Molecular Approaches to Recognize Relevant and Emerging Infectious Diseases in Animals

© Springer Science+Business Media New York 2015
Mónica V. Cunha and João Inácio (eds.)Veterinary Infection Biology: Molecular Diagnostics and High-Throughput StrategiesMethods in Molecular BiologyMethods and Protocols124710.1007/978-1-4939-2004-4_7

7. Molecular Approaches to Recognize Relevant and Emerging Infectious Diseases in Animals

Fredrik Granberg1, 2  , Oskar E. Karlsson1, 2, Mikael Leijon1, 3, Lihong Liu1, 3 and Sándor Belák1, 2, 3

OIE Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

Section of Virology, Department of Biomedical Sciences and Veterinary Public Health (BVF), Swedish University of Agricultural Sciences (SLU), 7028, Uppsala, 750 07, Sweden

The National Veterinary Institute (SVA), Uppsala, Sweden



Fredrik Granberg


Since the introduction of the first molecular tests, there has been a continuous effort to develop new and improved assays for rapid and efficient detection of infectious agents. This has been motivated by a need for improved sensitivity as well as results that can be easily communicated. The experiences and knowledge gained at the World Organisation for Animal Health (OIE) Collaborating Centre for Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine, Uppsala, Sweden, will here be used to provide an overview of the different molecular approaches that can be used to diagnose and identify relevant and emerging infectious diseases in animals.

Key words
Infectious diseasesPathogen detectionMolecular diagnosisTransboundary animal diseasesEndemic diseasesZoonosesPCRIsothermal amplificationHybridizationProximity ligation assay (PLA)MicroarraysNanotechnology

1 Introduction

The increased occurrence and emergence of devastating infectious diseases, in both domestic and wildlife animal populations, are causing very serious socioeconomic losses at both global and regional levels. This increase has been attributed to several contributing factors, the most prominent being the accelerated movements of humans and animals due to increased globalization and international trade, the climatic changes, and the larger and larger populations kept together in animal husbandry and breeding. Some of these diseases, termed transboundary animal diseases (TADs), such as foot-and-mouth disease and classical swine fever, have a high capacity to spread very rapidly over countries and borders, having a devastating impact on animal productivity and trade, as well as causing other losses in the animal husbandries and in wildlife. Other diseases, such as anthrax, bovine tuberculosis, and rabies, have more endemic character, establishing themselves in limited areas and showing slower tendency of spread. Considering their importance, many of these infectious diseases are listed by the World Organisation for Animal Health (OIE) as notifiable animal diseases, collectively referred to as OIE-listed diseases. The OIE is also determining and updating the international animal disease status on a regular basis. The current OIE-listed diseases and the latest disease status reports are available at the OIE website (www.​oie.​int).

Zoonoses, veterinary, and human public health. Of special importance among the animal infectious diseases are the ones that have the capacity to cross the species barriers and establish infections in a wider range of hosts including humans, causing zoonotic infections. It has been estimated that approximately 75 % of the new and emerging human infectious diseases over the past 10–20 years have been caused by pathogens originating from animals or from products of animal origin [1, 2]. Many of these diseases have the potential to spread through various means, over long distances, and to become global problems.

Accurate and rapid diagnosis. Considering the extremely high direct and indirect losses and other consequences caused by the TADs and the other infectious diseases, it is very important to develop and apply a wide range of diagnostic methods. These should preferably allow rapid detection and identification of the infectious agent(s), with high specificity and sensitivity, while still being affordable and readily available. When outbreaks do occur, rapid and accurate diagnosis is needed to screen susceptible populations and monitor the spread of the infectious pathogens, therefore helping with epidemiological investigation and implementation of necessary control measures, such as vaccination, stamping out, and quarantine restrictions, in order to prevent further spread.

