Mycobacterium salmoniphilum infection in farmed Atlantic salmon, Salmo salar L

Multiple greyish‐white visceral nodules containing abundant rapidly growing and acid‐fast bacteria, subsequently identified as Mycobacterium salmoniphilum, were detected in moribund and newly dead market‐sized fish during a period of increased mortality in an Atlantic salmon, Salmo salar, farm in western Norway. Isolates cultured from diseased fish were phenotypically consistent with Mycobacterium sp. previously isolated from Atlantic salmon [MT 1890 (= NCIMB13533), MT1892, MT1900 and MT1901] in the Shetland Isles, Scotland. Partial sequences of 16S rDNA, ribosomal RNA internal transcribed spacer (ITS1), 65‐kDa heat‐shock protein (Hsp65) and β subunit of RNA polymerase (rpoB) revealed 97‐99% similarity with M. salmoniphilum type strain ATCC 13758^T. The source of infection was not confirmed. Koch’s postulates were fulfilled following experimental challenge of Atlantic salmon with field isolate NVI6598 ( FJ616988 ). Mortality was recorded in experimentally infected fish; however, the infection remained subclinical in the majority of affected fish over the 131‐day challenge period.     

A simulated surveillance program for bovine paratuberculosis in dairy herds in Norway

Monte Carlo simulation models were used to evaluate the feasibility and potential results of a proposed national survey of the prevalence of bovine paratuberculosis (PTB) in dairy herds in Norway. The expected herd prevalence was assumed to be 0.2% in the simulations. The low sensitivity of the ELISA test, the assumed low herd prevalence, the typical low within-herd prevalence of PTB and the small herd sizes all present problems in detection of the disease. Simulations with 500, 1000, 2500 and 6000 herds tested were done. Our results suggest that a national survey would not be feasible at present, due to the low probability of detecting infected herds and because of the high number of false-positive reactions that would be expected to occur.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

副结核分枝杆菌DNA探针的制备与应用

以溶菌酶、ＳＤＳ、高氯酸钠等处理提取副结核分析杆菌Ｃ２株染色体ＤＮＡ，经限制性内切酶Ｐｓｔ Ｉ消化后，以质粒ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ为载体，通过Ｔ４ＤＮＡ连接酶连接，转入Ｅ。ｃｏｌｉ ＤＨ５ａ受体菌中，构建了副结核菌Ｃ２株的ＤＮＡ基因文库。应用反向杂交试验，从基因文库中筛选出４个重组克隆：ＰＴＰ１２、ＰＴＰ１９、ＰＴＰ３８。对这４个重组克隆进行酶切、电泳分析，结果表明４个插入片段长度分别为：４     

A case of mycobacteriosis associated with Mycobacterium pseudoshottsii in aquarium-reared fish in Japan

In 2019, several aquarium-reared fish died at a sea life park in Japan. Necropsy revealed micronodules on the spleen in the dotted gizzard shad (Konosirus punctatus). Seven of 16 fish exhibited microscopic multifocal granulomas associated with acid-fast bacilli in the spleen, kidney, liver, alimentary tract, mesentery, gills, and/or heart. Bacterial cultures yielded isolates from the dotted gizzard shad and a Japanese sardine (Sardinops melanostictus). Microbiological and molecular biological examinations revealed the isolates as Mycobacterium pseudoshottsii. To our knowledge, this is the first isolation of M. pseudoshottsii from aquarium-reared fish.     

应用基因分型技术研究牛结核分枝杆菌分子流行病学

牛分枝杆菌是引起牛结核病(bTB)的病原体,侵染宿主广泛,可感染多种家畜和野生动物,对人类和动物健康构成巨大危害。随着多年不懈的研究,有关分枝杆菌分子流行病学的知识积累不断增加。作者对近年来应用新的基因分型技术研究牛结核分枝杆菌分子流行病学方面的一些研究进展。这些技术包限制性片段长度多态性分析(RFLP)、PCR介导的间隔区寡核苷酸分析(spoligotyping)、数目可变串联重复位点(VNTR)等。主要用于对人、牛、家畜以及野生动物的结核病分子流行病学进行分析和监测。另外,还利用一系列敏感的基于PCR的技术从分枝杆菌杆菌复合体(NTM)中对菌型进行鉴别。对分枝杆菌感染分子流行病学的全面了解,有助于对本病扩散传播机制的深入研究。可为制定科学防治结核病的措施做出贡献。     

从许氏平鲉体表分离海分枝杆菌的鉴定及其脂多糖的抗原性分析

从许氏平鲉粘液、背鳍、鳃等部位分离筛选到同一菌株FZ09,经形态学观察、生理生化特性及热激蛋白65(HSP65)基因序列分析,鉴定菌株FZ09为海分枝杆菌。分别用酚-氯仿-甲醇法、乙醇-酚水法和传统热酚水法提取菌株FZ09的脂多糖(LPS),分析了LPS的组成和抗原性差异。SDS-PAGE结果表明,3种方法提取的LPS条带分布基本相同,主要条带分子量分别为14、16和23 ku,其中以乙醇-酚水法提取的LPS含量和纯度最高,热酚水法提取的LPS条带最多,抗原性最好。Western-blotting显示,海分枝杆菌抗血清主要与LPS的14 ku条带发生特异性免疫反应。高碘酸氧化免疫印迹结果表明,仅传统热酚水法提取的LPS在14 ku分子量区域有弱的阳性条带出现,其他方法提取LPS与抗血清的免疫反应呈阴性。高碘酸氧化ELISA反应显示,LPS经高碘酸氧化后与海分枝杆菌FZ09抗血清的反应活性降低,OD₄₀₅明显减弱。     

LRRK2/NF-κB signaling modulates inflammatory responses in BCG-infected RAW264.7 macrophages

AIM: To investigate the biological function and potential mechanism of leucine-rich repeat kinase 2 (LRRK2) in RAW264.7 macrophages during Mycobacterium tuberculosis infection. METHODS: The bacillus Calmette-Guerin (BCG)-infected RAW264.7 cell model was established. Colony-forming unit (CFU) analysis was used to determine the mycobacterial viability. The releases of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in the RAW264.7 cells were detected by ELISA. qPCR and Western blot were used to measure the mRNA and protein expression levels, respectively. RESULTS: LRRK2 was robustly enhanced in the RAW264.7 cells in response to BCG infection. Additionally, silencing of LRRK2 suppressed intracellular growth of mycobacteria during BCG challenge. Moreover, silencing of LRRK2 dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and IFN-γ induced by BCG infection. More importantly, LRRK2 modulated BCG-induced inflammatory responses by positively regulating the nuclear factor-κB (NF-κB) signaling pathway. CONCLUSION: LRRK2/NF-κB signaling pathway positively modulates inflammatory responses during BCG infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.     

牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。