Occurrence and seasonal variation of Perkinsus sp. Infection in wild mollusk populations from coastal waters of Qingdao, northern China.

Perkinsosis has been recognized as one of the major threats to natural and farmed bivalve populations, many of which are of commercial as well as environmental significance. Three Perkinsus species have been identified in China, and the Manila clam (Ruditapes philippinarum) was the most frequently infected species in northern China. Although the occurrence and seasonal variation of Perkinsus spp. have previously been examined, the pathological characteristics of these infections in wild Manila clams and sympatric species in China have seldom been reported. In the present study, the prevalence and intensity of Perkinsus infection in wild populations of Manila clams and 10 sympatric species from three sites were investigated by Ray's fluid thioglycolate medium (RFTM) assay seasonally across a single year. Perkinsus infection was only identified in Manila clams, with a high prevalence (274/284 = 96.48 %) and low intensity (89.8 % with a Mackin value ≤ 2, suggesting generally low-intensity infections) throughout the year. Heavily infected clams were mainly identified in Tianheng in January, which displayed no macroscopic signs of disease. An overview of the whole visceral mass section showed that the trophozoites mostly aggregated in gills and connective tissue of the digestive tract, to a lesser extent in the mantle and foot, and even less frequently in adductor muscle and connective tissues of the gonad. PCR and ITS-5.8S rRNA sequencing of 93 representative RFTM-positive samples revealed a 99.69 to 100 % DNA sequence identity to Perkinsus olseni. Unexpectedly, significantly higher infection intensities were usually identified in January and April when the Condition Index (CI) was relatively high. We propose that factors associated with the anthropogenic harvesting pressure and irregular disturbances should be responsible for the uncommon seasonal infection dynamics of perkinsosis observed in the present study.

The Probiotic Bacillus hwajinpoensis Colonizes the Digestive System of Crassostrea gigas Larvae and Protects Them from Vibrio alginolyticus Infection.

The Pacific oyster Crassostrea gigas is one of the most important cultured marine species around the world. Production of Pacific oysters in China has depended primarily on hatchery produced seeds since 2016, with the successful introduction and development of triploid oysters. However, the seed supply of Pacific oysters is threatened by recurring mass mortality events in recent years. Vibriosis is the most commonly encountered disease associated with intensive oyster culture in hatcheries and nurseries. Vibrio alginolyticus and Bacillus hwajinpoensis were the two strains with pathogenic and probiotic effects, respectively, identified during the Pacific oyster larvae production. To monitor their colonization process in Pacific oyster larvae, green fluorescent protein (GFP) and red fluorescent protein (RFP) were labeled to the pathogenic V. alginolyticus and the probiotic B. hwajinpoensis stain, respectively. The pathogenic and probiotic effects of the two strains during the colonization process were then assessed. Stabile expression of GFP and RFP were observed in corresponding stains, and the capabilities of growth, biofilm formation and in vitro adhesion of GFP- and RFP- tagged stains were not significantly different from those of the wild-type strains. Usage of probiotics of 105 CFU/mL significantly inhibited the growth of pathogenic V. alginolyticus and reduced the mortality of D-sharped larvae. Both the pathogenic and probiotic strains employed a similar route to enter and colonize the oyster larvae, which indicates that competing with pathogens for binding and spreading sites were one of the mechanisms of B. hwajinpoensis to provide the probiotic effects to oyster larvae. In summary, employment of fluorescence-tagged pathogenic and probiotic strains simultaneously provides us with an excellent bioassay model to investigate the potential mechanisms of probiotics.

Identification and Characterization of Infectious Pathogens Associated with Mass Mortalities of Pacific Oyster (Crassostrea gigas) Cultured in Northern China.