Collection of clinical samples and sample preparation. Identification of the relevant groups of animals, showing clinical signs or at stages of infection when the presence of infectious agents is likely to be sufficiently high, and correct sampling are the first two crucial steps in the diagnostic process. The next steps of great importance are the sample preparation procedures, such as cleanup and target enrichment, which are performed in order to reduce possible contaminants and retain concentrated materials from the target agents, most commonly nucleic acid and/or proteins, for further analysis. If any of these steps are not properly considered and carried out, all diagnostic methods, even the most powerful and sensitive, will be unable to detect and identify the infectious agents, and this is leading to false diagnosis, which could have very serious consequences.

The OIE Collaborating Centre (OIE CC) for Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine. Since the authors’ institutes in Uppsala, Sweden, are well-recognized centers of excellence in molecular diagnostics, the OIE has granted them the mandate to work together as its only collaborating center focused on biotechnology-based diagnostics (www.​sva.​se/​en/​About-SVA/​OIE-Collaborating-Centre). In this chapter, the experiences and knowledge gained at the OIE CC will be used to provide an overview of the molecular approaches capable of recognizing relevant and emerging infectious diseases in animals.

Detection and identification of the infectious agents. The diagnostic laboratories can apply two basic ways for a proper diagnosis: (a) direct detection and identification and (b) indirect detection and identification methods. Direct detection and identification means that the infectious agents and/or their components, such as nucleic acids or proteins, are detected in the collected samples. Commonly used classical diagnostic methods for direct detection include identification of microorganisms by culture techniques and immunofluorescence, and the most widely applied molecular diagnostic methods are the various assays of nucleic acid hybridization, e.g., polymerase chain reaction (PCR) and isothermal amplification methods, such as the loop-mediated isothermal amplification (LAMP), among others. When running indirect diagnosis and identification, the immune responses of the host are investigated, looking for antibodies against various infectious agents, which indicate the occurrence of the infections in the hosts. In this chapter we focus on direct diagnosis, with special regard to molecular diagnostic methods, as well as some considerations regarding the interpretation, understanding, and communication of the diagnostic results.

2 PCR-Based Approaches

Molecular approaches become increasingly important in infectious disease diagnostics and, with the exception of isolation by culturing, may supersede all other direct detection methods. The main reasons are that a unique signature of every microorganism is encoded in its genome, which in principle enables perfect specificity, and that various enzymatic mechanisms can be utilized to manipulate and amplify the genetic material, yielding an exquisite sensitivity of the molecular DNA-based assays. While bacteria have their genome encoded in the form of DNA, some viral genomes are composed of RNA, and an initial reverse transcription step is therefore required before further manipulations and amplification can be carried out. Enzymes typically utilized are polymerases, reverse transcriptases, ligases, glycosylases, and nucleases. Of these, the polymerases require a pair of sequence-specific primers, which enables selective target amplification.

2.1 PCR Assays

PCR employs thermostable polymerases to enable amplification by continuous thermocycling and is currently the most commonly used method for amplification of genetic material [3]. The highly charged phosphodiester backbone of DNA makes the PCR product amenable to high-resolution visualization on agarose gel electrophoresis utilizing DNA-binding fluorescent dyes such as ethidium bromide. Electrophoresis both provides a means for detection by band visualization and enables at least a tentative verification of specificity by estimation of the amplicon length.

Shortly after the introduction of PCR, attempts were made to enhance sensitivity of detection of target nucleic acid sequences by running a second PCR assay targeting the internal region of the amplicon resulting from the first reaction, so-called nested PCR [4, 5]. The greater sensitivity has been attributed to both a dilution effect of any inhibitory compounds present in the sample, since only a minor fraction of the first reaction volume is used in the second reaction, and the fact that the primer-driven reaction is run twice, using four specific primers, rather than two. An intermediate situation is obtained if one of the primers from the first reaction is retained in the second, which yields a semi-nested PCR format.

The drawback of using PCR, and in particular the nested PCR formats, is that conserved regions must exist on the genome, and this might be a serious problem for highly variable RNA viruses. Although more recently the convenient and less laborious real-time PCR methods have been developed (see below) and are mostly used today in clinical practice, nested PCR assays are still used due to their high sensitivity and robustness.

