key: cord-0035977-ak68ufst
authors: Liu, Dongyou
title: Technical Advances in Veterinary Diagnostic Microbiology
date: 2012-04-05
journal: Advanced Techniques in Diagnostic Microbiology
DOI: 10.1007/978-1-4614-3970-7_35
sha: 9e8d79809403a50d41d72a569ba63fd8d758a6ff
doc_id: 35977
cord_uid: ak68ufst

Forming a significant part of biomass on earth, microorganisms are renowned for their abundance and diversity. From submicroscopic infectious particles (viruses), small unicellular cells (bacteria and yeasts) to multinucleate and multicellular organisms (filamentous fungi, protozoa, and helminths), microorganisms have found their way into virtually every environmental niche, and show little restrain in making their presence felt. While a majority of microorganisms are free-living and involved in the degradation of plant debris and other organic materials, others lead a symbiotic, mutually beneficial life within their hosts. In addition, some microorganisms have the capacity to take advantage of temporary weaknesses in animal and human hosts, causing notable morbidity and mortality. Because clinical manifestations in animals and humans resulting from infections with various microorganisms are often nonspecific (e.g., general malaise and fever), it is necessary to apply laboratory diagnostic means to identify the culprit organisms for treatment and prevention purposes.

Forming a signi fi cant part of biomass on earth, microorganisms are renowned for their abundance and diversity. From submicroscopic infectious particles (viruses), small unicellular cells (bacteria and yeasts) to multinucleate and multicellular organisms ( fi lamentous fungi, protozoa, and helminths), microorganisms have found their way into virtually every environmental niche, and show little restrain in making their presence felt. While a majority of microorganisms are free-living and involved in the degradation of plant debris and other organic materials, others lead a symbiotic, mutually bene fi cial life within their hosts. In addition, some microorganisms have the capacity to take advantage of temporary weaknesses in animal and human hosts, causing notable morbidity and mortality. Because clinical manifestations in animals and humans resulting from infections with various microorganisms are often nonspeci fi c (e.g., general malaise and fever), it is necessary to apply laboratory diagnostic means to identify the culprit organisms for treatment and prevention purposes.

Veterinary diagnostic microbiology is devoted to the identi fi cation and detection of microorganisms that cause diseases in animals. Considering the close similarity among microorganisms causing diseases in humans and animals, many laboratory techniques that have been developed for the identi fi cation and detection, subtyping and phylogenetic analysis, virulence determination, and drug resistance assessment of human pathogens, have been thus readily adopted for the investigation of animal pathogens, or vice versa. Furthermore, apart from zoonotic pathogens that occur in both human and animals, animals of different classes and categories often have unique pathogens of their own. Therefore, veterinary diagnostic microbiology faces even greater challenges than its medical counterpart in achieving accurate, sensitive, and rapid identi fi cation and detection of pathogenic microorganisms in animals.

In view of the fact that many human pathogens have originated/evolved from microorganisms commonly occurring in animals, accurate identi fi cation and tracking of animal pathogens are crucial for the control and prevention of zoonotic infections in human populations. The threat of zoonotic pathogens (e.g., Bacillus anthracis ) being used in bioterrorism attacks, and the emergence of rapidly evolving antibiotic-resistant microorganisms and animal pathogens causing severe diseases in humans (e.g., SARS coronaviruses, and avian in fl uenza viruses), have made the development and application of improved diagnostic methods for animal pathogens increasingly important.

Accurate identi fi cation and detection of pathogenic microorganisms in animals have been and will remain the primary objective for veterinary diagnostic microbiology.

Similar to its medical counterpart, veterinary diagnostic microbiology has traditionally relied on various phenotypic procedures for microbial characterization. These procedures assess the morphological, biological, biochemical, serological, in vitro and in vivo characteristics as well as other phenotypic properties of microorganisms, and have played an essential role in the identi fi cation and detection of microbial pathogens affecting humans and animals. More recently, molecular techniques have been increasingly applied for identi fi cation and detection of microbial pathogens (Table 35 .1 ) [ 1 ] .