The Pacific oyster (Crassostrea gigas) aquaculture industry increased rapidly in China with the introduction and promotion of triploid oysters in recent years. Mass mortalities affecting different life stages of Pacific oysters emerged periodically in several important production areas of Northern China. During 2020 and 2021, we conducted a passive two-year investigation of infectious pathogens linked to mass mortality. Ostreid herpesvirus-1 (OsHV-1) was detected to be associated with mass mortalities of hatchery larvae, but not juveniles and adults in the open sea. Protozoan parasites, such as Marteilia spp., Perkinsus spp. and Bonamia spp. were not detected. Bacterial isolation and identification revealed that Vibrio natriegens and Vibrio alginolyticus were the most frequently (9 out of 13) identified two dominant bacteria associated with mass mortalities. Pseudoalteromonas spp. was identified as the dominant bacteria in three mortality events that occurred during the cold season. Further bacteriological analysis was conducted on two representative isolates of V. natriegens and V. alginolyticus, designated as CgA1-1 and CgA1-2. Multisequence analysis (MLSA) showed that CgA1-1 and CgA1-2 were closely related to each other and nested within the Harveyi clade. Bacteriological investigation revealed faster growth, and more remarkable haemolytic activity and siderophore production capacity at 25 °C than at 15 °C for both CgA1-1 and CgA1-2. The accumulative mortalities of experimental immersion infections were also higher at 25 °C (90% and 63.33%) than at 15 °C (43.33% and 33.33%) using both CgA1-1 and CgA1-2, respectively. Similar clinical and pathological features were identified in samples collected during both naturally and experimentally occurring mortalities, such as thin visceral mass, discolouration, and connective tissue and digestive tube lesions. The results presented here highlight the potential risk of OsHV-1 to hatchery production of larvae, and the pathogenic role of V. natriegens and V. alginolyticus during mass mortalities of all life stages of Pacific oysters in Northern China.

A biocompatible bacterial cellulose/tannic acid composite with antibacterial and anti-biofilm activities for biomedical applications.

Biofilm-associated infections are in a high rate of recurrence and biofilms show formidable resistance to current antibiotics, making them a growing challenge in biomedical field. In this study, a biocompatible composite was developed by incorporating tannic acid (TA) and MgCl2 to bacterial cellulose (BC) for antimicrobial and anti-biofilm purposes. The morphology was investigated by scanning electron microscopy (SEM), and chemical structure were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectra (XPS). In vitro release profiles of tannic acid revealed that the Mg2+ cross-links help impede the release of TA from BC matrix, while composite BC-TA lacked Mg2+ ionic cross-links, thus more TA was released from the hydrogel. The BC-TA-Mg composites also displayed strong antibacterial activity against S. aureus, E. coli and P. aeruginosa. Moreover, the composites significantly reduced biofilm formation of S. aureus and P. aeruginosa after 24 h incubation by ∼80% and ∼87%, respectively. As a consequence, the BC-TA-Mg composites are a very promising material for combating biofilm-associated infections in biomedical and public health fields.

[Repairing segmental radial bone defect with poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/sol-gel bioactive glass composite porous scaffold].

OBJECTIVE: To investigate the capability of the bone regeneration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate/sol-gel bioactive glass (PHBV/SGBG) composite porous scaffold. METHODS: PHBV/ SGBG composite porous scaffold was implanted into the segmental radial bone defect of the New Zealand white rabbits, PHBV/hydroxylapatite (PHBV/HA) as experimental control. The degradability, biocompatibility, and bone regeneration capability of the implants were evaluated through radiological, histological, computerized graphic, and biomechanical analysis. RESULTS: The new bone formation occurred as early as 4 weeks after implantation of PHBV/SGBG composite porous scaffold. The defect was filled with new bone 8 weeks after the implantation, and was completely repaired 12 weeks after operation. The new bone had normal bone structure and the medullar cavity regenerated. The biomechanical study showed that the anti-compression force of radial specimen in PHBV/SGBG groups was significantly higher than in PHBV/ HA groups (P < 0.05), but no significant difference existed between PHBV/SGBG group and autograft bone group (P>0.05). The PHBV/SGBG composite porous scaffold degraded no sooner than 4 weeks after the implantation and most of scaffold was absorbed after 12 weeks. The proportion of the scaffold to new bone decreased from 60% by week 4 to 8% by week 12. CONCLUSIONS: PHBV/SGBG composite porous scaffold is a degradable bone substitute. It can achieve early bone generation and complete repair. It can be used as an ideal scaffold for tissue-engineering bone.