2.2 Real-Time PCR Assays

Gel-based PCR is a heterogeneous, relatively laborious, detection method. Furthermore, it only reflects the end point of the PCR and, for this reason, doesn’t allow the determination of the initial quantity of the detected material, e.g., determination of the viral load. Since it lacks specific markers for the targeted amplicon, unspecific amplification yielding similar product sizes may lead to false positive detection. Nested PCR has the further disadvantage of being prone to cross-contaminations since reaction tubes with potentially very high quantities of target DNA are opened between the two reactions. Many of these drawbacks were solved by the advent of real-time PCR [6]. With this technique, the PCR product is monitored in the course of the reaction using DNA-binding moieties that alter their fluorescence upon binding to the amplified DNA. This allows a closed tube, homogeneous assay format, which reduces the risk for cross-contamination and also removes the laborious gel electrophoresis step. In addition, the cycle number where the fluorescence reaches a defined threshold level will depend on the initial quantity of target DNA or RNA (before reverse transcription).

Three main approaches have been taken to monitor fluorescence alteration in real time due to the buildup of the PCR product, which can be ordered according to the level of specificity the methods provide. The simplest method is to add a fluorescent dye to the PCR mixture with the property that the fluorescence intensity changes upon DNA binding. Typical dyes are asymmetric cyanine dyes, such as SYBR green or tiazole orange, that exhibit a fluorescence increase when bound to DNA [7, 8]. These types of real-time PCR have no better specificity than gel-based PCR, rather the opposite, since no information is provided about the product length. New possibilities are given by tethering the dye to one of the PCR primers that are constructed so that incorporation of the primer into the amplicon leads to an alteration of dye fluorescence. Several chemistries have been devised to this end, for example, scorpion primers [9], LUX primers [10], and Plexor primers [11]. Although in principle not providing a better specificity in regard to spurious amplification than the pure dye approach, fluorescent primers enable multiplexing by co-adding several primer pairs, each with a distinct fluorophore. The third approach includes addition of a third fluorescently labeled oligonucleotide, located between the primers, called a probe. The probe can also be labeled with a quencher (dual-labeled probe) but not always, e.g., not for the LightUp probes [7] or in the PriProET approach [12, 13]. Prominent examples of methods based on dual-labeled probes include TaqMan [14] and molecular beacons [15].

The signal that can be obtained from a probe-based real-time PCR experiment is often limited by the competing reannealing of the double-stranded PCR amplicon. Asymmetric PCR can be used to overcome this problem since it allows preferential amplification of one strand in a double-stranded DNA template. This is achieved by manipulating primer properties, most critically concentration, as well as other factors influencing primer melting temperature, such as length and nucleotide sequence. In the LATE-PCR method [16], asymmetric PCR has been combined with molecular beacons for readout to achieve a detection format that allows quantification from the end-point fluorescence. This format is suitable for simpler portable PCR instruments designed for detection in the field and has recently been commercialized by various companies.

The application of real-time PCR techniques and other methods in molecular diagnostics in veterinary medicine have recently been extensively reviewed [17, 18] and will be further discussed later in this section. To conclude this subsection, it is suitable to mention a recently developed method for the rapid molecular pathotyping of avian influenza [19] and Newcastle disease [20] viruses that combines several of the themes discussed here. This technique employs a three level semi-nested PCR format that utilize Plexor [11] fluorogenic primers as a detection mechanism. Furthermore, the assay format allows a highly multiplex interrogation of the sample by using primers in two vastly different concentration regimes. Instead of, as hitherto has been the case, requiring nucleotide sequencing over the hemagglutinin and fusion protein genes of avian influenza and Newcastle disease viruses, a much faster diagnosis can be obtained by a simple PCR-based method. This method could even be implemented on field PCR instruments for rapid on-site diagnosis and thereby providing means for faster containment of disease outbreaks.

