In silico prediction of Gallibacterium anatis pan-immunogens

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0080-0) contains supplementary material, which is available to authorized users.     

Early weaning of winter flounder (Pseudopleuronectes americanus Walbaum) larvae on a commercial microencapsulated diet

Like most small marine fish larvae, the stomachs of winter flounder Pseudopleuronectes americanus are undeveloped at first feeding and have relatively reduced digestive capacity. This work was undertaken to test whether larvae at the onset of stomach differentiation (larval size about 5.5 mm) could be early weaned onto a commercial microencapsulated diet. We assessed the effect of early weaning by first comparing growth performance (standard length, total protein content and age at metamorphosis) of larvae fed enriched live prey from first feeding to a size of 5.5 mm and then reared on three different feeding regimes until metamorphosis: (1) live prey (LP) as a control group; (2) mixed feeding of live prey and microencapsulated diet (LP‐ME); (3) exclusively microencapsulated diet (ME) after fast weaning over 4 days (to a larval size of 6.2 mm). No differences were observed between larval development in the two first groups, which began metamorphosis at 40 days old. The larvae of the third group showed significantly slower growth that resulted in a delay of 4 days in the onset of metamorphosis. Differences in live prey availability between the treatments and the short transition period to allow the larvae to adapt to the new diet were identified as possible contributing factors to the slower growth and to the delay in metamorphosis of early weaned larvae. In a second experiment, the transitional weaning period was increased until the larvae were 6.6 mm in length. Weaning at that size resulted in no slowing of growth or delay in metamorphosis, suggesting that the feeding schedule was adequate.     

Optimal conditions for egg storage, incubation and post-hatching growth for the freshwater turtle, Chelodina rugosa: Science in support of an indigenous enterprise

Incubation of northern snake-necked turtle (Chelodina rugosa) eggs and subsequent sale of hatchlings for the pet industry has the potential to provide culturally suitable employment for indigenous communities in northern Australia. Developmental arrest in response to egg inundation is unique to C. rugosa. Eggs can be stored under water for up to 10 weeks without appreciable impact on egg or embryo survival, allowing the transport and sale of eggs into niche markets without high levels of mortality, and permitting eggs to accumulate in diapause until there are sufficient numbers to incubate as batches. Eggs that are not inundated or inundated for short periods experience similar survival rates to eggs inundated for lengthy periods. Incubation temperature influences embryo survival and development period in C. rugosa. Embryonic survival is greatest at 26 °C, steadily declining as temperature increases to 32 °C. A similar increase in incubation temperature decreases incubation period by approximately 40 days, however almost half of this variation is attributed to the increase in incubation temperature from 26 to 28 °C. Hatchling growth in C. rugosa is characterized by two phases. There is an initial phase of relatively slow growth under the partial influence of initial egg size and incubation duration, followed by a second phase of relatively rapid growth under the partial influence of water temperature and mass at hatching. Post-hatching survival is negatively correlated with duration of egg inundation and water temperature. Evidence suggests that inundation of C. rugosa eggs for 6 weeks, incubation of embryos at 28 °C and raising hatchlings in 28 °C water will yield the best overall outcomes.     

The diving behaviour of little penguins in Western Australia predisposes them to risk of injury by watercraft

1. The most western little penguin colony globally, and the most northern in Western Australia (WA) is found on Penguin Island, WA. The penguins use coastal bays that are also used extensively by recreational watercraft. These penguins have been found to either dive predominantly to shallow depths of 1–5 m or to depths >8 m. It is thus hypothesized that (a) both the shallow and deeper diving penguins can potentially be disturbed or injured by these watercraft but that the risk will differ between the two diving strategies, and (b) that risk of injury for both is greater during the summer and autumn, when people are more likely to use watercraft.
2. This was tested by attaching data loggers to little penguins during chick rearing and by investigating necropsy records. Diving activity was studied for the very shallow and relatively deeper diving penguins separately, and we considered the penguins were vulnerable to interactions with watercraft when they were within the top 2 m of the water column or at the surface.
3. Shallow‐diving penguins executed >1,200 dives per day, 64% of dives occurred within the top 2 m, and they were vulnerable for approximately two‐thirds of their time at sea. The deeper diving penguins executed fewer dives. Almost half of dives were to ≥10 m, yet they were vulnerable for almost one‐third of their time at sea. Their post‐dive recovery was also longer. Thus, the risk of interaction from watercraft differs depending on the diving behaviour.
4. This study highlights the potential impact to little penguins throughout Australia and New Zealand.

