Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

Abstract

The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines.     

Wheat pasture bloat dynamics, in vitro ruminal gas production, and potential bloat mitigation with condensed tannins

The aim of this study was to determine the effect of winter wheat (Triticum aestivum L.) forage growth stage, forage allowance, time of day, and commercial condensed tannins (CT) on steer bloat dynamics and in vitro ruminal gas production. Twenty-six crossbreed steers (Angus x Hereford x Salers; average initial BW = 194 +/- 26 kg) were used. Wheat forage allowances were either 18 kg (high forage allowance) and 6 kg (low forage allowance) of DM/(100 kg BW.d). In each bloat observation period, fresh wheat forage samples were hand-clipped to ground level in all study pastures for nutrient and in vitro ruminal gas production analyses. In vitro ruminal gas accumulation was measured at 0, 1, 2, 3, 4, 5, 6, and 12 h. Commercial CT was added at 0, 10, 15, and 20 mg of CT/g of DM. Bloat was scored once per week on two consecutive days at 0800 and 1500 during the vegetative stage and once every 2 wk during the reproductive stage of wheat development. Mean bloat score was calculated for each steer by time of day, stage of plant growth, and forage allowance. Bloat was detected in 65.8% of the observation periods. Average bloat scores were four and 2.5 times greater (P < 0.05) in cattle grazing at a high forage allowance than at a low forage allowance in the vegetative and reproductive growth phases of wheat, respectively. Rate of gas production was greater (P < 0.001) in the vegetative stage than in the reproductive stage. Steer bloat score was positively correlated with forage CP (r = 0.22; P < 0.05) and IVDMD (r = 0.32; P < 0.05). Rate of ruminal gas production was positively correlated (P < 0.01) to forage CP (r = 0.48), NPN (r = 0.40), soluble protein (r = 0.32), and IVDMD (r = 0.47). Conversely, negative correlations were found for forage DM (r = -0.20; P < 0.05), insoluble protein (r = -0.40), NDF (r = -0.69), and forage height (r = -0.49; P < 0.01) on the rate of ruminal gas production. Addition of CT at levels greater than 10 mg of CT/g of DM decreased (P < 0.05) the rate of in vitro ruminal gas and methane gas production after 5 h of incubation. Wheat pasture bloat is a complex disorder that varies across an array of forage and environmental conditions. Condensed tannins have the potential to decrease bloat by altering ruminal gas production and soluble protein digestibility from wheat forage.     

圆眼燕鱼哈维弧菌的分离鉴定及药敏试验

从患病圆眼燕鱼(Platax orbicularis)体内分离到一批形态一致的优势菌株,通过细菌鉴定并检测其药物敏感性,以期为圆眼燕鱼的疾病防治提供参考。对分离菌株进行形态观察、生理生化试验以及16S rRNA基因序列分析,结果显示该菌株与弧菌表型特征一致,16SrRNA与GenBank中哈维弧菌(Vibrio harveyi)的同源性高达99%,在系统进化树中与哈维弧菌(KU245727)聚为一分支,结合该菌的生理生化特征和基因序列分析,鉴定该优势菌株为哈维弧菌。药敏试验结果显示,该分离菌对头孢噻肟、强力霉素、恩诺沙星、萘啶酸、卡那霉素、妥布霉素、氯霉素、复方新诺明等14种药物敏感,对阿莫西林、青霉素G、乙酰螺旋霉素、杆菌肽、多黏菌素B、利福平、红霉素和磺胺异恶唑等8种药物表现为耐药。采用PCR方法检测该菌株携带TEM、Sul2、ANT(3′′)-1耐药基因。研究结果可为哈维弧菌的鉴定及圆眼燕鱼的疾病诊治提供参考。     

Histamine in lambs with abomasal bloat, haemorrhage and ulcers

The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls. In group 2, there was also a strong correlation (R2 = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others. The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups. Five out of eight tested strains of Lactobacillus spp. and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine. Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.     

