广西地区禽致病性大肠杆菌的耐药性分析

禽致病性大肠杆菌(APEC)是一种能引起鸡、火鸡和其他鸟类肠外感染的致病性大肠杆菌,可以导致肉鸡气囊炎、败血型全身感染、蜂窝织炎和蛋鸡输卵管炎、腹膜炎。为了了解广西地区禽致病性大肠杆菌的耐药表型以及耐药基因的携带情况,本实验室对2019年从广西分离到的69株APEC采取K-B药敏纸片法进行药敏试验,药敏结果显示,69株APEC对氧氟沙星(56.5%)、恩诺沙星(69.6%)、氟苯尼考(79.7%)、氨苄西林(91.3%)、四环素(98.6%)耐药率较高,而对美罗培南、丁胺卡那霉素、呋喃妥因均不耐药;其中,多重耐药现象严重,对10种抗菌药物以耐4种、5种、6种的情况居多。同时用PCR扩增的方法对其耐药基因,包括碳青霉稀类、β-内酰胺类、氨基糖苷类、黏菌素类、喹诺酮类、四环素类在内的6大类共计17种耐药基因进行了检测。特别值得关注的是,发现了7株携带mcr-1基因的多黏菌素耐药APEC。药敏纸片法检测菌株的耐药表型和耐药基因存在一定关联度。本研究可为养禽场临床用药提供参考,同时为减缓耐药菌传播、降低对人类健康和公共卫生安全威胁提供依据。     

Avian pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli (APEC) cause aerosacculitis, polyserositis, septicemia and other mainly extraintestinal diseases in chickens, turkeys and other avian species. APEC are found in the intestinal microflora of healthy birds and most of the diseases associated with them are secondary to environmental and host predisposing factors. APEC isolates commonly belong to certain serogroups, O1, O2 and O78, and to a restricted number of clones. Several experimental models have been developed, permitting a more reliable evaluation of the pathogenicity of E. coli for chickens and turkeys. Hence, virulence factors identified on APEC are adhesins such as the F1 and P fimbriae, and curli, the aerobactin iron sequestering system, K1 capsule, temperature-sensitive hemagglutinin (Tsh), resistance to the bactericidal effects of serum and cytotoxic effects. Experimental infection studies have shown that the air-exchange regions of the lung and the airsacs are important sites of entry of E. coli into the bloodstream of birds during the initial stages of infection and that resistance to phagocytosis may be an important mechanism in the development of the disease. They have also demonstrated that F1 fimbriae are expressed in the respiratory tract, whereas P fimbriae are expressed in the internal organs of infected chickens. The role of these fimbrial adhesins in the development of disease is not yet, however, fully understood. The more recent use of genetic approaches for the identification of new virulence factors will greatly enhance our knowledge of APEC pathogenic mechanisms. Diagnosis of APEC infections is based on the clinical picture, lesions and isolation of E. coli. This may be strengthened by serotyping and identification of virulence factors using immunological or molecular methods such as DNA probes and PCR. Approaches for the prevention and control of APEC infections include the control of environmental contamination and environmental parameters such as humidity and ventilation. Antibiotherapy is widely used, although APEC are frequently resistant to a wide range of antibiotics. Vaccines containing killed or attenuated virulent bacteria protect against infection with the homologous strain but are less efficient against heterologous strains. Hence, vaccination for colibacillosis is not widely practised because of the large variety of serogroups involved in field outbreaks.     

Avian pathogenic Escherichia coli (APEC)