Morphological characterization is based on the premise that various classes of microorganisms often show distinct morphological features (e.g., size, shape, internal and external components, colony morphology) which allow their initial identi fi cation upon macroscopic and microscopic examination. Application of light microscopy or transmission and scanning electron microscopy (EM) together with stains/dyes helps reveal additional morphological details. Hematoxylin and eosin (H&E), Gram, Giemsa, crystal violet stains are common stains used to enhance the contrast of microbes to their background. Additionally, Gimenez' and Pinkerton's stains are useful for detection of rickettsial organisms in tissue sections; Ziehl-Nielsen, Kinyoun, or auramine O stains for initial detection of mycobacteria; KOH, lactophenol cotton blue, India ink, and Southgate's mucicarmine stains for detection of fungi; periodic acid-Schiff (PAS), Grocott's methenamine silver (GMS), Fontana-Masson, Gridley's, and H&E stains for detection of mycotic elements in tissue biopsies. In general, morphological characterization is rapid and inexpensive, but has relatively low sensitivity and speci fi city, and its result interpretation is somewhat subjective. To improve the sensitivity and speci fi city of microscopic detection of pathogenic microorganisms (especially viruses), fl uorescently labeled antibodies may be utilized. Application of highly sensitive and speci fi c fl uorescent sensor molecules in electron microscopy, fl uorescence microscopy, or time-lapse microscopy has further enhanced morphological characterization of microorganisms. Besides unraveling paradigms of pathogen entry and pinpointing the exact intracellular location, these new techniques permit direct monitoring of the intracellular lifestyle of microbial pathogens and yield insights into the underlying mechanisms of their pathogenicity [ 2, 3 ] . Furthermore, atomic force microscopy (AFM) techniques offer a powerful platform for analyzing the structure, properties and functions of microbial pathogens as well as the localization, mechanics, and interactions of the individual cell wall constituents, contributing to the elucidation of the molecular bases of cell adhesion (nanoadhesome) and mechanosensing (nanosensosome) [ 4, 5 ] .

Biochemical characterization focuses on the metabolic or enzymatic products of microorganisms, including distinct patterns of carbohydrate, protein, amino acid, fat metabolisms and production of particular enzymes. Biochemical tests are often conducted to distinguish between aerobic and anaerobic breakdown of carbohydrates, to show carbohydrates that can be attacked, to detect speci fi c breakdown products of Macroscopic and microscopic examination of morphological features (e.g., size, shape, internal and external components, colony morphology) allows for rapid, inexpensive identi fi cation of microorganisms. Use of general or specialized stains/dyes further enhances the contrast of microbes to their background. Nonetheless, morphological characterization often lacks desired sensitivity and speci fi city Biochemical

Examination of metabolic or enzymatic products of microorganisms (e.g., carbohydrate, protein, amino acid, fat, and enzyme) using biochemical techniques permits their discrimination at genus-and species-levels. However, the performance of biochemical tests is impacted by factors that affect microbial growth and metabolism Serological

Detection of speci fi c interactions between host antibodies and microbial antigens (e.g., protein or carbohydrate) by serological techniques provides indirect evidence for causal relationships between diseases and microbial pathogens. Serological tests have a relatively high sensitivity, speci fi city and quick turn-around time, but may show cross-reactivity with closely related microbial species Biological, in vitro and in vivo Assessment of biological features (e.g., host range, transmission pattern, pathological effects, geographical origin) of microorganisms helps diagnose microbial infections in cases where other relevant data are scarce In vitro culture techniques using laboratory media and cell lines facilitate isolation and propagation of target microorganisms for subsequent morphological, biochemical, serological, and molecular characterization In vivo assays using laboratory animals and chicken embryos allow for recovery of microorganisms that fail to grow on culture media or cell lines, and help determine host susceptibility and immune response to, and pathogenic effects of microorganisms Molecular