Abomasal bloat, haemorrhage and ulcers in young Norwegian lambs

An abomasal syndrome affecting mainly three- to four-week-old lambs was studied by using a case-control design involving 88 cases and 85 controls. The principal clinical signs were tympany and colic. The cases were divided into three groups according to the main gross pathological findings in 82 of them. The 28 lambs in group 1 had abomasal tympany, the 29 lambs in group 2 had severe damage to the abomasal mucosa, and the 25 lambs in group 3 consisted mainly of lambs with various other diseases in combination with abomasal changes. The lambs in group 1 had a significantly (P<0.05) lower mean (se) abomasal pH (2.7 [0.19]) than those in group 2 (4.1 [0.32]), group 3 (3.7 [0.39]) or the controls (3.3 [0.13]). The ruminal pH values ranged from 3.5 to 7.4 but there were no significant differences between the groups. Lambs with ulcers had a significantly (P<0.05) higher frequency of trichophytobezoars, than the cases without ulcers or the controls. The tympanic lambs in group 1 had a significantly higher mean packed cell volume, and higher mean red and white blood cell counts, and a significantly lower mean cell volume and mean cell haemoglobin concentration than the healthy control lambs.     

Sarcina-like bacteria associated with bloat in young lambs and calves

Sarcina species are fastidious Gram-positive anaerobic bacteria that occur in cubical packets of eight or more cells. In 2006 to 2007, they were associated with cases of acute abomasal bloat in young lambs and calves. Two incidents were in lambs aged three to six weeks that were found dead, with one or two cases in each of a group of 15 and 100 lambs. Three incidents were recorded in small groups of calves up to 10 days of age, two cases in each incident, with the calves found dead or dying after a short illness characterised by bloat. Their gross lesions included emphysema and oedema of the abomasal wall, mucosal hyperaemia and haemorrhage, and rupture of the abomasum. Histological lesions included abomasitis with congestion, haemorrhage, emphysema and oedema. Bacteria characteristic of Sarcina species were observed in sections associated with the superficial mucosa of these cases, but the bacteria were not detected in cultures.     

噬菌体防治副溶血弧菌感染研究进展

副溶血弧菌（Vibrio parahaemolyticus）不仅是我国水产养殖中引发细菌性疾病的重要病原菌,也是引发水产品食源性疾病的重要致病菌。近年来,噬菌体（bacteriophage）作为抗生素替代品在防治细菌性疾病方面的研究得到广泛关注,未来可能是破解抗生素滥用导致的微生物耐药难题的新型生物制剂,具有广阔的应用前景。噬菌体具有高度宿主特异性和宿主菌依赖性,可有效控制软体动物、甲壳类动物、鱼类动物副溶血弧菌感染;结合基因工程技术、鸡尾酒疗法可解决噬菌体在防治副溶血弧菌感染中的一些不足,但噬菌体安全风险仍需进一步评估。今后,深入研究噬菌体的结构与功能,揭示噬菌体与宿主之间相互作用关系,是推动噬菌体应用于生产实践的重要基础;优化噬菌体鸡尾酒疗法、噬菌体基因编辑等,将成为噬菌体治疗研究领域的重要方向。噬菌体将为水产养殖绿色发展和生物安保提供重要技术手段。     

Cloning and expression of Vibrio harveyi OmpK* and GAPDH* genes and their potential application as vaccines in large yellow croakers Pseudosciaena crocea

Vibrio harveyi is a causative agent of vibriosis in the large yellow croaker Pseudosciaena crocea and causes severe losses to the aquaculture industry in China. The vaccines based on the outer membrane proteins (OMPs) of the pathogens are considered to be the optimum intervention for this disease. In this study, two V. harveyi OMP genes, OmpK* and glyceraldehyde-3-phosphate dehydrogenase (GAPDH*), were cloned, sequenced, and characterized. The recombinant proteins (r-OmpK and r-GAPDH) were expressed by the prokaryotic expression vector pET-30a(+) and purified with nickel-nitrilotriacetic acid affinity chromatography. Western blots showed that rabbit antisera against purified r-OmpK and r-GAPDH specifically reacted with the native OMP of V. harveyi. Large yellow croakers were immunized with r-OmpK and r-GAPDH. Specific antibody titer assessed by enzyme-linked immunosorbent and phagocytosis assays demonstrated that specific and innate immunity was stimulated in response to the OMPs of V. harveyi. Challenge results indicated that vaccination of large yellow croakers with r-OmpK and r-GAPDH increased relative survival (37.7% and 40.0%, respectively) against wild V. harveyi.     