Infections with avian pathogenic Escherichia coli (APEC) cause colibacillosis, an acute and mostly systemic disease resulting in significant economic losses in poultry industry worldwide. Avian colibacillosis is a complex syndrome characterized by multiple organ lesions with airsacculitis and associated pericarditis, perihepatitis and peritonitis being most typical. Environmental factors as well as the constitution of poultry or initial viral infections influence the outcome of APEC-infections. However, several challenge experiments in chickens proofed the role of virulent APEC strains as the single aetiological agent. Currently serotypes O1:K1, O2:K1 and O78:K80 are recognized as the most prevalent, however the number of published serotypes is increasing. In addition, single APEC isolates vary profoundly in virulence, and knowledge about the molecular basis of this variability is still scarce. Known virulence factors of APEC are adhesins (F1- and P-fimbriae), iron acquisition systems (aerobactin and yersiniabactin), hemolysins (hemolysinE and temperaturesensitive hemagglutinin), resistance to the bactericidal effects of serum and phagocytosis (outer membrane protein, iss protein, lipopolysaccharide, K/1)-capsule and colilcin production) as well as toxins and cytotoxins (heat stable toxin, cyto-/verotoxin and flagella toxin). Esperimental studies have shown that the respiratory tract, principally the gas-exchange region of the lung and the interstitium of the air sacs are the most important sites of entry for avian pathogenic E. coli. APEC strains adhere to the epithelial cells of air sacs presumably through F1-fimbriae. After colonization and multiplication the bacteria enter the bloodstream, and the temperature-sensitive hemagglutinin (tsh) seems to be important int his step. After invading the bloodstream APEC cause a septicemia resulting in massive lesins in multiple internal organs and in sudden death of the birds. The ability of the bacteria to acquire iron and the resistance to the bactericidal effects of serum, predominantly conferred by the increased serum survival (iss)--protein, enables APEC to multiply quickly in their hosts. Iss is regarded a specific genetic marker for avian pathogenic E. colistrains. A critical review of the literature published so far on APEC reveals, that these pathotypes are not defined appropriately. This findings urge investigations on the population structure of APEC, enabling the establishment of appropriate diagnostic tools and avoiding the obsolete use of serotyping for APEC diagnosis. So far more than 20 APEC strains have been investigated in animal experiments, explaining contrary published results. Thus, the lack of knowledge in pathogenicity and in immunity of APEC infections urges further experimental studies. As APEC share not only identical serotypes with human pathogens but also specific virulence factors, their zoonotic potential is under consideration.     

Avian pathogenic Escherichia coli (APEC)

Escherichia coli infections are being increasingly detected among poultry flocks, indicating the growing importance of this pathogen to the industry. The infection begins as a respiratory infection of the trachea, followed by colonization of the air sacs and lungs, from where it invades the blood-stream, leading to infection of the deeper organs (liver, heart, oviduct, and peritoneum). A number of factors play a crucial role in the virulence and pathogenesis of infection. The F1 and P pili are particularly important in establishing the infection at the level of the tracheal epithelium cell. Other important factors are aerobactin, capsule, and serum resistance. Treatment is with antibiotics, but the growing bacterial resistance of avian E. coli and stricter regulations mean that attention is turning to prophylactic, preventative, measures, such as vaccination. Current vaccines provide limited homologous protection against the pathogen. Research is needed to develop a good, broad-spectrum vaccine.     

Four distinct neurologic syndromes in Marek's disease: effect of viral strain and pathotype

A chronological study of central nervous system disorders induced by Marek's disease virus (MDV) has been conducted. Neurologic clinical signs were recorded daily for individual chickens of two genetic lines after inoculation of 13 serotype 1 MDV strains representing all three pathotypes. In addition to classical transient paralysis (TP) previously described by many workers, and acute TP, described in the companion paper, we have identified for the first time two other neurologic syndromes, persistent neurologic disease (PND) and late paralysis (LP). PND designates birds that showed a variety of neurologic signs (ataxia, torticollis, and nervous tics) after recovery from paralysis (12-15 days postin-oculation [DPI]) that either persisted through the observation period or presented a cyclic pattern. LP was a rare syndrome characterized by the late onset of the paralytic stage (about 20 DPI), perhaps indicating occasional failure of the initial intraabdominal inoculation to induce infection. Clinical signs and histopathologic alterations of the brain were also evaluated sequentially in chickens of two genetic lines after inoculation with two MDV strains (virulent MDV and very virulent plus MDV). Although clinical response differed greatly among treatment groups, types of lesions (endotheliosis, mononuclear perivascular cuffing, vasculitis, vacuolization, and increase in cellularity of the neuropil) were similar. However, early onset of lesions (by 6 days) appeared to be associated with a greater severity of clinical signs. We also found that neurologic response was greatly influenced by viral pathotype (virulence). This study thus confirms that the central nervous system is an important target organ for MDV resulting in several distinct clinical manifestations and suggests that neurologic responses in antibody-free chickens might be a useful criterion for virus pathotyping.     