Detection of nucleic acids using molecular techniques offers direct evidence on the presence of microorganisms. Application of nucleic acid ampli fi cation technologies further improves the speed, sensitivity, and speci fi city of microbial identi fi cation and detection carbohydrates (e.g., the formation of acids, alcohols and gases when grown in selective liquid or solid media), to determine the ability of carbohydrates to utilize substrates such as citrate and malonate, to examine the metabolism of protein and amino acids (e.g., gelatine liquefaction, indole production, amino acid decarboxylase test, and phenylamine deaminase test) as well as of fats (e.g., hydrolysis of tribtyrin), and detect production of enzymes (e.g., catalase test, oxidase test, urease test, ONPG test, and nitrate reduction). Assessment of fungal primary metabolites such as ubiquinones (coenzyme Q) is useful for the taxonomy of black yeasts and fi lamentous fungi, whereas examination of fungal secondary metabolites (e.g., steroids, terpenes, alkaloids, cyclopeptides, and coumarins) by chromatographic techniques provides another means for fungal identi fi cation. A recent approach for biochemical characterization of microorganisms centers on the characteristic outer surface charges of microbes that contribute to their distinct migration under a direct-current electric fi eld such as capillary electrophoresis (CE), leading to rapid and ef fi cient separation, identi fi cation, quantitation, and characterization of intact microorganisms (i.e., bacteria, viruses, and fungi) [ 6 ] . Another useful technique for biochemical characterization of microbes is matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF), which has been shown to be useful for speci fi c identi fi cation of Francisella and other microbial pathogens [ 7 ] . Serological characterization on the basis of speci fi c reactions between host antibodies and microbial antigens (usually protein or carbohydrate) provides highly sensitive, speci fi c, and rapid identi fi cation of microorganisms. Detection of rising levels of speci fi c IgA, IgM, and IgG antibody titers or seroconversion in blood, urine, and fecal materials offers indirect evidence for causal relationships between diseases and microbial pathogens [ 8 ] . An interesting development in the serological characterization of disease-causing microorganisms is the use of chemically synthesized peptides. Generated by chemical approaches, these peptides are composed of two or more amino acids linked together by peptide bonds. By mimicking naturally occurring peptides or segments of proteins, these peptides serve as synthetic antigens in peptide microarrays as potential diagnostic tools in high-throughput immunoassays [ 9 ] . Other new developments in serological characterization of microorganisms include biosensors and nanotechnology (nanoarrays and nanochips). Biosensors involve the use of a microbe-speci fi c antibody and a transducer (e.g., electrochemistry, re fl ectometry, interferometry, resonance, and fl uorimetry) to convert a biological interaction into a measurable signal. In the particle concentration fl uorescence immunoassay (PCFIA) for brucellosis, submicron polystyrene particles are coated with antigen and placed in a 96-well vacuum plate. After addition of fl uorescent conjugate followed by vacuum fi ltration to remove unbound conjugate, the total particle-bound fl uorescence is measured by front surface fl uorimetry. Nanotechnology (nanoarrays and nanochips) offers small scale platforms to identify an array of infectious agents or serotypes on a single chip.

Biological characterization focuses on the issues related to the host susceptibility, transmission patterns, pathological effect(s), and geographical origin of microbial pathogens, which are critical in helping achieve correct diagnosis of microbial infections in cases where other relevant data are scarce [ 10 ] . In vitro isolation and propagation on laboratory media and cell lines offers a valuable tool for identi fi cation and diagnosis of microbial infections. The size, color, shape and form of colonies formed by microorganisms on nutritional agar and other selective media are diagnostically informative. In case of viral pathogens, the formation of a region of dead cells resulting from viral growth (called a "plaque") suggests their cytopathogenic effects (CPE). Parasitic protozoa may also be cultivated as a means of identi fi cation [ 11 ] . However, because not all microorganisms will grow in laboratory media and cell lines, embryonated eggs, insect vector, and laboratory animals (e.g., rodents) may be utilized. For example, Trypanosoma cruzi , the causal agent for Chagas disease, is grown in the guts of its vector triatomine bug for con fi rmation and diagnosis. The availability of cultured isolate/strain permits further antigenic studies, antibiotic susceptibility testing, and genetic studies. Despite their relatively high expense and length of time required, in vitro and in vivo techniques have contributed to the studies of microbial taxonomy, biology, epidemiology, pathogenesis, and treatment response. A recent development in the use of in vivo techniques for microbial characterization relates to the in vivo bioluminescence imaging or biophotonic imaging (BPI). Based on genetically engineered bioluminescent/ fl uorescent microorganisms, this technique enhances the study of microbial infections and host immune responses [ 12 ] . Application of genetically engineered mice with luciferase reporters for speci fi c microbial or host genes helps overcome the limitations of in vivo bioluminescence imaging for assessment of microbial replication, activation of key genes in host immunity, and response to tissue damage in vivo [ 13 ] .

