Pathologic and immunohistochemical findings in an outbreak of systemic toxoplasmosis in a mob of red kangaroos

Toxoplasma gondii is a zoonotic protozoan pathogen that infects many endothermic vertebrates, including humans; the domestic cat and other felids serve as the definitive host. Macropodids are considered highly susceptible to toxoplasmosis. Here, we describe the clinical, pathologic, and immunohistochemical findings of an outbreak of systemic toxoplasmosis in a mob of 11 red kangaroos (Macropus rufus), with high morbidity (73%) and mortality (100%) rates. Affected animals had either severe and rapidly deteriorating clinical conditions or sudden death, which was correlated with widespread necrotizing lesions in multiple organs and intralesional T. gondii organisms identified via MIC3-specific immunohistochemistry and confirmed by REP529-specific rtPCR. Quantification of parasites demonstrated the highest parasite density in pulmonary parenchyma compared with other tissues. Our study highlights the continued importance of this severe condition in Australian marsupials.     

猪戊型肝炎病毒ORF3蛋白影响HepG2细胞核黄素代谢的lncRNA-mRNA调控网络的鉴定

为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪 HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。     

人工养殖西伯利亚狍肠道微生物组成的雌雄差异

肠道微生物是一个复杂的生态系统,与宿主的健康、疾病以及其他因素有着密切的关系。目前,关于肠道菌群结构和功能的研究较多,但是对西伯利亚狍肠道菌群的研究还未见报道。西伯利亚狍(Capreolus pygargus)属于反刍动物,有独特的消化特性和微生物群来帮助它们适应高纤维的食物。本研究利用高通量测序技术在Illumina MiSeq平台上对14只狍肠道微生物进行16S rRNA基因分析。结果表明:个体样本中门类组成的差异,雌性个体JY3和雄性个体JY9除Firmicutes,Bacteroidetes,Proteobacteria这3类菌群外,其他门类菌群所占比例都很低。在科水平上,雌性个体中Ruminococcaceae在JY7和JY13肠道内含量相对较少,Bacteroidaceae在JY7,JY10和JY13中含量较高。雄性个体中Ruminococcaceae在JY11和JY14肠道内含量较少,Prevotellaceae在JY4中含量较少。在属水平上,雌性个体特有的菌属为Roseburia,Alloprevotella,Lachnospiraceae_incertae_sedis和Coprococcus;雄性个体特有的菌属为Fusobacterium,Succiniclasticum,Streptococcus和Clostridium sensu stricto。Alpha多样性分析表明,Ace指数范围2128.93—772.98,Chao1指数范围2128.89—785.27,Shannon指数范围5.44—3.78,Simpson指数范围0.07—0.01。在不同物种分类水平上,雌雄狍肠道内的优势菌群有些不同。PCoA(principal co-ordinates analysis)分析显示,狍样本几乎混在一起,没有明显差异。     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 x 10(6) viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 x 10(5) viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 x 10(5) viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.     

Sampling and repeatability of radiometric faecal culture in bovine Johne's disease

The repeatability of detection of Mycobacterium avium subsp. paratuberculosis (Map) within and between samplings from 16 paratuberculous dairy cows (13 subclinical; 3 clinical) was investigated by radiometric culture of quadrants of faecal dung pats collected on four to seven occasions over a 10-16-day period. Results were compared to serological status and to pathological and bacteriological findings in multiple tissues obtained at slaughter from 15 of the animals 2-6 weeks after the faecal samplings. From faecal samples taken on 77 occasions over the 2-week period, 296/308 (96%) quadrants were culture positive, with samples from all cattle showing evidence of faecal shedding of Map. Histological lesions typical of paratuberculosis were present in 14 of the 15 cows examined at slaughter, varying in severity from mild (two animals) to moderate (4) and advanced (8), and all predilection tissue sites yielded Map. The negative faecal samples were derived from a single animal that was culture positive in two quadrants on each of the first two (of four) sampling occasions (i.e. culture positive in only 4 of 16 collected quadrants). This animal was found to be histologically negative at slaughter, and culture positive from three of five predilection tissue sites. Faecal samples from cows with subclinical and clinical paratuberculosis, with lesion severity ranging from mild to severe at multiple predilection sites, produced faeces with relatively consistent concentrations of Map within samples. There was significant variation in concentrations of Map between samples in individual animals over a period of 2 weeks, but this did not affect the dichotomous positive-negative culture status for 15 of the 16 cattle. A faecal sample collected non-randomly per rectum thus provides a representative specimen for detection of Map by radiometric culture on a single sampling occasion.