禽致病性大肠杆菌(APEC)感染对雏鸡脾脏miRNA表达谱的影响

基于miRNA测序技术研究感染禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)雏鸡脾脏microRNA(miRNA)的差异表达,为深入了解APEC的致病机制奠定理论基础。将14日龄罗曼鸡随机分为2组,分别腿部肌肉注射APEC分离株AE17和生理盐水后采集脾脏组织,制作病理切片并进行miRNA测序,用miRanda算法预测差异表达miRNA的靶基因,并进行GO和KEGG分析。切片结果显示,与对照组相比,AE17株感染组脾脏组织有病变。通过miRNA测序总共筛选了21个差异表达miRNA的靶基因,其中8个下调和13个上调;对预测靶基因进行GO分析发现其显著富集在细胞、细胞器、膜等功能上;KEGG分析显示其参与Toll样受体信号通路和MAPK信号通路等免疫系统过程。本研究在AE17株感染组脾脏病理切片中发现病理变化,并利用miRNA测序技术成功获取了APEC感染鸡脾脏的miRNA表达谱,发现雏鸡脾脏组织miRNA表达丰富且表达量各异,为探讨APEC致病机制提供新思路。     

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh_s) and a 33 kDa β-domain (Tsh_β). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh_s (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh_β (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.     

Equine herpesvirus-1, non-neurogenic pathotype, in a 9-year-old American Saddlebred with neurological signs

Equine herpesvirus 1 (EHV-1) causes rhinopneumonitis, abortion, and rarely, myeloencephalopathy. The neurovirulence of this virus is due to a point mutation in the DNA polymerase gene. Diagnosis by virus isolation has been replaced by real-time polymerase chain reaction (RT-PCR) assays that can detect strains, viral loads, and states; this may aid in control and management of the disease.     

Characterizing the Serum Proteome of Donkeys (Equus asinus)

Serum and plasma are commonly used in clinical practice considering the widely accepted fact that the “normal” protein expression pattern of a healthy animal changes under disease conditions. We herein used a label-free mass spectrometry–based quantitative proteomics approach to characterize the serum proteome of donkeys. A total of 277 unique proteins were identified from 2,388 unique peptides. Gene ontology analyses showed that the most frequent processes were related to metabolic activities and biological regulation, response to stimulus, and immune system processes. The main annotated areas of origin were the extracellular region, extracellular region part, and organelle, and their molecular functions included binding, catalytic activity, and molecular function regulator. Analyses using the Clusters of Orthologous Groups for Eukaryotic Complete Genomes database indicated that the identified proteins could be categorized into three main groups: signal transduction mechanisms, amino acid transport and metabolism, and defense mechanisms. Most of the unique proteins were associated with the complement and coagulation cascades, and they participated in several disease-related metabolic pathways. Our results should be crucial for further analyses of changes in different physiological and pathophysiological conditions in donkeys.     

APEC强毒力岛核心基因irp2、fyuA敲除对其致病性影响的研究

采用Red同源重组技术分别构建出APEC irp2单基因敲除株△AE17和irp2、fyuA双基因敲除株△△AE17。运用活菌计数法分别观察AE17、△AE17、△△AE17黏附鸡胚成纤维细胞DF-1的能力。并且应用Real-time PCR检测AE17、△AE17和△△AE17中的luxs,pfs,tsh,ibeA,stx2f,iss,ompA,fimC 8个毒力基因转录水平。结果成功构建出基因敲除株△AE17和△△AE17,它们黏附DF-1细胞的能力分别下降为AE17的66.04%和53.54%。同时,Real-time PCR结果显示,△AE17的luxs,iss,ompA和fimC等4个毒力基因的转录水平均极显著下降(P<0.01),△△AE17的luxs,pfs,tsh,iss,ompA和fimC等6个毒力基因的转录水平极显著的下降(P<0.01)。结果表明强毒力岛中核心基因的敲除能够减弱APEC黏附DF-1的能力,并且使其毒力基因的转录水平有所降低。irp2、fyuA双基因敲除较irp2单基因敲除对APEC的致病性影响更显著。     