Because of their time-consuming, occasionally variable nature, and/or their limited sensitivity and speci fi city, phenotypic approaches (e.g., morphological, biochemical, serological, and biological characterization) to the identi fi cation of microorganisms are increasingly supplemented with molecular techniques. Progresses in the areas of genetic target selection, template preparation, transition from nonampli fi ed to ampli fi ed approaches, and product detection over the past two decades have made molecular methods an indispensable tool in the laboratory diagnosis of microbial pathogens in veterinary medicine [ 1 ] .

With regard to the selection of genetic targets, the following three types may be considered: nonspeci fi c, shared, and speci fi c genetic targets. Nonspeci fi c genetic targets include the guanine and cytosine composition (or G + C content), short random primer sites, randomly dispersed repetitive extragenic palindromes (REP), enterobacterial repetitive intergenic consensus sequences (ERIC), variable-number tandem repeats (VNTR) (also known as simple sequence repeats (SSRs), or microsatellites), restriction enzyme sites, and so on. Shared genetic targets include ribosomal RNA (rRNA) genes (e.g., 16/18S rRNA, 23/25/28S rRNA), internal transcribed spacer (ITS) regions, mitochondrial DNA (mtDNA), and housekeeping genes, etc. Speci fi c genes are uniquely present and represent ideal targets for the identi fi cation of pathogenic bacteria, fungi and parasites.

Preparation of nucleic acid templates from cultured isolates and clinical specimens represents an important initial step for molecular identi fi cation and detection of microorganisms. This often involves (1) disruption of cell walls, (2) denaturation of nucleoprotein complexes, (3) inactivation of endogenous DNase/RNAse, and (4) removal of contaminating proteins, polysaccharides, polyphenolic pigments, and other compounds [ 14 ] . While enzymatic digestion (e.g., using lyticase, zymolase, chitinase, gluculase, and/or proteinase K) and occasionally acid and alkali treatments may be effective for breaking up bacterial and yeast cells, mechanical grinding, sonication or bead-beating is often necessary to disrupt the mycelial and helminth cell walls. Following extraction with organic solvents (e.g., phenol/chloroform) and detergents (e.g., sodium dodecyl sulfate, SDS; hexadecyltrimethylammonium bromide, CTAB; and N -lauroylsarcosine), which denatures cytosolic proteins and lipid membranes and inactivates endogenous DNase/RNAse, nucleic acids of high purity are obtained after precipitation with ethanol or isopropanol. The recent development of various easy-to-use commercial kits has negated the need to use hazardous organic solvents in the isolation of microbial DNA/RNA. Furthermore, automated nucleic acid extraction systems have become increasingly sophisticated and affordable, contributing to the streamlining of template preparation and reduction of potential cross-contamination during manual handling.

