Identification of Arcanobacterium (Trueperella) abortisuis, a novel species of veterinary importance, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)

In the present study A. (T.) abortisuis isolated from pigs and bovines could be reliably identified by determination of phenotypic properties, genotypically by polymerase chain reaction with the help of A. (T.) abortisuis 16s-23S rDNA intergenic spacer region specific oligonucleotide primer and by Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS). The latter appeared to be a promising tool for fast and cost effective identification of this species and might help to elucidate the role A. (T.) abortisuis plays in infections of pigs, bovines, possibly other animals or humans.     

Characterization of porcine Trueperella pyogenes by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), molecular typing and antimicrobial susceptibility profiling in Sao Paulo State

Trueperella pyogenes can be found as a commensal or pathogenic bacterium among animals causing a variety of pyogenic infections in several species. The agent appears to act primarily as an opportunistic pathogen but may disseminate and produce metastatic abscesses accompanied or not by mastitis, metritis or pneumonia. In this study, 30 porcine T. pyogenes strains were identified by MALDI-TOF MS and 16S rRNA sequencing and further evaluated in relation to their resistance and genetic profiles through broth microdilution and single enzyme AFLP. All strains were susceptible to β-lactams, florfenicol, gentamicin, spectinomycin and tiamulin. The highest resistance rates were observed for sulfonamides, tetracyclines and clindamycin. All isolates could be characterized by SE-AFLP presenting more than 80% of similarity, despite their distinct origins. Four genotypes were detected with the segregation of T. pyogenes ATCC 19411 from Brazilian T. pyogenes strains. No correlation between genotypes and isolates origins and susceptibility profile was observed.     

Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and,as novel target,by sequencing pluranimaliumlysin encoding gene pla

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.     

基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)在鸭源鸡杆菌快速鉴定中的应用

按照MALDI-TOF MS要求进行56株鸡杆菌分离株的样品制备,点靶并获取样品质谱图,运用Biotyper分析软件对所获取的质谱图鉴定分析;同时对这些菌株进行荧光定量PCR检测、gyrB基因的PCR扩增和序列分析,进行辅助分析鉴定。结果显示,56株鸡杆菌分离株经MALDI-TOF MS和gyrB基因序列分析均鉴定为鸭源鸡杆菌,阳性率为100.0%;荧光定量PCR鉴定出55株鸭源鸡杆菌,阳性率为98.2%(55/56)。结果表明,MALDI-TOF MS可用于鸭源鸡杆菌分离株的快速鉴定,操作简便,准确率高。     

山羊化脓隐秘杆菌的分离鉴定与分析

通过对重庆某羊场送检病死羊只进行病原菌的分离培养、形态学鉴定、生化鉴定、药敏试验、致病性试验及16S r RNA序列分析。结果从病变组织中分离到1株革兰阳性短棒状杆菌,其对多种抗生素敏感,能够引起小鼠肝脏和肺脏发生病变,PCR扩增出长为1 434 bp的目的序列,已被Gen Bank数据库收录,序列号为KJ842125,将该序列与NCBI中公布的序列进行比对,发现所得序列与化脓隐秘杆菌(Arcanobacterium pyogenes,登陆编号为HQ712123)同源性高达98%,将该序列与其相关性较高菌株的16S r RNA构建系统发育进化树,与化脓隐秘杆菌聚为一簇。本研究为化脓隐秘杆菌致病机理、诊断技术和疾病控制奠定了基础。     

Analysis of the bovine plasma proteome by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry

In this study, the bovine plasma proteome was analysed using a three step protocol: (1) plasma was treated with a combinatorial peptide ligand library (CPLL) to assimilate the differences in concentrations of different proteins in raw plasma; (2) CPLL-treated material was fractionated by three standard electrophoretic separation techniques, and (3) samples were analysed by nano-liquid chromatography (nLC) matrix-assisted laser desorption/ionisation (MALDI) time-of-flight tandem (TOF/TOF) mass spectrometry. The efficiencies of three fractionation protocols for plasma proteome analysis were compared.After size fractionation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), resolution of proteins was better and yields of identified proteins were higher than after charge-based fractionation by preparative gel-free isoelectric focussing. For proteins with isoelectric points >6 and molecular weights ?63 kDa, the best results were obtained with a ‘shotgun’ approach, in which the CPLL-treated plasma was digested and the peptides, rather than the proteins, were fractionated by gel-free isoelectric focussing. However, the three fractionation techniques were largely complementary, since only about one-third of the proteome was identified by each approach.     

