Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) for Identification of Bacterial Isolates From Horses

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is used for bacterial identification by analyzing the spectra of isolates and comparing them against a database of reference spectra; it is known for its rapidity and accuracy. Although MALDI-TOF MS is used for identification of bacterial isolates from animals, not all animal pathogens are identified correctly. In this study, we used a commercial MALDI-TOF MS identification system to examine 3724 bacterial isolates from horses and their environments. Isolates that could not be identified with MALDI-TOF MS were identified by 16S rRNA gene sequence taxonomic analysis. MALDI-TOF MS could identify 86.2% of the isolates from horses to the species level, showing that this method could be successfully applied for bacterial identification in horses. However, some species known to be equine pathogenic agents including Taylorella equigenitalis and Rhodococcus equi were difficult to identify with MALDI-TOF MS, which might be the result of an inadequate reference database. Some Prevotella, Staphylococcus, and Streptococcus isolates, which could not be identified with either MALDI-TOF MS or 16S rRNA gene sequencing analysis, formed clusters in the 16S rRNA phylogenic tree, and might be unknown species isolated from horses. Adding the spectra of isolates identified in this study to an in-house database might make MALDI-TOF MS a more useful tool for identifying equine isolates.     

Application of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Identification of Stallion Semen Bacterial Contamination

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the cutting-edge methods currently applied in medical bacteriologic diagnostics. The aim of the study was to prove the possibility of applying MALDI-TOF MS to identify bacterial contamination in the ejaculate of stud stallions, which may cause infections to reproductive organs of mares following artificial insemination with cooled semen. A partial aim was to determine changes in the total count of microorganisms in long-term storage of ejaculate after its treatment with gentamicin and also without antimicrobial medication. Aerobic cultivation on Columbia agar was used to examine 26 semen samples from 13 horses; 31 different species of bacteria were isolated, which were identified by MALDI-TOF MS. The most frequently detected species came from Aerococcaceae, Staphylococcaceae, and Micrococcaceae families. The results of our work confirm that MALDI-TOF MS is a quick alternative method for identifying bacterial species that may contaminate stallion semen.     

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.     

Streptococcus pluranimalium meningoencephalitis in a horse

A 3-y-old, female Quarter Horse with a history of acute neurologic signs was found dead and was submitted for postmortem examination. Areas of petechial and ecchymotic hemorrhage were present on cross-sections of the cerebrum, cerebellum, and brainstem. Histologic examination of the brain revealed severe, purulent meningoencephalitis and vasculitis with a myriad of intralesional gram-positive cocci. Streptococcus pluranimalium was identified from formalin-fixed, paraffin-embedded tissue obtained from sites with active lesions by PCR and nucleotide sequencing of bacterial 16S ribosomal RNA. S. pluranimalium should be considered as a cause of meningoencephalitis in a horse.     

Identification of flea species using MALDI-TOF/MS

In the present study, a molecular proteomics (MALDI-TOF/MS) approach was used as a tool for identifying flea vectors. We measured the MS spectra from 38 flea specimens of 5 species including Ctenocephalides felis, Ctenocephalides canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata. A blind test performed with 24 specimens from species included in a library spectral database confirmed that MALDI-TOF/MS is an effective tool for discriminating flea species. Although fresh and 70% ethanol-conserved samples subjected to MALDI-TOF/MS in blind tests were correctly classified, only MS spectra of quality from fresh specimens were sufficient for accurate and significant identification. A cluster analysis highlighted that the MALDI Biotyper can be used for studying the phylogeny of fleas.     

Detection of Bartonella spp. in Cimex lectularius by MALDI-TOF MS

Bed bugs are small hematophagous insects. They are found in temperate and tropical climates around the world. Their vectorial capacity for several pathogens, including Bartonella spp., has been suspected. An experimental study of artificial infection of Cimex lectularius with Bartonella quintana and Bartonella henselae bacteria was developed to evaluate the ability of MALDI-TOF MS to simultaneously identify bed bugs and their infectious status. This experimental study confirmed the ability of MALDI-TOF MS to identify bed bugs. In addition, it was able to differentiate between control bed bugs, bed bugs infected with Bartonella quintana and bed bugs infected with Bartonella henselae, with an identification percentage above 90%.     

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections

Background

Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC.A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.

Results

The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.

Conclusions

The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.     

