Molecular Testing for Infectious Diseases in Cattle, Sheep, and Goats

Beate M. Crossley • Sharon K. Hietala

The availability of molecular-based diagnostic technologies designed to identify infection or genetic conditions by detecting

specific genome targets in the host or agent DNA or RNA has expanded logarithmically in the past decades.

Whole genome sequencing, the ability to rapidly generate the genetic sequence of an entire pathogen, is increasingly available as a diagnostic tool and provides a high level of both diagnostic sensitivity and specificity, although at the expense of rapid turn-around and high technical costs that are most often passed on to the user. PCR-based technologies and assays that target a single gene or part of a gene remain the most common of the molecular-based diagnostic assays currently available for routine diagnostic services. These services are provided by state and federal diagnostic laboratories, university laboratories, and commercial laboratories; due to the technical complex­ity of molecular-based diagnostics, paired with the wide range of assays available to diagnose genetic and infectious diseases of food animals; however, no single laboratory can feasibly provide comprehensive testing for all species and all diseases. Instead, laboratories tend to focus on the diseases or genetic conditions of primary significance to their allied industries and use the technologies that provide the most rapid, reproducible, and economic results for their particular group of clients. Largely based on this specialization within laboratories, molecular-based assays may be developed and put into use without significant effort toward assay standardization and validation.

Sample Submission

As with any diagnostic technology, the specimen used must be appropriate for the disease or genetic condition and take into consideration the timing and pathogenesis of the disease process and availability of the intact genetic material being targeted by the diagnostic assay. Among the important criteria in the selection and use of molecular-based assays are onset and duration of agent replication in the host, specific tissue(s) affected, and the relative abundance of target DNA or RNA over the course of the infection. In addition to tissues obtained from postmortem investigation or surgical sampling, antemor­tem diagnostic testing can target genetic material recovered from swabs of mucous membranes; blood, urine, milk, or skin scrapings; or fecal material as appropriate to the disease process.

Because all molecular-based diagnostics detect genomic material in one form or another, the isolation and purification of nucleic acids (DNA or RNA) from the diagnostic specimen are critical to the success of the assays. Dependent on the tissue or specimen used for diagnostic testing, recovery of sufficient quantities and quality of nucleic acid can be prob­lematic. Common sources of assay failure include substances in the sample that can interfere with the chemistries used in detection or PCR-based amplification steps. Examples include, but are not limited to, heparin, iron, peroxidases, hemoglobin, and myoglobin, as well as the relative abundance of total DNA or RNA associated with the different specimen types. This type of assay error is controlled in an increasing number of PCR assays by the inclusion of internal controls before initiating the assay in order to monitor for specimen-based interference or related-assay failure. Another common source of assay failure is destruction of target nucleic acids in the specimen before testing, commonly as a result of enzymes associated with postmortem autolysis or those chemistries used in preserving the sample, such as formalin fixation.¹ Because of the complexi­ties of nucleic acid recovery and target nucleic acid detection, assays developed for use with one matrix, such as whole blood, may not be suitable for use with other matrices, such as milk or feces.

Molecular-Based Diagnostic Technologies

Nucleic acid fragments, either DNA or RNA, can be detected by binding to a complementary fragment of nucleic acid that has been marked with a reporter dye or enzyme. The short strands of DNA or RNA, generally chemically synthesized and referred to as oligonucleotides, are identified as hybridization probes when marked with a reporter dye or enzyme. Radioactively labeled probes, although once the standard, have almost uniformly been replaced by enzymatic tags, such as avidin-biotin, peroxidase, or chemiluminescent tags. Target nucleic acids can be detected directly in the diagnostic specimen when sufficiently abundant, or they may be detected following PCR-based amplification of the nucleic acid target.

SOUTHERN BLOTS, NORTHERN BLOTS, AND DOT-BLOTS. Various techniques are used to immobilize genetic material before detection steps, with or without prior PCR amplification. As a group, the techniques are referred to as “blots.” Dot-blots and Southern blots refer to DNA bound to nitrocellulose membranes, whereas Northern blotting is the terminology used when RNA is immobilized onto membranes. In Southern and Northern blots, nucleic acid fragments are first separated by size using an electric current running through a semisolid gel, a technique referred to as gel electrophoresis. The nucleic acid detection step for DNA and RNA blots may include a specific hybridization probe or may simply use the molecular weight of the DNA or RNA fragment. Related terminology includes Western blot, which describes protein immobilized on a membrane, and immunoblot, referring to detection of the immobilized protein using specific antibody as an immunologic marker. Variations of this approach, using proteins or capture antibodies immobilized on specialized paper strips, form the basis for lateral flow (“dip-stick”) pen-side tests including those developed for the rapid field detection of foot-and-mouth disease virus.