3 Isothermal Amplification

Isothermal amplification of nucleic acids is an alternative method to PCR. The reaction is performed at a constant temperature in simple devices, such as water baths or heating blocks, which eliminates the need for high-end equipment and system maintenance. It can be used to test for infections in regions where resources are limited and logistic chains are impossible, but a rapid answer is needed. Isothermal amplification normally takes about an hour or less to complete, providing a fast specimen-to-result diagnosis at the point of care (POC). To make the best use of isothermal amplification, a system should ideally integrate the upstream sample preparation and the downstream detection steps and be operated by personnel without extensive training. Several platforms utilizing isothermal technology are commercially available or close to market [21].

Recently, the field of isothermal amplification technologies has advanced dramatically, resulting in several different amplification systems. These have been summarized by Niemz et al. [21] and include transcription-mediated amplification (TMA) [22], helicase-dependent amplification system [23], loop-mediated isothermal amplification (LAMP) [24], and rolling-circle amplification [25]. Of those methods, LAMP has gained the greatest interest because of its high specificity, efficiency, and rapidity. By addition of a reverse transcriptase in the reaction, RNA targets can also be amplified and detected by LAMP, which is referred to as RT-LAMP. The LAMP utilizes four primers that bind to six distinct regions of the target DNA to specifically amplify a short region and is catalyzed by Bst DNA polymerase with strand-displacement activity [24]. Addition of loop primers may accelerate the reaction [26]. As of 8 February 2014, PubMed listed 990 publications with the search term “loop-mediated isothermal amplification.” LAMP technology has been applied for the detection of viral pathogens such as classical swine fever virus [27] and foot-and-mouth disease virus (FMDV) [28], bacteria such as Clostridium difficile [29], and parasites such as malaria [30]. Commercial developments have progressed: a total of eight LAMP kits are approved in Japan for the detection of SARS coronavirus, Mycobacterium tuberculosis (TB), Mycoplasma pneumoniae, Legionella species, influenza A virus, H1 pdm 2009 influenza virus, H5 influenza virus, and human papilloma virus, as reviewed by Mori et al. [31]. Future development would need to consider simplification of sample preparations, reaction mix in a dried down formation and integration of all three steps in a compact, disposable, and inexpensive system.

4 Detection by Hybridization-Based Approaches

Identification and classification of bacteria and viruses using DNA hybridization-based approaches rely on the use of oligonucleotide probes that selectively bind to target sequences based on the degree of complementarity. This was early utilized in fluorescence in situ hybridization (FISH), which became a valuable tool for localization of infectious agents in clinical samples without cultivation [32]. However, to overcome limitations in multiplex capacity, sensitivity, and signal intensity, there has been an ongoing development of the initial approach. This has resulted in high-throughput methods such as DNA arrays but also interesting new hybridization-based methodologies combined with signal amplification, such as padlock probe (PLP) [33] and proximity ligation assay (PLA) [34]. PLP belongs to the methodologies of genomic partitioning where one specific region of the genome is massively replicated, and thereby detectable, even though it normally is masked by the presence of other genomes or in too low amount to be detected. PLA relies on the primary detection of antigens followed by oligonucleotide amplification and subsequent detection by fluorescent probes or by RT-PCR.

4.1 DNA Array Technologies

With the development of DNA macro- and microarray technologies, it became possible to detect and characterize a wide variety of bacteria and viruses through simultaneous hybridization against large numbers of DNA probes immobilized on a solid support [35, 36].