     

Comparison of traditional and environmental DNA survey methods for detecting rare and abundant freshwater fish

1. Detecting rare species is often a necessity for conservation and management, yet challenging for many field survey methods. Environmental DNA (eDNA) is a highly promising solution that has been shown to outperform many established survey methods.
2. Macquarie perch (Macquaria australasica) is an endangered native species that has declined significantly in range and abundance. Detection of M. australasica was compared with an abundant alien fish species (Oncorhynchus mykiss) using eDNA and three conventional survey methods: gill nets, electrofishing and fyke nets.
3. eDNA occupancy estimates for both fish species were compared using four different models to investigate what effect these differences have on false positives and false negatives for the rare and common fish species. These models used unadjusted eDNA detections in water samples, eDNA detections that have been screened using a limit of detection method to remove potential false positives, eDNA data supplemented with a second survey method, or eDNA data augmented with sequencing of positive polymerase chain reaction replicates.
4. eDNA surveying as a single detection method was found to be more efficient and sensitive compared with each capture method separately and combined. Occupancy estimates for the common and rare species did not vary significantly between the four site occupancy-detection models, suggesting that supplementary data may not have as much effect on occupancy estimates compared with other approaches such as temporal or spatial sampling.
5. We conclude that eDNA outperforms the three established survey methods for both a rare and common freshwater fish species. Although there was no significant effect of augmenting eDNA survey methods with other survey data, additional data may improve confidence in detection, and provide confirmatory evidence for unexpected or new detections of a species.

     

Salinity tolerance during early development of threatened Murray hardyhead (Craterocephalus fluviatilis) to guide environmental watering

1. The worldwide reduction in wetlands has led to the large‐scale decline of wetland‐dependent species. In Australia, to redress some of the decline, partial restoration of the hydrology of a small number of wetlands has been attempted using allocations of environmental water.
2. A common goal of the watering is the maintenance and enhancement of native fish communities, which historically have included populations of the salt tolerant Murray hardyhead (Craterocephalus fluviatilis), a small, short‐lived fish, endemic to the lower Murray–Darling Basin.
3. Despite the addition of environmental water to several sites at which the species is known to persist, populations continue to decline. This decline is, at least in part, suspected to be a consequence of salinities that conflict with the breeding ecology and survival of early life stages.
4. Here the effect of salinity on egg hatch rate and the upper salinity tolerance of larval and juvenile Murray hardyhead was determined under laboratory conditions. It was found that eggs were vulnerable to elevated salinities, whereas juveniles were capable of tolerating salinities up to 105 ppt.
5. Based on the results of the experiment, brackish wetlands managed for Murray hardyhead should be maintained, where possible, between 12 and 45 ppt. Such a salinity regime will necessitate less intensive management of salinity, and a reduced volume of environmental water, providing both environmental and fiscal benefits. The research highlights the benefits of investment in targeted research.

