Distribution of Vibrionaceae in farmed Asian sea bass, Lates calcarifer in Thailand and their high prevalence of antimicrobial resistance.

This study describes the etiological agent of Vibriosis along with its distribution and antimicrobial resistance profiles among farmed Asian sea bass (Lates calcarifer) in Thailand. The study isolated 283 Vibrionaceae from 15 Asian sea bass farms located around the provinces of the Andaman Sea and Gulf of Thailand coasts to uncover the distribution and antimicrobial resistance profiles. Bacterial identification based on a combination of the biochemical characteristics, Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, and the species-specific PCR demonstrated the predominant Vibrionaceae were Vibrio harveyi (n = 56), Photobacterium damselae (n = 35), and V. vulnificus (n = 31), respectively. According to a laboratory challenge experiment, among the six isolates, only V. harveyi was found to cause clinical signs of muscle necrosis and scale loss in Asian sea bass. Antibiotics resistance test results exhibited high resistance to antibiotics such as metronidazole (100%), streptomycin (97%), clindamycin (96%), colistin sulphate (70%) and amoxicillin (59%). Remarkably, 100% of Vibrionaceae isolates are susceptible to florfenicol. The 28 of 29 resistance profiles were multidrug resistances (MDR), with V. vulnificus having the highest MAR value (0.66). The findings of this study advise that a surveillance program, as well as preventive and control measures, be developed for Vibrionaceae to reduce production loss, pathogen proliferation, and antibiotic abuse, whereas AMR data indicate substantial health problems for aquatic animals and humans.

Dissecting the localization of Tilapia tilapinevirus in the brain of the experimentally infected Nile tilapia, Oreochromis niloticus (L.).

Tilapia tilapinevirus or tilapia lake virus (TiLV) is an emerging virus that inflicts significant mortality on farmed tilapia globally. Previous studies reported detection of the virus in multiple organs of the infected fish; however, little is known about the in-depth localization of the virus in the central nervous system. Herein, we determined the distribution of TiLV in the entire brain of experimentally infected Nile tilapia. In situ hybridization (ISH) using TiLV-specific probes revealed that the virus was broadly distributed throughout the brain. The strongest positive signals were dominantly detected in the forebrain (responsible for learning, appetitive behaviour and attention) and the hindbrain (involved in controlling locomotion and basal physiology). The permissive cell zones for viral infection were observed mostly to be along the blood vessels and the ventricles. This indicates that the virus may productively enter into the brain through the circulatory system and widen broad regions, possibly through the cerebrospinal fluid along the ventricles, and subsequently induce the brain dysfunction. Understanding the pattern of viral localization in the brain may help elucidate the neurological disorders of the diseased fish. This study revealed the distribution of TiLV in the whole infected brain, providing new insights into fish-virus interactions and neuropathogenesis.

Synergistic infection of Ichthyophthirius multifiliis and Francisella noatunensis subsp. orientalis in hybrid red tilapia (Oreochromis sp.).

Francisella noatunensis subsp. orientalis (Fno) and Ichthyophthirius multifiliis (Ich) are deadly infectious pathogens in farmed tilapia, particularly during cold season when the water temperature drops to under 25 °C. We hypothesized that infection of the ectoparasite Ich might enhance susceptibility of hybrid red tilapia (Oreochromis sp.) to the facultative intracellular bacterium Fno. To prove the hypothesis, the experiment was designed as follows. Hybrid red tilapia naturally infected by Ich at 9 ± 6 theronts/fish gills and 4 ± 3 theronts/fish skin were distributed into 5 distinct groups exposed to different concentrations of Fno. In parallel, the same number of Ich-free tilapia were challenged to only Fno in the same manner. The results showed that cumulative mortality in the Fno single infection with 2.88 × 106 CFU mL-1 of water was 25 ± 7%, whereas 100% mortality was found in the coinfection treatment at dose of 1.93 × 105 CFU mL-1 of water. No mortality was observed in both control groups (Ich-infected and Ich-free fish). The coinfected fish revealed typical clinical signs and histopathological manifestations of francisellosis and ichthyophthiriasis. This study revealed synergistic effect of the Ich and Fno infection in hybrid red tilapia leading to the exacerbated mortality. Thus, farming management of fish to be free from the Ich ectoparasite might reduce risk of francisellosis and probably other bacterial diseases in farmed tilapia.

Transmission of Francisella noatuensis subsp. orientalis from subclinically infected hybrid red tilapia broodstock (Oreochromis sp.) to their offspring.