In vitro bacterial growth and in vivo ruminal microbiota populations associated with bloat in steers grazing wheat forage

The role of ruminal bacteria in the frothy bloat complex common to cattle grazing winter wheat has not been previously determined. Two experiments, one in vitro and another in vivo, were designed to elucidate the effects of fresh wheat forage on bacterial growth, biofilm complexes, rumen fermentation end products, rumen bacterial diversity, and bloat potential. In Exp. 1, 6 strains of ruminal bacteria (Streptococcus bovis strain 26, Prevotella ruminicola strain 23, Eubacterium ruminantium B1C23, Ruminococcus albus SY3, Fibrobacter succinogenes ssp. S85, and Ruminococcus flavefaciens C94) were used in vitro to determine the effect of soluble plant protein from winter wheat forage on specific bacterial growth rate, biofilm complexes, VFA, and ruminal H2 and CH4 in mono or coculture with Methanobrevibacter smithii. The specific growth rate in plant protein medium containing soluble plant protein (3.27% nitrogen) was measured during a 24-h incubation at 39 degrees C in Hungate tubes under a CO2 gas phase. A monoculture of M. smithii was grown similarly, except under H2:CO2 (1:1), in a basal methanogen growth medium supplemented likewise with soluble plant protein. In Exp. 2, 6 ruminally cannulated steers grazing wheat forage were used to evaluate the influence of bloat on the production of biofilm complexes, ruminal microbial biodiversity patterns, and ruminal fluid protein fractions. In Exp. 1, cultures of R. albus (P < 0.01) and R. flavefaciens (P < 0.05) produced the most H2 among strains and resulted in greater (P < 0.01) CH4 production when cocultured with M. smithii than other coculture combinations. Cultures of S. bovis and E. ruminantium + M. smithii produced the most biofilm mass among strains. In Exp. 2, when diets changed from bermudagrass hay to wheat forage, biofilm production increased (P < 0.01). Biofilm production, concentrations of whole ruminal content (P < 0.01), and cheesecloth filtrate protein fractions (P < 0.05) in the ruminal fluid were greater on d 50 for bloated than for nonbloated steers when grazing wheat forage. The molecular analysis of the 16S rDNA showed that 2 different ruminal microbiota populations developed between bloated and nonbloated animals grazing wheat forage. Bloat in cattle grazing wheat pastures may be caused by increased production of biofilm, resulting from a diet-influenced switch in the rumen bacterial population.     

Effects of iron dextran injections on the incidence of abomasal bloat, clinical pathology and growth rates in lambs

A preliminary study revealed significantly lower serum iron concentrations in lambs that developed abomasal bloat about one week later, than in lambs that did not develop bloat. In a subsequent trial, with 754 naturally reared twin lambs from five flocks, iron dextran injections were found to have a preventive effect on the development of abomasal bloat. Clinical signs of abomasal bloat were observed in the placebo-treated lamb of 16 couples and in the iron-treated lamb of six couples (P<0.05). In three further couples, both lambs developed bloat. The iron-treated group had significantly (P<0.001) better average weight gains, both from birth to summer, and from birth to autumn of approximately 0.5 and 1 kg, respectively. There was a reduction in red blood cell values and iron saturation in the placebo group 14 days after treatment but not in the iron-treated lambs. In one of the flocks, there was a decrease in the cumulative incidence of abomasal bloat from 37 per cent to 3 per cent during the period of four years after measures such as later lambing, earlier turnout and iron injections were introduced.     

The application of hCG, CPH and Ovopel in successful artificial reproduction of goldfish (Carassius auratus auratus) under controlled conditions

Artificial reproduction of fish is one of the main goals of aquaculture production. The aim of this study is to optimize the method of goldfish reproduction under controlled conditions by comparing the effectiveness of carp pituitary homogenate (CPH), Ovopel and human chorionic gonadotropin (hCG), administered as a one-off dose and inducing two spawns in the same fish within a short time period. Goldfish spawners were stimulated with hCG, CPH and Ovopel, and the results were compared to the fish from the control group, comprised of unstimulated fish. In another experiment, spawn were induced twice within an interval of 21 days with the same group of fish. The best results in the first experiment in terms of the percentage of ovulating females and survival to the eyed-egg stage were achieved after administering hCG (100% and 88.7%, respectively). However, the highest fecundity was observed in fish stimulated with Ovopel (89,960 eggs/kg). It was shown in the second experiment that female goldfish produce higher weight of eggs during the first spawning, but the number of eggs/BW ratio was higher during the next reproduction process. Survival, both that of embryos to the eyed-egg stage and that of spawners, is higher during the first reproduction act.