The laboratory technique referred to as in situ hybridization allows for localization and observation of a DNA or RNA target within a histologic tissue section.^2-5 The hybridization probes are typically end-labeled with a reporter molecule. Examples of hybridization assays used in veterinary diagnostics include FISH to identify genetic abnormalities and RNA probes (ribo­probes) used as assay controls or for selected infectious disease detection in tissue samples. In situ hybridization technology can be applied to frozen or formalin-fixed tissues, with best results provided when the tissue is fixed as soon as possible postmortem to avoid loss of target nucleic acids during tissue autolysis. Nucleic acids from veterinary pathogens have been shown to be stable in formalin-fixed, paraffin-embedded tissues for up to 15 years.^2,6 Because of technical complexity, in situ hybridization is not widely applied to routine diagnostics but appears to be advancing in that direction.

POLYMERASE CHAIN REACTION. PCR is the most commonly applied of the molecular technologies and can be grouped roughly into standard PCR and the more rapid real-time and quantitative PCR. Both techniques amplify minute quantities of target DNA to levels that are detectable using laboratory instrumentation or special staining procedures. The principal difference in the two approaches is that standard PCR is divided into two major processes in the laboratory: 1) amplification of the DNA target, followed by 2) detection of the amplified target DNA. Real-time PCR combines the two steps so that target detection occurs during the DNA amplification process. The advantages of PCR over standard isolation and identifica­tion techniques is the significantly reduced time required to obtain a result, with PCR results generally provided in hours instead of days. Real-time PCR results can be reported as dichotomous results (positive, negative) or on a continuous numeric scale, generally as a cycle threshold (Ct) with the lower numeric values correlating with higher concentrations of the target pathogen or genetic marker.

Standard PCR. Standard PCR is used for detection of DNA or RNA and can be applied to mammalian, microbial, or viral genomes. The PCR technique is based on the natural cross-linking of complementary (matching) nucleic acids to form double-stranded DNA. When DNA is sufficiently heated, double-stranded DNA separates into single strands, each of which can then be used as a template for chemically (or biologi­cally) generating an exact copy. Specific heat-stabile polymerase enzymes are used in the laboratory for the process of DNA replication. The PCR reaction amplifies DNA using multiple cycles of heating double-stranded DNA to generate a single­strand template and cooling sufficiently to allow the polymerase enzyme to generate a new double-stranded DNA made up of the original strand and its test-tube synthesized copy. For diagnostic assay purposes, it is necessary to copy only a small but diagnostically characteristic fragment of any particular genome. This fragment is generally referred to as the PCR target or target sequence. PCR primers are short chemically synthesized fragments typically 10 to 30 nucleic acids in length that are used to mark the beginning and end of the selected target sequence and, as their name implies, are used to prime or start the initial PCR amplification process. The choice of target sequence and appropriate primer design are critical to the accuracy and efficiency of PCR-based diagnostic assays. For detecting target RNA, as is needed for the PCR diagnosis of an RNA virus, the technique of reverse transcriptase PCR (RT-PCR) is used. The reverse transcriptase enzyme converts target RNA into DNA, identified as cDNA or copy DNA for use in the PCR steps. The PCR amplification process typically takes 2 to 6 hours to complete and is followed by detection steps that require additional hours to days to complete. Detec­tion of the amplified DNA fragment, often referred to as an amplicon, uses techniques that identify the DNA fragment by molecular weight, by marking a specific target sequence in the amplified DNA with hybridization probes, or by a combination of both (PCR/probe). PCR amplification followed by hybridiza­tion probe detection offers increased specificity, thus less chance of a false-positive finding, and often provides a 10-fold or greater increase in detection sensitivity. Dependent on the stringency designed into the assay by balancing such measures as temperature, length of replication cycles, and enzyme concentrations, false-negative results will occur when the sequence of the primers or probe does not precisely match the sequence of the target. In some cases, PCR assays are very precise and designed to recognize a single nucleic acid poly­morphism (SNP analysis) in the target sequence, and in other cases they are intentionally designed with lower stringency to allow some degree of variability among isolates or strains being detected by the assay. Because RNA viruses in general lack the proofreading enzymes found in DNA viruses, bacteria, and protozoa, they are at greater risk for appearance of random mutations at primer or probe sites. PCR false-negative results associated with primer or probe mismatches resulting from random genomic mutations at the primer or probe binding sites are of particular concern for RNA viruses, and they provide ample justification for a laboratory to investigate with alternative or additional technologies when negative PCR-assay results clearly do not match a clinical or epidemiologic pattern of disease.