Phenotypic characteristics and virulence genotypes of Trueperella (Arcanobacterium) pyogenes strains isolated from European bison (Bison bonasus)

In a breeding and fattening pig farm an increasing number of cases of abortion and generalized mycobacteriosis at slaughter occurred. Pathological findings compatible with mycobacteriosis, acid-fast organisms in tissues, and isolation of mycobacteria from tissue samples including fetuses, lungs and reproductive organs from sows, genital swabs, mesenteric lymph nodes, and from a sperm sample revealed the cause of the disease. Bacterial cultures were identified as Mycobacterium avium subsp. hominissuis using IS901-/IS1245-specific PCR. Genotyping of selected isolates from animals as well as from their environment by MIRU-VNTR analysis showed that the herd was infected with one single outbreak strain. The same genotype was also isolated from pigs of two other farms which showed comparable symptoms and were in direct contact with the index farm as well as from their environment. Immunological host responses detected by tuberculin skin test and ELISA gave positive results at herd level only. Despite the detection of other potential pathogens mycobacteria were regarded as the causative agent of the reproductive disorders. To our knowledge this is the first report of an epidemic mycobacterial infection in a pig holding associated with reproductive disorders, which could be attributed to one single virulent strain, and the first report of detection of M. avium subsp. hominissuis in pig sperm.     

Comparative electrocardiography in four species of macaws (genera Anodorhynchus and Ara)

Electrocardiograms (ECGs) were recorded during isoflurane anaesthesia from 52 macaws of four species of the genera Anodorhynchus and Ara in order to establish electrocardiographic reference values. The birds examined were clinically healthy macaws of the following species: hyacinth macaw (HM; Anodorhynchus hyacinthinus, n = 14); green-winged macaw (GWM; Ara chloroptera, n = 11); blue-throated macaw (BTM; Ara glaucogularis, n = 15); and red-fronted macaw (RFM; Ara rubrogenys, n = 12). All ECGs were recorded using a paper speed of 50 mm/s and a calibration of 10 mm = 1 mV. Significant differences were determined between species for the heart rate, duration and amplitude of the P wave, amplitude of the T wave, and amplitude of the QRS complex, specially comparing the RFM to the other macaw species. No significant differences were found between two species of similar body weight: the HM and the GWM.     

液相色谱串联质谱测定水产品中吩噻嗪类药物残留的研究

建立了水产品中氯丙嗪、乙酰丙嗪、丙酰丙嗪、异丙嗪、甲苯噻嗪等5种吩噻嗪类药物多残留的液质联用确证方法。用碱性乙睛萃取样品中的5种吩噻嗪类药物,然后用HLB固相萃取柱净化,以YMC-PackProC18色谱柱为分离柱,在正离子模式下以电喷雾电离串联质谱仪进行测定。在1.0、2.0、4.0ng/g3个质量浓度水平进行验证试验,方法的线性范围为0.5～5.0ng/g,总体平均回收率为75.7%～87.7%,相对标准偏差为9.0%～15.2%。该方法简便、快速、准确,各项技术指标满足国内外法规的要求,可用于水产品中吩噻嗪类药物残留的确证检测。     

Rapid identification of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has been reported to be an easy and rapid identification method for many human pathogens, but applications for common veterinary pathogens are lacking. Gene probes for FISH of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes were designed to provide probes for a specific identification of these bacteria from cultures. Specific FISH probes for these species have so far not been published. Both probes recognized all isolates of the target species correctly. With the S. uberis probe SUB 196 no false-positive results were obtained for reference strains as well as for clinical isolates. Probe APYO 183 for A. pyogenes produced false-positive reactions with so far rarely described Arcanobacterium species from animals at standard hybridization conditions. In order to avoid any incorrect classifications of microorganisms as A. pyogenes, two non-labelled competitor probes were designed and successfully evaluated.     

Identification and determination of antigenic proteins of Korean ranavirus-1 (KRV-1) using MALDI-TOF/TOF MS analysis

Ranaviruses are serious pathogens of fish, amphibians, and reptiles, and pose a major threat to global biodiversity. A ranavirus isolated from tissues of diseased tadpoles and frogs in Gangwon province, Korea, in 2006 and 2007, was designated Korean ranavirus-1 (KRV-1) and was infectious in a variety of fish cell lines with highest titers (10¹⁰ TCID₅₀/ml) in Epithelioma papulosum cyprini cells (EPCs) and baby hamster kidney-21 (BHK-21) cells. Bullfrog (Rana catesbeiana) tadpoles challenged by immersion in 10⁵ TCID₅₀/ml of KRV-1 showed 60% mortality within 10 days. SDS-PAGE of frog virus 3 (FV3) and KRV-1 proteins yielded several bands 35-49 kDa in size, which were identified as major capsid proteins (MCPs) by MALDI-TOF MS. Immunoblotting of FV3 proteins showed antigenic bands 34 kDa and 93 kDa in size which were identified by MALDI-TOF/TOF MS as MCP and neurofilament triplet H1-like protein (NF-H1), respectively. In KRV-1, antigenic bands at 32 kDa, 69 kDa, and 72 kDa were identified as MCP, Hypothetical protein, and NF-H1, respectively. The genes encoding these KRV-1 proteins were sequenced. KRV-1 appeared to be closely related to the soft-shelled turtle iridovirus (STIV), based on alignments of amino acid sequences from various ranaviruses. Variability in ranavirus antigenic proteins was apparent in an earlier study. It is expected that use of the methods employed here, together with the results of the present work, will contribute to an understanding of the pathogenesis of ranaviruses, and will further the development of DNA- or protein-based bait vaccines for conservation of natural habitats.     

