Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

Morphological changes in carp epithelial cells infected with Aeromonas hydrophila

The in vitro interaction of Aeromonas hydrophila with epithehoma papillosum cells of carp (EPC) was studied. All the virulent and type strains invaded and multiplied inside EPC cells. Morphological changes were induced by the virulent and type strains during the processes of invasion and intracellular replication. Among the virulent group, strain PPD134/91 required the shortest time (30 min post-infection) to induce cytopathic effects in the EPC monolayers. The EPC cells infected with PPD 134/91 became retracted and condensed, lost their attachment abilities, and eventually disintegrated. Confocal microscopy revealed that tubular structures measuring 0.77 ± 0.19 μm appeared to connect the retracted EPC cells. The cytopathic effect was attributed to the growing and metabolically active bacteria. Avirulent strains such as L37 did not multiply nor induce cytopathic effects in the EPC monolayers.     

Recent developments and improvements in ornamental fish packaging systems for air transport

Current ornamental fish packaging systems are characterized by very high fish loading densities and high metabolic wastes in the transport water after shipment. They focus mainly on management of the quality of transport water. Recent studies using the guppy as a model fish showed that post‐shipment mortality could be reduced through enhancement of the stress resistance of the fish, and hence emphases should also be placed on the preparation of the fish for transport and recovery of the fish after shipment. Farmers can contribute significantly by applying nutritional prophylaxis before harvesting. Exporters may use the salinity stress test to identify fish lots of good quality for transport, apply health prophylaxis to eradicate parasites and optimize other techniques such as starvation of the fish or addition of salt to the transport water to enhance the stress resistance of the fish. Importers may adopt proper acclimation procedure and allow fish to recover in low salinity water to reduce post‐shipment mortality. As the main bulk of post‐shipment mortality is stress‐mediated and occurs during the 1‐week recovery period, the industry should consider revising the basis of the current warranty system for their customers, from death on arrival to cumulative mortality at 7 days post shipment (or death after 7 days, DA7), in order to cut down fish losses after shipment.     

Riboflavin Requirement of Fingerling Red Hybrid Tilapia Grown in Seawater

Red hybrid tilapia Oreochromis mossambicus × O. niloticus fingerlings were fed diets containing 0, 2.5, 5.0, 10.0, 20.0 and 40.0 mg/kg, and 0, 2.5, 5.0, 7.5, 10.0 and 20.0 mg/kg of riboflavin in separate 8 and 12 wk feeding studies, respectively. The dietary riboflavin level required to provide maximum growth and survival, and prevent deficiency symptoms in red hybrid tilapia fingerlings was found to be approximately 5 mg/kg of diet. In both trials, fish fed the diet devoid of supplemental riboflavin exhibited anorexia, reduced growth and nervous symptoms after 4–6 wk. Mortality began to occur after the sixth week. None of these abnormalities were observed during the first 6 wk in fish fed the riboflavin supplemented diets. However, by the seventh week, fish fed the diet supplemented with 2.5 mg/kg of riboflavin showed reduced appetite and growth rate. In both experiments, short body dwarfism was observed during week 8 for fish fed the diet without riboflavin supplementation. In experiment 1, fish fed the riboflavin-deficient diet had lens cataracts at week 8. This deficiency sign was not observed in experiment 2. Histological studies of liver, kidney, spleen, lateral muscle, gill and gastrointestinal tract revealed no major histopathological changes.     

Feed deprivation of channel catfish, Ictalurus punctatus (Rafinesque), influences organosomatic indices, chemical composition and susceptibility to Flavobacterium columnare

Withholding feed has been suggested as a strategy to manage infectious disease of channel catfish, Ictalurus punctatus (Rafinesque). In a previous study, we demonstrated that deprivation of feed for as little as 7 days reduced innate resistance of catfish to Flavobacterium columnare. This study was conducted to evaluate the effect of feeding regimens [no feeding (NF), fed once every other day to satiation (FEOD) and fed once daily to satiation (FD)] on organosomatic indices, physiological changes and susceptibility of channel catfish to F. columnare. Fish that were not fed for 2 and 4 weeks had a significant increase (P < 0.05) in gutted weight:-wet weight ratio and decrease in other organosomatic indices [gut index (GI), mesenteric fat index (MFI) and hepatosomatic index (HSI)]. Haematology was not effected by feeding regimen except at week 4, when a significantly higher haemoglobin level was observed in the NF fish. Serum protein did not differ at week 2, but the level at week 4 of the NF fish (35.91 mg mL(-1)) was significantly lower than that of the FD fish (41.77 mg mL(-1)). Significantly lower (P < 0.05) blood glucose (39.5 and 40.3 mg dL(-1)) and liver glycogen (1.7 and 1.8 mg g(-1)) were seen in the NF fish at weeks 2 and 4, respectively, as compared with blood glucose and liver glycogen levels of FD fish (67.5 and 92.8 mg dL(-1) and 46.5 and 52.6 mg g(-1) at weeks 2 and 4, respectively) and FEOD (82.8 and 85.5 mg dL(-1) and 45.1 and 51.4 mg g(-1) at weeks 2 and 4, respectively). Mortality in the NF fish caused by F. columnare (78%) was significantly higher (P < 0.05) than mortality in the FD and FEOD treatments (0.0 and 1.7%, respectively). Blood glucose and liver glycogen showed the same trend of low values for NF fish following challenge (week 6). Blood glucose, liver glycogen, GI and HSI are sensitive indicators for channel catfish deprived of feed (NF) for 4 weeks. Blood glucose and liver glycogen levels around 40 mg dL(-1) and 2 mg g(-1), respectively, are indicative of starvation in juvenile channel catfish. Moreover, NF fish were susceptible to F. columnare infection. Thus, it is suggested that in the absence of natural food, juvenile channel catfish should be fed at least once every other day to apparent satiation to maintain normal physiological function and improve resistance to F. columnare.     