Nested PCR. Nested PCR is a PCR technique modifica­tion generally used to increase the detection sensitivity of an assay.⁹ During nested PCR, an initial PCR reaction amplifies a selected fragment of DNA, which is then used as a template in a second round of PCR. The second or nested PCR reac­tion targets one or more unique fragments of DNA within the initially amplified region. The two-step process has the advantages of increasing detection sensitivity and detection specificity and of diluting the impact of interference from potential PCR inhibitors found in some tissues and clinical materials. Nested PCR assays have been described for WNV, malignant catarrhal fever virus, and Chlamydia spp., among others. The significant disadvantage of the two-step process comes from handling the first round of amplified DNA in the laboratory. With the high concentrations of target DNA available following PCR amplification, laboratories must take rigorous precautions to mediate the risk of contaminating laboratory workspaces, equipment, and reagents with the amplified DNA. The significant risk and associated costs of laboratory contamination and resulting false-positive results are used in many diagnostic laboratories as justification for charging additional fees or not offering nested PCR techniques.

Real-time PCR. Diagnostic assays originally developed using standard PCR approaches have over the years been converted to real-time PCR and real-time qPCR technology, to take advantage of the speed, technical efficiency, and cost reduction often associated with this approach.¹⁰ Because real-time PCR technology completes both amplification and detection in the same reaction tube or well, there is no need for the laboratory to handle amplified DNA and risk workspace contamination. Importantly, the combination of amplification and detection in a single-reaction vessel not only decreases the time required to complete the test, but importantly has allowed expansion of PCR-based assays into high-throughput, portable, and on-site formats as described for foot-and-mouth disease virus, anthrax detection, and others. Because real-time PCR targets shorter genetic sequences, the methodology is more effective than standard PCR for testing formalin fixed tissue.¹ The preservation method tends to destroy longer strands of genetic material. Real-time PCR offers an additional advantage of supporting quantitative approaches for measuring and reporting the amount of the original target in the sample tested. qPCR answers a frequent criticism of standard PCR, specifically that detection limits, potentially as low as a single cell or viral particle, may produce a positive finding that is biologically irrelevant to clini­cal disease. qPCR allows the relative amount of target nucleic acid to be correlated with the associated clinical presentation. Despite the significant benefits, real-time PCR is, however, less efficient than standard PCR in producing amplified genetic material that can be used for subsequent nucleic acid (genome) sequence analysis, as is often employed to confirm positive PCR findings, or further characterize a pathogen on the basis of genetic material obtained from the diagnostic case materials.

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Multiplex PCR. Multiplex PCR expands the use of PCR and real-time PCR-based detection from a single target to multiple targets simultaneously from a single specimen.^11-13 The ability to include multiple genome targets for a single virus or bacteria in a PCR assay minimizes the risk of a PCR false-negative as a result of genetic diversity within the agent of interest. Technically, the inclusion of an internal control, which is rapidly becoming a standard quality practice in veterinary diagnostic laboratories, is a simple form of multiplexing. Inclusion of targets for multiple closely related viruses has been used to differentially detect related pathogens, such as the closely related sheep and wildebeest-associated forms of malignant catarrhal fever; to subtype BVD viruses; and to differentially detect virulent Escherichia coli. Multiplex PCR also has broad application in syndrome-based testing where several pathogens may produce similar to identical clinical pictures. A single diagnostic specimen can be used in a multiplex format to detect one or more agents associated with a particular clinical presentation, such as the multiple viral and bacterial agents in shipping fever complex. Prototypes of this approach have been developed but are not yet widely deployed, and there are technologic limits to the number of PCR assays that can be combined without significant loss of assay performance. In real-time PCR formats, three to five unique assays have been the practical limit, based on the technical limitations of commonly available equipment and detection dyes.

ISOTHERMAL AMPLIFICATION. Isothermal techniques use alternate chemistries to PCR that allow amplification of nucleic acids to be carried out at a single temperature. Among the isothermal amplification technologies that have been used for the detection of veterinary pathogens are nucleic acid-based amplification, loop-mediated isothermal amplification (LAMP), rolling circle amplification, and direct signal amplification systems.^14,15 Although there are increasing efforts, especially for the development of LAMP-type diagnostic assays, none of these technologies has seen widespread diagnostic application, largely based on the complexity in initial assay design. However, LAMP-type assays may offer future PCR alternatives that can be developed into automated portable diagnostic tools, or possibly be developed as fully integrated self-contained testing units for use in the field rather than limited to a laboratory environment.