The early generation molecular procedures rely on nonampli fi ed, hybridization approaches, such as DNA-DNA hybridization (for estimation of guanine-cytosine ratio or G-C content), and use of gene probes in dot blot, Southern blot, and fl uorescence in situ hybridization (FISH), etc. [ 15 ] . A more recent development in the DNA hybridizationbased approach is DNA microarray, in which high-density oligonucleotide probes (or segments of DNA) are immobilized on a solid surface, and used to hybridize (catch) any complementary sequences (labeled with fl uorescent nucleotides) in a test sample. Subsequent detection and quanti fi cation of fl uorescence signal permits identi fi cation and determination of the relative abundance of nucleic acid sequences in a sample [ 16 ] . Although these nonampli fi ed procedures have adequate speci fi city, they are relatively insensitive, often requiring large quantity of starting materials for reliable detection. Nonetheless, some of these nonampli fi ed techniques remain valuable for comparison of microbial genomes and for identi fi cation of species-and virulencespeci fi c gene regions. For example, dot blot hybridization was employed for screening genomic DNA libraries of Dichelobacter nodosus strains causing virulent and benign footrot, and several virulent-and benign-speci fi c gene regions for subsequent differentiation of virulent and avirulent D. nodosus strains were identi fi ed as a result [ 17, 18 ] . This approach was also applied for identi fi cation of novel virulence-speci fi c gene regions in zoonotic bacterial pathogen Listeria monocytogenes and novel speciesspeci fi c gene in animal bacterial pathogen Listeria ivanovii [ 19, 20 ] .

The mid-1980s witnessed the advent of a novel, highly ef fi cient in vitro nucleic acid ampli fi cation technique known as polymerase chain reaction (PCR). This technique has the capacity to synthesize billions of copies from a single nucleic acid template within 3-4 h, and demonstrates superior sensitivity, exquisite speci fi city, rapid turnover time and amenableness to automation for high throughput testing. Since then, PCR and its variants (e.g., nested PCR, multiplex PCR, real-time PCR, quantitative PCR, reverse transcription PCR (RT-PCR), arbitrarily primed PCR (AP-PCR), random ampli fi ed polymorphic DNA (RAPD), degenerate oligonucleotide primed PCR (DOP-PCR), sequence-independent single primer ampli fi cation (SISPA), and rolling circle ampli fi cation (RCA)) have been widely applied in research and clinical laboratories for identi fi cation and phylogenetic analysis of microorganisms [21] [22] [23] [24] . Apart from PCR, other nucleic acid ampli fi cation procedures include nucleic-acid-sequence-based ampli fi cation (NASBA), ligase chain reaction (LCR), strand displacement ampli fi cation, Q-b replicase-mediated ampli fi cation, linearlinked ampli fi cation, and loop-mediated isothermal ampli fi cation (LAMP), etc.

Conventional methods for detection of nucleic acid products are based on electrophoretic separation followed by staining with ethidium bromide, gelstar, or SYBR Green. Whereas agarose gel electrophoresis provides a convenient, inexpensive way for separation and semiquantitation of DNA and RNA, polyacrylamide gel electrophoresis (PAGE) is useful for separating small nucleic acid fragments (<500 bp). Among the various PAGE-based procedures, single strand conformational polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are widely applied. SSCP is capable of detecting single nucleotide variations, and in combination with capillary electrophoresis (CE), SSCP-CE provides an automated system for rapid separation of nucleic acid products. Recent advances in instrument automation and fl uorescent dye chemistry permit real-time monitoring of PCR amplicons (so-called real-time PCR). Besides the use of double-stranded DNA intercalating dye (e.g., SYBR Green), speci fi cally designed probes such as hydrolysis dual-labeled probes (TaqMan ® ), hybridization probes (LightCycler), molecular beacons, peptide nucleic acid (PNA) probes, TaqMan minor groove binding (MGB™) probes, locked nucleic acid (LNA ® ) primers and probes, and scorpions™ may be utilized [ 25 ] .