The probes represent known sequences that may serve as markers for identification and/or genotyping of bacterial strains, resistance genes, viruses, etc. These are commonly arranged in an ordered array of spots (or features), and hybridization with a labeled target, i.e., the sample to be investigated, will therefore result in a hybridization profile in which individual probe results also can be assessed. As the names imply, the main difference between macro- and microarrays is the number and size of spots on the support. Macroarrays typically have larger and fewer spots and have proven particularly effective for detecting smaller subsets of genes, such as genes involved in antibiotic resistance [37]. Microarrays can contain thousands, and even up to many hundred thousands, of spots with different oligonucleotide probes and have successfully been used for detection and genotyping of bacterial and viral pathogens [38, 39]. The main advantages of microarray technology are high throughput, parallelism, miniaturization, and speed. However, microarrays are still considered to be an expensive technology and usually require large amounts of nucleic acid targets. Furthermore, unless it has been completely automated, the data analysis procedure might be time-consuming, and the results can be difficult to translate into information that is clearly communicable and decision supportive.

4.2 Genomic Partitioning

Genomic partitioning refers to the methodologies used for capture and enrichment of target regions. Within these methodologies, PLP has been used repeatedly for genotyping, localization, and array-based diagnostics. The earliest version of PLP consisted of two oligonucleotide probes of 20 nt connected by a linker region of 40 nt [33]. As the probes hybridize towards the target, the construct is ligated into a circular detector that can be replicated isothermally by Phi29 polymerase [40]. The detection can then be performed through incorporation of fluorophore tagged nucleotides. The PLP concept was further expanded with the introduction of the molecular inversion probe (MIP) technology. Where PLP leaves no gap after hybridization to the target region, MIP aims at leaving a single nucleotide gap. This gap is then filled in by addition of a single type of nucleotide into the assay. This approach enables substitutions on nucleotide level to be detected using just four reactions easily set up in a normal lab environment. It also provides a possibility of highly multiplexed designs of assays [41, 42]. Building on the same principle as PLP and MIP, the connector inversion probe (CIPer) technology extends the gap up to a few hundred nucleotides. Using DNA polymerase to fill the gap generates a product that can be sequenced, revealing the content of the target region [43]. Applications of PLP methodology and its derivatives for infectious diseases in animals include detection of all hemagglutinin and neuraminidase subtypes of AIV [44], as well as multiplex detection of FMDV, swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV) [45]. In addition, by designing different probes for the genomic and replicative form of the virus, it is possible to not only detect a virus but also localize it in relation to the host cells and perform semiquantitative analysis of the amount of replicative viruses, as demonstrated with porcine circovirus type 2 [46].

4.3 Proximity Ligation Assay

Although PLA is designed for detection of protein interactions and localization using antibodies for target recognition, hybridization events are required to generate a detectable signal [34]. Two sets of antibodies are designed: one targets the protein/s of interest and the other target the first set. The antibodies in the second set carry short oligonucleotide strands that can hybridize with special connector oligonucleotides and thereby enable the formation of circular DNA constructs. These are amplified and detected by PCR and fluorescent probes. The methodology combines dual antibody specificity with the signal amplification power of DNA amplification to produce a versatile and sensitive method for detection of very low amounts of targets. It also enables in situ localization studies of protein targets within cells [47]. Furthermore, PLA requires little to no sample preparation, making it ideally suitable for screening of massive amounts of samples, and can be used with a solid support to capture antigens for detection, similar to ELISA. The use of a solid support may also facilitate the removal of contaminants from the sample, thereby enabling PCR-based detection without the problem of inhibition. By combining the solid-support approach with RT-PCR detection, great sensitivity was demonstrated in a study of avian influenza virus [48]. Other applications of PLA technology include detection of several viruses, among them FMDV, with detection levels close to those of RT-PCR and 100-fold more sensitive than ELISA [49], as well as localization of influenza virus proteins within cells [50].

5 Further Trends, New Tools in Molecular Diagnostics

In the development of new molecular diagnostic methods, there has been a continuous effort to enable efficient and rapid detection of infectious agents from ever-smaller volumes of complex fluids without the need for a skilled operator. As a result, microfluidic analysis systems and nanotechnology-based detection devices have gained increased popularity, as previously reviewed [51, 52]. These systems and devices have been employed to construct a wide range of integrated tools, capable of semiautomated complex diagnostic procedures, which also allow rapid, portable field-based testing [53].

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