Characterizing the in vitro capacitation of motile bull sperm by the hyamine-induced acrosome reaction

Described in this paper is a method to characterize capacitation of bull spermatozoa. Involved is the action of 0.005 per cent of hyamine 2389 on selected motile cells at 37 degrees C for 30 minutes to detect the number of spermatozoa capable of acrosomal reaction. The latter amount results from the percentual difference between acrosome-free spermatozoa prior to hyamine action and those after hyamine action. Acrosome-free spermatozoa can be easily identified by means of simple staining under optical light microscopy. The value thus obtained is related to the amount of spermatozoa in progressively motile in the unselected original suspension and is called "Percentage Acrosome Reaction Induction" (% ARI). The ARI capacitation-specific acts: Only small amounts of acrosome-free spermatozoa are recordable among incapacitated spermatozoa or from spermatozoa incubated in the presence of decapacitation factors (1.9 or 2.3 per cent, respectively). On the other hand, clearly increased values of 17.6 ot 26.1 per cent are obtainable from treatments with capacitation effect (high ionic strength or addition of heparin). The method is claimed to be suitable for adequate choice of conditions for improved capacitation and in vitro fertilization as well as for elucidation of specific individual differences in capacitation.     

青海省三江源地区退化草地蒸散特征

了解三江源地区退化草地的蒸散状况有助于认识该地区退化生态系统水循环,对该地区的生态安全有着重要的意义。本研究利用涡度相关法观测青海省玛沁县小嵩草退化草地（３４．３５°Ｎ;１００．５０°Ｅ,海拔３９６３ｍ）的水热通量,对该地区２００６－２００８年全年的蒸散情况进行了特征分析。研究表明,２００６－２００８年三江源退化草地的蒸散值分别为４５２．２４,４７４．２４,４５９．５７ｍｍ,降水量分别为４６０．７,４９６．１,４８０．１ｍｍ,其中有７５％以上的降水分布在生长季,全年水平上蒸散量与降水量之比（ＥＴ／ＰＰＴ）为９５％ 以上,蒸散量与降水量基本持平。生长季日蒸散量１．８～１．９ｍｍ／ｄ,而非生长季日蒸散量＜０．６ ｍｍ／ｄ。蒸散日变化和年变化均为单峰型,每年６－７ 月蒸散量最大,为２．０～２．２ｍｍ／ｄ;日蒸散峰值出现在正午左右,其最大值生长季为０．２１ｍｍ／ｈ,非生长季为０．１０ｍｍ／ｈ。退化草地蒸散的主要环境因子是净辐射,其次为饱和水汽压亏和空气温度。随着生长季的推进,潜热对这些因素的敏感性逐渐增大。与未退化的矮嵩草草甸相比,退化草地生长季蒸散量较小,而非生长季蒸散量较大,这一结果表明,高寒草地的植被盖度驱动草地生态系统的蒸散。     

SNPpath: Characterizing cattle SNPs by enriched pathway terms

High‐density single nucleotide polymorphism (SNP) microarrays have made large‐scale genome‐wide association studies (GWAS) and genomic selection (GS) feasible. Valuable insight into the genetic basis underlying complex polygenic traits will likely be gained by considering functionally related sets of genes simultaneously. SNPpath, a suite of computer‐generated imagery‐based web servers has been developed to automatically annotate and characterize cattle SNPs by enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms. The SNPpath allows users to navigate and analysis large SNP sets and is the only web server currently providing pathway annotations of cattle SNPs in National Center for Biotechnology Information's dbSNP database and three commercial platforms. Hence, we describe SNPpath and provide details of the query options, as well as biological examples of use. The SNPpath may be favorable for the analysis of combining SNP association analysis with pathway‐driven gene set enrichment analysis and is freely available at http://klab.sjtu.edu.cn/SNPpath .     