Other nucleic acid detection approaches include DNA microarray (also known as DNA chip, gene or genome chip, or gene array), biochips (biosensors), line probe assay (LiPA), enzymatic signal ampli fi cation (e.g., ELISA and fl ow cytometry), and DNA sequencing. Biochips (biosensors) are small analytical devices designed for nucleic acid-based electrical/optic detection ( fl uorescence or chemiluminescence) [ 26 ] . DNA sequencing analysis provides a most accurate way to determine the identity of microbial organisms. While the classic Sanger method (also known as "chain termination method" or "dideoxy sequencing") can read up to 900 bp per and produce 100 kb of sequence data per run, the "next generation sequencing" technologies (e.g., the 454 pyrosequencing-based instrument (Roche Applied Sciences), genome analysers (Illumina), and the SOLiD system (Applied Biosystems)) show improved ef fi ciency for DNA sequencing analysis. Although Illumina and 454 sequencing technologies read 76-106 bp and 250-400 bp, they have the capacity to generate 20 Gb and 400 Mb of sequence data per run, respectively.

Microbial pathogens are noted for the diversity and their ability to adapt and survive in challenging environments. The ability to identify and track microbial strains and varieties involved in disease outbreaks is crucial for their control and prevention. For this reason, a number of phenotypic and molecular procedures have been developed and applied for subtyping and phylogenetic analysis of microbial strains and varieties causing animal diseases (Table 35. 2 ) [ 27 ] . Biotyping separates microbial strains into "biotypes" on the basis of their metabolic and enzymatic activities (e.g., sugar fermentation, amino acid decarboxylation/deamination, urease activity, hydrolysis of compounds, hemagglutination, and hemolysis), colonial morphology, and environmental tolerances (e.g., tolerance to pH, chemicals, dyes, and heavy metals). Biotyping is generally reproducible and easy to perform and interpret. However, it has poor discriminatory power due possibly to variation in gene expression and point mutation Phage typing Phage typing distinguishes microbial strains into "phage types" by their patterns of resistance or susceptibility to a standard set of bacteriophages, depending on the presence or absence of particular receptors on the bacterial surface for phage (virus) binding. Phage typing shows good reproducibility, discriminatory power and ease of interpretation, but requires maintenance of biologically active phages and demands technical skills. In addition, many strains are nontypeable Serotyping

Serotyping differentiates microbial strains into serotypes (serovars) according to the antigenic variations present on the surface structures (e.g., lipopolysaccharides, membrane proteins, capsular polysaccharides, fl agella and fi mbriae). Agglutination, latex agglutination, coagglutination, or fl uorescent and enzyme labeled assays may be used for serotyping. Serotyping has good reproducibility, and ease of interpretation and performance. However, serotyping depends on the availability of good quality reagents, and some autoagglutinable (rough) strains are nontypeable. Additionally, the technique tends to have poor discriminatory power due to cross-reactive antigens Bacteriocine typing Bacteriocine typing assesses microbial strains for their susceptibility to a set of bacterial peptides (bacteriocine), and has been employed to type stains of Pseudomonas aeruginosa , Escherichia coli , and Yersinia pestis , etc. The technique has good reproducibility, discriminatory power, and ease of interpretation, but it is technically demanding and many strains are nontypeable Multilocus enzyme electrophoresis (MLEE) typing MLEE typing separates strains into "electromorphs" (typically re fl ecting amino acid substitution that alters the charge of the protein) in accordance with their distinct electrophoretic mobilities of a set of metabolic enzymes. The technique has excellent reproducibility and ease of interpretation, but shows moderate discriminatory power, and requires expensive equipments Antibiogram typing Antibiogram typing compares different microbial isolates in their susceptibility to a set of antibiotics. The technique has ease of performance and interpretation and reasonable reproducibility. However, it has poor discriminating power Restriction endonuclease analysis (REA) or restriction fragment length polymorphism (RFLP)

Digestion of chromosomal DNA with certain restriction endonuclease produces various fragments whose number and sizes (from 0.5 to 50 kb) are distinct among microbial strains and varieties. This technique has good reproducibility, but generates complex pro fi le of hundreds of bands that may be dif fi cult to interpret (continued) MGE-PCR uses a single primer in PCR to amplify particular MGEs followed by electrophoresis to discriminate amplicon pro fi les. This technique has been utilized to characterize different isolates of Trypanosoma brucei by targeting RIME which has a relatively high copy number in the genome

