The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1–6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1β, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

In October 2023, a disease outbreak in pufferfish (Takifugu obscurus) farms in Zhongshan City, Guangdong, China, caused high mortality. Diseased fish (mean length: 15 ± 1 cm) exhibited swimming disorders, fin rot, hemorrhage, and an enlarged spleen. Histopathological observations generally revealed inflammation, necrosis, and congestion in the spleen, kidneys, and brain tissues. The most severe pathological changes included interstitial edema and tubular atrophy in the kidneys, hemosiderin deposition in the spleen, massive red blood cell infiltration, and a decrease in lymphocytes. A single strain of bacteria (Tol-1) was isolated from the diseased pufferfish and identified as a Gram-positive streptococcus strain, exhibiting α-hemolysis on sheep blood agar plates. Through biochemical characterization, 16S rDNA sequencing, morphological analysis, and specific primer-based identification, the Tol-1 strain was identified as Lactococcus garvieae, serotype I. Antimicrobial susceptibility testing indicated that Tol-1 was sensitive to Chloramphenicol, Ampicillin, Cephalexin, and Doxycycline, but resistant to Kanamycin, Gentamicin and Ciprofloxacin. In addition, 15 common virulence factors were detected in the Tol-1 strain, including adhPav, adhPsaA, adhC I–II, adh, and hly 1–3. Pufferfish (mean length: 17 ± 1 cm) subjected to artificial infection via intraperitoneal injection (IP) with the Tol-1 strain exhibited clinical symptoms and histopathological damage similar to those observed in naturally infected fish. An infection dose of 1 × 10⁵ CFU/fish resulted in 80% mortality. The study fulfilled Koch’s postulates, indicating that the disease outbreak in pufferfish was caused by L. garvieae, which exhibited a high mortality rate in pufferfish despite the subtle clinical symptoms. These results serve as a warning for pufferfish farming areas and provide a scientific basis for future prevention and control efforts.

Flavobacterium columnare, which causes columnaris disease, is responsible for significant mortality in grass carp. Vaccination is a safe and effective measure to combat this disease, and this study aimed to investigate the immune protective effects of different treatments using an inactivatedF. columnarevaccine. The vaccine was prepared by inactivating the bacteria with 0.05% formaldehyde at 4°C for 24 hours. The experiments involving grass carp were divided into two parts. In Experiment 1, the immune effects of two isolates, JX-01 (genomovar I) and MU-04 (genomovar II), were compared, along with the impact of white oil adjuvant and the number of immunizations. The results showed that when the white oil adjuvant was used as a booster, the relative percent survival (RPS) of the JW2 group and MW2 group after 8 weeks of the first immunization was 34% and 61%, respectively. In Experiment 2, only the MU-04 (genomovar II) isolate was used as an antigen, with the white oil adjuvant as a booster. The effects of different doses (CFU=10⁸, 10⁷, and 10⁶bacteria/mL) on immune responses were compared, and the RPS values in the MW6, MW7, and MW8 groups after 4 weeks of the first immunization were found to be 38%, 57%, and 71%, respectively. Furthermore, in the cross-antigen protection experiment, the MW2 group exhibited an RPS of 55% against the JX-01 isolate, which was significantly higher than the control group (33%). These findings suggest that an inactivated vaccine comprising an appropriate antigen isolate when administered with a white oil adjuvant as a booster, can provide effective protection in grass carp.