Sequence analysis demonstrates the conservation of fimH and variability of fimA throughout avian pathogenic Escherichia coli (APEC)

In this study we sequenced and analysed the fimH and fimA genes of 24 avian pathogenic Escherichia coli (APEC) isolates, in order to investigate their possible conserved nature. Additional parameters (serotype, presence of aerobactin receptor, expression of F1 pili and virulence for chickens) were investigated to look for correlations with the obtained sequences. The sequence analysis demonstrated that FimH is highly conserved among all investigated APEC strains (>99% homology), whereas the major subunit FimA is less conserved, presenting 6 variable regions distributed along the protein. A hydrophilicity analysis suggested several variable domains of FimA to be potential epitopes. We were able to classify the investigated strains into three main groups, on the basis of the amino-acid sequences of the variable regions. This grouping was consistent throughout all variable regions and was independent of serotype, leading to an improved classification of the F1 pili. No correlation was found between the fimH and fimA sequences and the following parameters: avian species, organ of isolation, serotype, presence of aerobactin receptor and virulence for chickens. This study elucidated the molecular structure and the degree of conservation of FimH and FimA among various avian pathogenic E. coli strains.     

Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry

The molecular biology and epidemiology of 150 avian pathogenic Escherichia coli strains (APEC) isolated from septicemic poultry in Germany was investigated by serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction (PCR). Only 49.6% of the isolates could be grouped to serogroups O1, O2, and O78. Macrorestriction analyses data revealed two large clonal groups (clusters I and II) among the APEC strains with a similarity of 60.9% to each other. An association between restriction pattern and serogroup or origin of the strains was only present in a few subgroups of each clusters I and II, but was not evident. In contrast, our data revealed distinct combinations of virulence-associated genes in that 51.2% of the O2-strains harboured a combination of the genes fyuA, irp2, iucD, tsh, vat, fimC, and colV and 36.4% of the O78-strains possessed the same gene combination with exception of vat. With 34 different gene combinations the non-O1, -O2, -O78 isolates revealed a higher variability in their virulence gene pattern than O1-, O2-, and O78-strains with 6, 13, and 9 patterns, respectively. Our data indicate only a limited association between the virulence gene pattern and the serogroup of APEC strains and question the sensitivity of O-typing for APEC identification without the application of further diagnostic tools. Although a limited number of APEC clones exist, horizontal gene transfer seems to be common in these pathogens. These findings strengthen further research on the population structure of APEC and may be the reason for the lack of clear definition of this common E. coli pathotype.     

Characterizing rumination patterns of dairy cows using spectral analysis

Spectral analysis techniques were used to characterize the cyclical variation in rumination behavior of cows. Four Holstein cows were fed twice daily a diet of 60% high-moisture shelled corn-based concentrate, 15% first-cut alfalfa-grass hay and 25% second-cut alfalfa silage. The number of minutes that each cow spent ruminating was determined for 15-min intervals during six consecutive days. Rumination data then were characterized using Fourier harmonic analysis to decompose the total sum of squares into 288 orthogonal components due to different rumination wavelengths. Rumination patterns for all cows consisted mainly of wavelengths that were harmonics of a 24-h cycle, indicating a circadian pattern of rumination. Differences in rumination patterns between cows occurred mainly at wavelengths of less than 2 h. Rumination patterns of two of the four cows were more complex, and consisted of high-frequency, non-24-h harmonic wavelengths in addition to the circadian pattern. Spectral analysis can be used to identify the component cycles of rumination patterns of individual animals, which can then be used to determine the effects of dietary or other manipulations on rumination behavior.     

Characterizing the Economic Value and Impacts of Wild Pig Damage on a Rural Economy

Wild pigs are a free-ranging invasive species capable of inflicting significant damage on rural property. Wildlife management personnel may benefit from understanding the negative societal impact of wild pigs. A statewide mail survey of randomly selected landowners was conducted in rural Tennessee counties known to have wild pigs. The economic value of damage caused by wild pigs in 2015 in these counties was estimated at $26.22 million, whereas the cost incurred in control and eradication was $2.09 million. Input-output modeling of damage in these counties on the state’s economy showed $32.8 million in lost industrial output, $4.6 million in lost labor income, and 332 jobs or job equivalents affected. Findings are useful in understanding the types of damage, and the extent of these impacts on the rural economy. They could also facilitate comparing the expected benefit with the cost of control programs in Tennessee and comparable areas facing similar invasions from wild pigs.