The common phenotypic subtyping procedures include biotyping, phage typing, serotyping, bacteriocine typing, multilocus enzyme electrophoresis (MLEE) typing, and antibiogram typing (Table 35. 2 ). The genotypic subtyping ( fi ngerprinting) approach targets the microbial chromosome and plasmid DNA such as their composition, homology, and presence or absence of speci fi c genes. The genotypic subtyping techniques consist of two categories: nonampli fi ed techniques (e.g., restriction endonuclease analysis (REA) or restriction fragment length polymorphism (RFLP), pulse-fi eld gel electrophoresis (PFGE), and ribotyping) and ampli fi ed techniques (e.g., ampli fi ed fragment length polymorphism (AFLP), PCR-restriction fragment length polymorphism (PCR-RFLP), and multilocus sequence typing (MLST), and mobile genetic element-PCR (MGE-PCR)) (Table 35. 2 ) [ 28 ] .

Many microbial species encompass a diversity strains with varied virulence potential. The availability of laboratory techniques to accurately assess the pathogenic potential of these microorganisms is vitally important to their control and prevention. For example, gram-negative bacterium Dichelobacter nodosus harbors strains that cause virulent, intermediate or benign footrot in sheep. As virulent and some intermediate footrot induces lameness and severe pain in affected sheep, leading to ill-thrift and reduced weight gain, it is necessary to apply control measures to stem the economic losses. On the other hand, benign footrot causes minimal harm to affected sheep and has the tendency to self-cure, it is unnecessary and indeed wasteful to treat benign footrot. Traditionally, the virulence of D. nodosus strains is determined by elastase test and gelatin gel test, which may take up to 4 weeks to complete, and often demonstrate notable variability. After comparative analysis of recombinant DNA libraries from D. nodosus virulent and benign strains, a panel of virulent-and benign-speci fi c genes was identi fi ed. Use of gene probes and primers derived from these genes facilitate rapid and sensitive determination of D. nodosus virulence [ 17, 18 ] .

Gram-positive bacterium L. monocytogenes is a zoonotic pathogen that encompasses a spectrum of strains with various pathogenic inclination. While some L. monocytogenes strains are highly pathogenic and sometimes deadly, others are relatively avirulent and cause little harm in the host. The current laboratory techniques for assessing the virulence of L. monocytogenes strains include the mouse virulence assay and in vitro cell assays. While the mouse virulence assay is capable of providing an in vivo measurement of all virulent determinants, its high expense limits its application. Representing a low-cost alternative to the mouse virulence assay for assessing L. monocytogenes virulence, in vitro cell culture techniques measure the ability of L. monocytogenes to cause cytopathogenic effects in the enterocyte-like cell line Caco-2, to form plaques in the human adenocarcinoma cell line HT-29, or to cause death in chicken embryos. Several other cell lines (e.g., hepatocyte Hep-G2, macrophage-like J774, epithelial Henle 407 and L2) are also useful for studies on L. monocytogenes ability to adhere, invade, escape from vacuoles, grow intracellularly and spread to neighboring cells. However, these techniques are timeconsuming, and occasionally variable. Following recent identi fi cation of novel virulence-speci fi c genes (e.g., inlJ ), the virulence of L. monocytogenes strains can be rapidly and speci fi cally determined by PCR [ 19, 29 ] .

Microorganisms have the ability to acquire resistance to drugs that are used for their treatment. As drug are often used in animals (e.g., such as cows, pigs, chickens, fi sh, etc.) that provide an important source of human food, microorganisms exposed to these drugs can develop antibiotic resistance through horizontal gene transfer events (e.g., conjugation, transduction, or transformation) and point mutations [ 30 ] . The resistant bacteria in animals due to antibiotic exposure can be transmitted to humans through the consumption of meat, from close or direct contact with animals, or through the environment. For example, use of fl uoroquinolone in poultry production has been linked to the emergence of fl uoroquinolone resistant campylobacter infections in humans. Some bacteria (e.g., Staphylococcus aureus , enterococci, gonococci, streptococci, salmonella, and Mycobacterium tuberculosis ) have developed multidrug resistance [ 31 ] . For example, methicillin-resistant S. aureus (MRSA) are resistant to non-beta-lactam antimicrobial drugs as well. Identi fi cation of MRSA has implications not only for treatment of infected animals, but for potential zoonotic transmission [ 32, 33 ] . While application of in vitro culture technique facilitates determination of MIC (medium inhibition concentration) of the strains, detection of speci fi c gene mutations provides an alternative approach for assessment of antimicrobial drug resistance in microorganisms such as B. hyodysenteriae [ 34 ] .