     

Effects of cage netting colour and density on the skin pigmentation and stress response of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

The unnaturally dark pigmentation of cultured Australian snapper Pagrus auratus can be improved through dietary astaxanthin supplementation and by holding fish in tanks with a white background. The practical application of these laboratory‐based findings was examined with two experiments to establish if the advantages of transferring fish to light coloured tanks before harvest could be achieved on‐farm using white cages and to determine the effects of fish density on skin colour. For the first experiment, snapper (mean TL=29.7 cm) were transferred from a commercial snapper sea cage to black or white netted cages and fed diets supplemented with unesterified astaxanthin (supplied as Lucantin^® Pink, BASF) at 0 or 39 mg kg^?1 for 42 days. Skin colour was measured using the CIE (black–white), (green–red), (blue–yellow) colour scale. Snapper held in white netting cages became significantly lighter (higher ) than snapper held in black cages; however, values were not as high as previous laboratory‐based studies in which snapper were held in white plastic‐lined cages. Snapper fed astaxanthin displayed significantly greater and values, and total carotenoid concentrations after 42 days. In addition, total carotenoids were higher in fish from black than white cages. The second experiment was designed to investigate whether density reduced the improvements in skin colour achieved by holding fish in white coloured cages and whether cage colour affected stress. Snapper (mean weight=435 g) were acclimated to black cages and fed 39 mg kg^?1 astaxanthin for 44 days before transferring to black or white plastic‐lined cages at 14 (low), 29 (mid) or 45 (high) kg m^?3 for 7 days after which time skin colour, plasma cortisol and plasma glucose concentrations were measured. Skin lightness () was greater in snapper transferred to white plastic‐lined cages with the lightest coloured fish obtained from the lowest density after 7 days. Density had no effect on plasma cortisol or glucose levels after 7 days, although plasma cortisol was elevated in snapper from black cages. For improved skin colouration we recommend feeding unesterified astaxanthin at 39 mg kg^?1 for approximately 6 weeks and transferring snapper to white plastic‐lined cages or similar at low densities for short periods before harvest rather than producing fish in white netting sea cages subject to biofouling.     

瘤胃乙酸与丙酸摩尔比例的改变对瘤胃发酵及血液指标的影响

通过向瘤胃灌注乙酸：丙酸：丁酸的摩尔比例分别为75：15：10，65：25：10，55：35：10和45：45：10溶液，并在连续灌注的作用下，以期达到最终改变瘤胃液中乙酸：丙酸：丁酸摩尔比例的目的。研究表明：（1）随着灌注液中丙酸浓度的提高，最终瘤胃液中丙酸的比例增加，乙酸的比例逐渐下降，与对照组差异显著（P＜0.05），丁酸的摩尔比例无论与对照组，还是各处理间均无差异；（2）向瘤胃灌注的VFA溶液提供高于维持能量水平30％以上的能量，使瘤胃中总的VFA浓度、VFA产生速率、丙酸的产生速率及VFA的总产量均显著增加（P＜0.01）；但是对基础混合日粮的干物质（DM）、有机物（OM）、中洗纤维（NDF）、酸洗纤维（ADF）、半纤维素（HCF）等养分的消化率不构成显著影响；（3）当灌注溶液中丙酸所占摩尔比例增加到35％，粪氮和尿氮的排出量显著减少（P＜0.05），从而提高日粮氮的沉积能力。进一步研究表明，当灌注液中丙酸的摩尔比例进一步提高至45％时，血糖浓度及胰岛素的浓度才显著增加（P＜0.05）；但胰岛素与胰高血糖素的比值对于不同的处理或对照组，基本保持稳定。     

The genomic sequence of the bovine T cell receptor gamma TRG loci and localization of the TRGC5 cassette

The bovine and ovine TRG genes have previously been shown to be located in two loci, TRG1 and TRG2, in contrast to human and mouse TRG genes that are located in a single locus. The bovine TRG1 and TRG2 loci are located on chromosome 4 at 4q3.1 and 4q1.5-2.2, respectively. The complete genomic organization of the two bovine loci is described: each locus comprises three cassettes, each one includes one or several variable genes (TRGV) and one or several joining genes (TRGJ) preceding a constant (TRGC) gene. The location of the TRGC5 cassette is conclusively described in 5' of the TRG1 locus. Analysis of 17 TRGV belonging to 10 different subgroups, 8 TRGJ and 6 TRGC genes is conducted which comprises the most comprehensive list to date.