Influence of Dietary Levels of Magnesium on Growth, Tissue Mineral Content, and Resistance of Channel Catfish Ictalurus punctatus Challenged with Edwardsiella ictaluri

Juvenile channel catfish were fed purified diets supplemented with magnesium (Mg) from Mg sulfate at levels of 0, 200, 400, 600, 800, and 1,000 mg/kg and 0, 200, 400, 600, and 800 mg/kg in two separate feeding studies. In study I, the effect of dietary levels of Mg on growth response, vertebral mineral content, and macrophage chemotaxis were evaluated. Study II had similar objectives except that whole body mineral content was measured, and resistance of channel catfish to Edwardsiella ictaluri challenge was also determined. Fish with an average weight of 10.89 g were stocked at a rate of 50 fish/110‐L aquarium (study I). In study II, fish with an average weight of 4.14 g were stocked at rates of 40 fish/110‐L aquarium. Prior to stocking, each batch of fish was acclimated to laboratory conditions and fed the basal diet for 2 wk. The concentration of Mg in rearing water was 1.8 mg/L. Each diet was fed to fish in quadruplicate and triplicate aquaria to apparent satiation for 10 wk for studies I and II, respectively. Fish fed the basal diet started to die as early as 3 d after the study began (17 d of feeding the diet without Mg supplementation). In both studies, weight gain, survival, and feed efficiency were lowest for fish fed the basal diet but increased with increasing dietary levels of Mg. However, the differences between the values of each of these parameters for fish fed diets containing supplemental Mg were not always significant. Magnesium‐deficiency signs observed were anorexia, sluggishness, convulsions, deformed snout, vertebral curvature, muscle flaccidity, and high mortality. Vertebral and whole body ash concentrations were high, but Mg content was low for fish fed the basal and the 200‐mg Mg diets. Bone Ca content did not differ among fish fed different diets (study I), but whole body Ca tended to increase for fish fed the basal diet, suggesting the possibility of calcification of soft tissues. Macrophage chemotaxis in the presence of exoantigen was highest for fish fed diets supplemented with Mg at 400 and 200 mgkg for studies I and II, respectively. When expressed in terms of chemotaxis index, however, maximum or near maximum value was observed at a dietary Mg level of 400 mg/kg. Thus, a dietary level of Mg of 400 mg/kg from Mg sulfate was required for optimum growth and survival, maintaining high tissue levels of Mg, prevention of muscle flaccidity and skeletal deformity, and stimulating macrophage chemotaxis. Dietary levels of Mg had no effect on the resistance of juvenile channel catfish to Edwarsiella. ictaluri challenge.     

The conservation status of the Nee Soon freshwater swamp forest of Singapore

1. The most important area in Singapore for native aquatic fauna and flora is a relatively intact 5 km² patch of freshwater swamp forest in Nee Soon. 2. The Nee Soon Swamp Forest contains 48% of the primary freshwater fish, 71% of the amphibian, 28% of the reptile and 34% of the bird fauna of Singapore. The swamp also has the highest percentage of native and threatened freshwater fish and crustacean species on the island, as well as being the main (if not the only) habitat of the aquatic plant Hydrostemma. It is also the last refuge of two mammals, the Banded Leaf Monkey, Presbytis femoralis femoralis, and the Cream-coloured Giant Squirrel, Ratufa affinis affinis. 3. The threats to the swamp forest are discussed, and proposals are made to establish the swamp forest as Singapore's second major nature reserve. 4. Study of the swamp forest is essentially still at a survey and discovery phase. The research priorities are to establish detailed species lists, the status of endangered species and the extent of buffer zones. 5. The swamp forest is administered primarily by the newly established National Parks Board, but several other government organizations also have jurisdiction over the land, causing some problems in the management of the swamp. The roles of the university and non-governmental organizations in conducting surveys, drafting recommendations and educating the general public are detailed.