Given the diversity of animal hosts that are susceptible to a wide variety of microbial infections, veterinary diagnostic microbiology faces a greater challenge than its medical counterpart in achieving a correct and timely identi fi cation of culprit microorganisms causing signi fi cant economic losses in agricultural production. Although phenotypic procedures are useful for microbial identi fi cation, their time-consuming nature and occasional variability have provided the impetus for the development of nucleic acid detection methodology. With a high sensitivity, exquisite speci fi city and speed, molecular procedures especially those involving nucleic acid ampli fi cation (e.g., PCR) have been widely adopted in clinical and research laboratories for identi fi cation, typing, virulence determination, and drug resistance assessment of microorganisms of veterinary and medical importance. Further improvement through miniature and multiplexing will help reduce the cost of conducting molecular testing in diagnostic microbiology.

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Spotting the right location-imaging approaches to resolve the intracellular localization of invasive pathogens

Microbial nanoscopy: a closer look at microbial cell surfaces

Frontiers in microbial nanoscopy

Capillary electrophoresis separation of microorganisms

Identi fi cation of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of fl ight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels

The development of oral fl uid-based diagnostics and applications in veterinary medicine

Diagnostic and immunoprophylactic applications of synthetic peptides in veterinary microbiology

Traditional and molecular techniques for the study of emerging bacterial diseases: one laboratory's perspective

Examining heterogeneity in the diagnostic accuracy of culture and PCR for Salmonella spp. in swine: a systematic review/meta-regression approach

Noninvasive biophotonic imaging for studies of infectious disease

Bioluminescence imaging of reporter mice for studies of infection and in fl ammation

Preparation of Listeria monocytogenes specimens for molecular detection and identi fi cation

Species-speci fi c probes for the identi fi cation of the African tsetse-transmitted trypanosomes

Microarray-based molecular detection of foot-andmouth disease, vesicular stomatitis and swine vesicular disease viruses, using padlock probes

Development of gene probes of Dichelobacter nodosus for differentiating strains causing virulent, intermediate or benign ovine footrot

A polymerase chain reaction assay for improved determination of virulence of Dichelobacter nodosus , the speci fi c causative pathogen for ovine footrot

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR ampli fi cation of putative transcriptional regulator and internalin genes

PCR detection of a putative N-acetylmuramidase gene from Listeria ivanovii facilitates its rapid identi fi cation

A quantitative polymerase chain reaction assay for detection and quanti fi cation of Lawsonia intracellularis

Pushing the envelope: advances in molecular techniques for the detection of novel viruses

The polymerase chain reaction in the diagnosis of infectious diseases

Metagenomics and the molecular identi fi cation of novel viruses

Mycoplasma bovis real-time polymerase chain reaction assay validation and diagnostic performance

Developing nucleic acid-based electrical detection systems

Advances in viral disease diagnostic and molecular epidemiological technologies

Genotyping of Francisella tularensis , the causative agent of tularemia

A multiplex PCR for species-and virulence-speci fi c determination of Listeria monocytogenes

Methicillin-resistant Staphylococcus aureus in animals

Methicillin-resistant Staphylococcus aureus and animals: zoonosis or humanosis?

A veterinary perspective on methicillin-resistant staphylococci

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Trends towards lower antimicrobial susceptibility and characterization of acquired resistance among clinical isolates of Brachyspira hyodysenteriae in Spain