Francisella noatunensis subsp. orientalis (Fno) has been reported as an important bacterial pathogen causing significant mortality (30-95%) in farmed tilapia in broad geographic areas. However, we found that there was a proportion of broodfish in our laboratory that appeared to be healthy but which tested positive for Fno. We therefore hypothesized that Fno might be able to be transmitted from subclinically infected tilapia mouthbrooders to their offspring through the current practice of fry production in tilapia hatcheries. To prove this, experimentally infected hybrid red tilapia broodstock were mated and their offspring were examined for the presence of Fno. In this study, three pairs of infected broodfish were mated for natural spawning and fertilized eggs from each couple were then collected from the female mouths for artificial incubation. The newly hatched larvae were cultured for 30 days and sample collection was performed at different developmental stages i.e. yolk-sac larvae, 5 and 30-day old fry. The results showed that the ovary and testis of all 3 pairs of the broodstock, as well as their fertilized eggs and offspring were Fno positive by Fno-specific PCR and in situ DNA hybridization. In summary, this study revealed that with the current practice in tilapia hatcheries, Fno might be able to transmit from subclinically infected tilapia mouthbrooders to their offspring. Therefore, using Fno-free broodfish in tilapia hatcheries should be considered in order to produce Fno-free tilapia fry.

Uptake ability of hepatic sinusoidal endothelial cells and enhancement by lipopolysaccharide.

The liver is one of the major organs that remove exogenous substances and waste products from the blood circulation. Hepatic macrophages (Kupffer cells) and sinusoidal endothelial cells are responsible for the scavenger function of the liver. The sinusoidal endothelial cells, called scavenger endothelial cells, are believed to take up only soluble substances and nanometer-sized particles under normal conditions, while Kupffer cells can ingest larger particles and whole cells. However, the sinusoidal endothelial cells may have the potential to take up considerably large particles under special conditions. In this morphological study, we compared the uptake ability between sinusoidal endothelial cells and Kupffer cells after intravenous injections of latex beads (20 nm, 100 nm and 500 nm in diameter), bovine serum albumin (BSA) and dextran. Under normal conditions, the sinusoidal endothelial cells vigorously took up 100-nm-sized latex beads as well as 20-nm latex beads. BSA and dextran were ingested by the endothelial cells but not the Kupffer cells. The administration of lipopolysaccharide (LPS), which mimics inflammation, stimulated the uptake by endothelial cells. The uptake of latex beads by Kupffer cells was also elevated under LPS-stimulated conditions, but the uptake of BSA and dextran by them was not. These findings suggest that the sinusoidal endothelial cells can ingest not only soluble substances but also larger particles than those expected, and their uptake ability is strengthened under inflammatory conditions.

The expression of src-suppressed C kinase substrate (SSeCKS) and uptake of exogenous particles in endothelial and reticular cells.

Src-suppressed C kinase substrate (SSeCKS), a potent tumor suppressor, plays a role in membrane-cytoskeletal remodeling to regulate mitogenesis, cell differentiation, and motility. Our previous study showed that lipopolysaccharide (LPS) induced a selective and strong expression of SSeCKS in the vascular endothelial cells of several organs, such as hepatic sinusoids, and in the reticular cells of lymphoid organs. In the present immunocyto-chemical study, we determined the detailed cellular and subcellular localization of SSeCKS in mouse tissues after LPS administration, and examined the involvement of SSeCKS in the uptake of exogenous particles. SSeCKS immunoreactivity in the liver and lymph nodes was below the detectable level under normal conditions. After LPS stimulation, an intense immunoreactivity for SSeCKS became noticeable in sinusoidal endothelial cells of the liver and medullary reticular cells of the lymph node. Electron-microscopically, the immunoreactivity was localized predominantly along the cytoplasmic membrane of both cell types. These cells in normal mice incorporated a small amount of injected particles (carbon particles and latex beads), while after LPS stimulation, the uptake of particles increased in terms of the amount and extent of the uptaking sites. Endothelial cells and reticular cells without SSeCKS expression could not incorporate any particles even after LPS stimulation. The subcellular localization of SSeCKS in endothelial cells correlated with some pinocytic pits and phago-lysosomes, although a diffuse distribution of SSeCKS in the cytoplasm was also visible. Taken together, these findings indicate that SSeCKS expression in endothelial cells and reticular cells is a functional index of the reticulo-endothelial system and is involved in the uptake of particles from blood and lymph circulation.