae, emerging pathogen isolated from cage-cultured cobia (Rachycentron canadum).

The diseased cage-cultured cobia (Rachycentron canadum) displayed clinical signs, haemorrhagic eyes, dorsal darkness and gross pathological lesions, enlargement of spleen and liver. Haemorrhages were found in brain, heart and liver with cumulative mortality rates ranging from 20% to 50%. Extensive congestion in the heart, liver, spleen, kidney and brain was observed histopathologically. Epicarditis and meningitis were also revealed in diseased cobia. All isolates recovered from the organs (liver, spleen, head kidney, posterior kidney, brain and muscle) of cobia were found to be gram-positive, non-motile, ovoid cocci, short-chain-forming (diplococci) and α-haemolytic. The API 32 strep system together with the polymerase chain reaction assay for species-specific primers (pLG1 and pLG2) and the internal transcribed spacer (ITS) region (G1 and L1 primers) confirmed all four selected isolates as Lactococcus garvieae. Partial 16S rDNA nucleotide sequence (~1,100 bp) of one representative L. garvieae isolate AOD109191 (GenBank accession number, MW328528.1) shared 99.9% identities with the 16S rDNA nucleotide sequence of L. garvieae (GenBank accession numbers: MT604790.1). Transmission electron microscopy (TEM) evaluation of one representative L. garvieae isolate (AOD109191) and the results of multiplex PCR did not reveal the presence of the capsular gene cluster (CGC), thus categorizing the isolate as the KG+ phenotype. Capsule staining and TEM observations confirmed the presence of a hyaluronic acid-like capsule, a possible virulence factor in KG+ phenotype L. garvieae isolates. The pathogenic potential of the representative isolate (AOD109191) was assessed through intraperitoneal injection challenges in cobia. The gross lesions and histopathological changes found in experimentally infected cobia were similar to those seen in naturally infected fish. This is the first report that confirms L. garvieae-induced 'warm water lactococcsis' can cause outbreaks of diseases in cage-cultured cobia.

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan.

Chinese softshell turtles (Pelodiscus sinensis) (CST) are susceptible to infections by bacteria belonging to the Bacillus cereus group (Bcg). Bcg includes several closely related species, two of which, B. cereus and B. thuringiensis, are pathogens of aquatic animals or insects. In the present study, we collected 57 Bcg isolates obtained from diseased CST from 2016 to 2019 in Kaohsiung and Pingtung, the areas with the most CST farms in Taiwan. All isolates were divided into four genotypes with two restriction enzymes, SmaI and NotI, by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Representative isolates from each genotype were subjected to phylogenetic tree analysis using 16S rDNA and pyruvate carboxylase genes as phylogenetic markers, and these CST isolates appeared in different clades. PCR was performed targeting six selected virulence genes, four of which were detected in CST isolates, including cytotoxin K (1/57), hblC of the haemolysin BL complex (46/57), nheA of the non-haemolytic enterotoxin complex (52/57) and enterotoxin FM (57/57), whereas cereulide synthetase and cereulide peptide synthase-like genes were not detected in any isolates.