Relationships among intramammary health,udder and teat characteristics,and productivity of extensively managed ewes

Mastitis is an economically important disease and its subclinical state is difficult to diagnose, which makes mitigation more challenging. The objectives of this study were to screen clinically healthy ewes in order to 1) identify cultivable microbial species in milk, 2) evaluate somatic cell count (SCC) thresholds associated with intramammary infection, and 3) estimate relationships between udder and teat morphometric traits, SCC, and ewe productivity. Milk was collected from two flocks in early (<5 d) and peak (30 to 45 d) lactation to quantify SCC (n = 530) and numerate cultivable microbial species by culture-based isolation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; n = 243) identification. Within flock and lactation stage, 11% to 74% (mean = 36%) of samples were culture positive. More than 50 unique identifications were classified by MALDI-TOF MS analysis, and Bacillus licheniformis (18% to 27%), Micrococcus flavus (25%), Bacillus amyloliquefaciens (7% to 18%), and Staphylococcus epidermidis (26%) were among the most common within flock and across lactation stage. Optimum SCC thresholds to identify culture-positive samples ranged from 175 × 10³ to 1,675 × 10³ cells/mL. Ewe productivity was assessed as total 120-d adjusted litter weight (LW120) and analyzed within flock with breed, parity, year, and the linear covariate of log₁₀ SCC (LSCC) at early or peak lactation. Although dependent on lactation stage and year, each 1-unit increase in LSCC (e.g., an increase in SCC from 100 × 10³ to 1,000 × 10³ cells/mL) was predicted to decrease LW120 between 9.5 and 16.1 kg when significant. Udder and teat traits included udder circumference, teat length, teat placement, and degree of separation of the udder halves. Correlations between traits were generally low to moderate within and across lactation stage and most were not consistently predictive of ewe LSCC. Overall, the frequencies of bacteria-positive milk samples indicated that subclinical mastitis (SCM) is common in these flocks and can impact ewe productivity. Therefore, future research is warranted to investigate pathways and timing of microbial invasion, genomic regions associated with susceptibility, and husbandry to mitigate the impact of SCM in extensively managed ewes.     

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.     

Rapid Identification of Mycoplasma pulmonis Isolated from Laboratory Mice and Rats Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.     

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.     

家蚕对浓核病毒(镇江株)抵抗性和感受性品种的中肠组织蛋白比较分析

为了探明家蚕对浓核病毒(镇江株)抵抗性和感受性品种间在分子机制上的差异,通过蛋白质双向电泳(2-DE)和基质辅助质量飞行时间质谱技术(MALDI-TOF MS)对两个不同抗性家蚕品种的中肠组织蛋白进行了比较分析。2-DE电泳结果表明,两个品种间的中肠组织蛋白斑点数及位置、形态等差异很小,进一步比较获得了9个差异蛋白斑点,其中感受性蚕品种JS有6个,抵抗性蚕品种NIL有3个。经MALDI-TOF MS鉴定结果显示,感受性品种JS有4个差异蛋白斑点可能分别为氢离子转运ATP合酶β亚基1、氢离子转运ATP合酶β亚基2、组织蛋白酶D或3-羟酰辅酶A脱氢酶;抵抗性品种NIL中有2个差异蛋白点可能是组织蛋白酶。     

Molecular Detection of Avian Pathogens in Poultry Red Mite (Dermanyssus gallinae) Collected in Chicken Farms

Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek’s disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms.     

基质辅助激光解析电离飞行时间质谱在动物病原菌检测中的应用

应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）技术,对厦门口岸进口的1000多份动物及其产品的样品进行致病菌的检测和鉴定,共检出20多种致病菌,并对这些菌株同时用传统生化鉴定方法进行确认。结果表明MALDI-TOF-MS对未知细菌进行鉴定,较传统方法更加快速、准确,而且可以进行高通量检测,可以广泛应用于口岸动物检疫以及微生物检验实验室的日常检验。此外,将国内分离鉴定的各种参考菌株建立的常见致病菌MALDI-TOF-MS数据库与布鲁克公司的MALDI Biotyper数据库进行病原菌鉴定的比较,结果表明,实验室自建的MALDI-TOF-MS数据库可以取得更加准确地结果。     

Charting the proteome of Cryptosporidium parvum sporozoites using sequence similarity-based BLAST searching

Cryptosporidium (C.) spp. are important zoonotic parasites causing widespread diarrhoeal disease in man and animals. The recent release of the complete genome sequences for C. parvum and C. hominis has facilitated the comprehensive global proteome analysis of these opportunistic pathogens. The well-known approach for mass spectrometry (MS) based data analysis using the BLAST tool (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins using peptide sequences produced by mass spectrometry. We have used several complementary approaches to explore the global sporozoite proteome of C. parvum with available proteomic tools. To optimize the output of the MS data, a sequence similarity-based MS BLAST strategy was employed for bioinformatic analysis. Most significantly, almost all the constituents of glycolysis and several mitochondrion-related proteins were identified. In addition, many hypothetical Cryptosporidium proteins were validated by the identification of their constituent peptides. The MS BLAST approach was found to be useful during the study and could provide valuable information towards a complete understanding of the unique biology of Cryptosporidium.     

Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.     

Synergistic and antagonistic hemolytic activities of bacteria of genus Arcanobacterium and CAMP-like hemolysis of Arcanobacterium phocae and Arcanobacterium haemolyticum with Psychrobacter phenylpyruvicus

A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium.A synergistic hemolytic reaction with staphylococcal β-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal β-hemolysin could be observed for A. phocae and A. haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.     

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.     

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.     

First description of Trueperella (Arcanobacterium) bernardiae of animal origin

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.