Isolation and identification of intestinal bacteria from Japanese tree frog (Hlya japonica) with the special reference to anaerobic bacteria.

The bacteria in the large intestines of eight Japanese tree frogs (Hlya japonica) were enumerated by using an anaerobic culture system. The microorganisms at approximately 3.1 x 10(9) bacteria per g (wet weight) of intestinal contents were present in the intestine of all the frogs tested. No difference of the total bacteria in the frog intestine was observed between two different incubation-temperatures (room temperature and 37 degrees C). Eleven genera and 16 species were isolated from the frog intestine. In most frogs, Bacteroides (B.) caccae and B. vulgatus were detected as the predominant organisms. Escherichia coli was also present in greater numbers in the intestine. Other bacteria isolated at high dilutions were strict anaerobes, including Fusobacterium and Clostridium. Enterococcus faecalis was frequently isolated from the frog intestine. However, four genera of Bifidobacterium, Eubacterium, Peptostreptococcus, and Lactobacillus were not isolated from the frog intestine.     

菊芋内生固氮菌分离、鉴定及特性研究

通过气相色谱,结合乙炔还原法,对菊芋根系的内生固氮菌进行了分离和鉴定。结果表明,筛选得到的7株具有固氮酶活性的菌株,分别属于根瘤菌属,寡养单胞菌属,假单胞菌属和肠杆菌属。菌株之间固氮酶活性相差较大,固氮酶活性较高的菌株较少,100 nmol/(mL·h)以上的只有cho1和cho9两株菌。菌株cho4的耐盐性较高,可以在盐浓度为7%的条件下正常生长。分离得到的菌株中,有5株具有解磷活性,其中以菌株cho7的活性最高,为63.84 μg/mL。菌株全部都具有分泌生长素的能力,但差异较大,分泌IAA浓度最高的是cho2,达到13.49 μg/mL。     

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus)

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.     

Morphological and molecular identification of three species of Sarcocystis in reindeer (Rangifer tarandus tarandus) in Iceland

Six Sarcocystis species have previously been described from reindeer in Norway based on sarcocyst morphology and DNA sequencing. The aim of this study was to determine whether reindeer in Iceland, which descend from reindeer imported from Norway in 1787, also were infected with Sarcocystis, and to identify and genetically characterise any species present. Muscle tissue from the heart, diaphragm and/or oesophagus was collected from 36 reindeer in Iceland. Pieces of all tissue samples were examined histologically. Frozen/thawed samples of cardiac muscle, oesophagus and/or diaphragm from 11 of the 36 reindeer were also examined under a stereoscopic microscope and sarcocysts present were identified to species either in situ or under a light microscope. Two cysts of each species, originating from two different reindeer were randomly selected for DNA analyses. The complete ssu rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. In addition, two sarcocysts that could not be classified by microscopic examination were selected for partial ssu rRNA gene sequence analysis. By histology, sarcocysts were found in the diaphragm and/or oesophagus of 8 of 36 (22.2%) animals. By examination of fresh tissue, sarcocysts of Sarcocystis rangi, S. tarandivulpes and S. hardangeri were found in the oesophagus of seven of nine (77.8%) animals, suggesting a high prevalence of Sarcocystis in the Icelandic reindeer population. Cyst morphology and the ssu rRNA gene sequence of each of the three species were identical to isolates of the same species from Norwegian reindeer. DNA sequencing was useful in order to identify cysts with an ambiguous morphology. This is the first record of these Sarcocystis species in reindeer outside Norway.     

Feeding cluster preferences in four genera of Lories and Lorikeets (Loriinae) that should be considered in the diet of nectarivorous psittacine species in captivity

Lories and lorikeets are popular birds in the pet bird trade, captured from the wild and exported worldwide. Their captive propagation has not been so successful for many species due to health issues, low breeding success and reduced longevity. As a result, uptake from the wild is currently the only way to meet the market’s demand. Field studies on Asian species of loris and lorikeets are limited; therefore, dietary recommendations are based on the well‐studied Australian species such as the rainbow lorikeet (Trichoglossus moluccanus). We aimed to provide an ad libitum diet to diverse Loriinae species at Jurong Bird Park (Singapore) which allowed for them to select between a low and moderate protein diet to compare their nutrient and energy intake with other Loriinae species. We measured the following variables: daily dry matter (DM) intake, nectar‐to‐fruit energy intake ratio (NF ratio), metabolisable energy (ME), protein and non‐protein energy (NPE)‐to‐protein energy (PE) ratio intake (all by kg metabolic body weight MBW, kg^0.75) for 36 pairs over a 1‐month period. A Kruskal–Wallis test revealed every genus had significantly different intakes of DM, NF ratio, NPE‐to‐PE ratio, ME and protein than each other. Post hoc Mann–Whitney U tests confirmed that the majority of variables were ingested in different amounts for each genus except for NF ratio, NPE/PE ratio which Lorius spp. are not different to Charmosyna sp. or Trichoglossus spp. and protein intake of Eos spp. does not differ from Trichoglossus spp. Our conclusion is that no species should be used as a model for a species from another genus of Loriinae; future studies should be species‐specific for each genus to increase captive propagation success.