MICROARRAYS. Arrays of DNA or RNA fragments, variously referred to as gene chips and microarrays, consist of thousands of oligonucleotides attached in a specific pattern to a solid surface, typically a glass slide or silica chip.^16-22 The sample nucleic acids compete with fluorescently labeled competitor oligonucleotides for binding to the chip. A fluorescent detector and computer software are then used to analyze the patterns of fluorescence, indicating where nucleic acids have (or have not) been bound to the chip. Microarrays have been used primarily as research tools focused on detecting and understanding gene expression. The practical application to veterinary medicine includes studies such as the evaluation of genetic resistance to naturally occurring nematode infections, including Hemonchus, Trichostrongylus, and Ostertagia in sheep. Microarray technology has not seen wide use in veterinary diagnostic laboratories, largely because of the large initial investment required for equipment. However, examples of diagnostic microarrays have shown promise, and it is expected that, once established, the technology will be cost effective on an individual animal basis. In human diagnostic medicine, microarrays designed with genetically conserved sequences for all major groups of microorganisms have been used to identify or “mine” for unknown pathogens. This approach was used to detect and identify the SARS virus, before it was initially recovered and characterized by virus isolation techniques. Alternately, genotyping arrays have been designed to target specific virulence-associated genes, allowing differentiation of disease-associated subtypes of organisms from other members of the same or closely related species, such as enterotoxigenic E. coli and multidrug-resistant Salmonella strains.

An alternative to gene chip technology is liquid array technology, in which the nucleic acids are captured by oligo­nucleotide probes bound to a microbead, rather than to a flat surface. Bead sets are internally labeled with different-colored dyes used to distinguish the specific agents being measured. Liquid array systems use flow cytometry, laser technology, and signal processing to recognize the internal color of the beads and measure associated surface-binding of a target-specific fluorescent hybridization probe. The bead-based modification to microarray technology allows a laboratory to create a flexible diagnostic-test panel in a single assay by adding or removing target beads on the basis of the unique clinical presentation, diseases suspected, or species' specificity of interest. In theory, 100 different targets are detectable in a single assay.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM. Molecu­lar techniques provide a valuable tool for genotypic charac­terization or subtyping of viral, bacterial, and parasitic agents. Subtype characterization^23-26 is essential for, among other things, taxonomic classification, differential detection of virulent strains, identification of genetic sources of antimicrobic resistance or virulence factors, recognition of vaccine escape mutants, epide­miologic investigation of disease outbreaks, and identification of interspecies transmission of specific pathogens. Restriction fragment length polymorphism (RFLP) is a technique useful for subtyping of pathogens based on genetic sequence variation within specific genes.²⁷ Target DNA, which may or may not previously have been PCR amplified, is digested using one or more well-characterized restriction enzymes. Characteristic profiles or patterns produced by the different-sized DNA frag­ments remaining after enzyme digestion are detected by gel electrophoresis. RFLP has been used to investigate interspecies transmission of enterotoxigenic Staphylococcus aureus associated with intramammary infections in cattle, sheep, and goats.²⁸ Random amplified polymorphic DNA (RAPD) analysis is a technique that has proved useful in detecting genetic varia­tion and for strain typing.²⁹ Rather than amplifying a specific region of a genome, RAPD relies on random amplification of genomic DNA using short arbitrary sequences as PCR primers. RAPD does not require prior knowledge of an organism's DNA sequence for specific primer design and can be applied to small amounts of template DNA. The RAPD technique has been applied, for example, to rapid differentiation of pathogenic from nonpathogenic coccidia of sheep including Toxoplasma gondii and Sarcocystis spp. Identification of nonculturable, slow-growing, and atypical bacterial species using sequence analysis of the 16s ribosomal RNA (rRNA) gene has become recognized as a valuable diagnostic tool in recent years. The basis of the 16s rRNA typing technique is the highly conserved nature of the 16s rRNA gene within bacterial species, as well as among species of the same genus.^13,30,31 Automation developed to support 16s rRNA amplification, sequencing, and data analysis is available to and currently in place in selected veterinary diagnostic laboratories. Although the technique is not currently cost effective for routine identification of all microbial isolates in a clinical setting, the technology is used for identification of atypical, slow-growing, and rarely encountered species.³² Because 16s rRNA databases continue to be updated to include pathogens of veterinary and zoonotic interest, it is likely that the technology is seeing broader application.

SEQUENCE ANALYSIS. Nucleic acid sequence analysis is used to identify and compare the exact nucleic acid sequence of a gene fragment, gene, or possibly entire genome for forensic purposes; to investigate disease outbreaks; to track evolutionary changes in rapidly mutable microorganisms; or for precise phenotype or genotype analysis of animals or organisms. The technology used for sequence analysis is progressing exponentially since initially introduced in the 1970s and most recently includes “next-generation sequencing,” high-throughput sequencing procedures, and metagenomics used for routine diagnostic applications, food safety, and detection and characterization of new and emerging pathogens. Equipment and computational strength used for sequence analysis were once limited to specialized research laboratories and reference centers, but they are increasingly available in well-equipped diagnostic laboratories. Next-generation, also called second-generation, sequence analysis approaches use bench-top commercialized equipment based on DNA synthesis, ligation, or ion semicon­ductor technology for the rapid and highly accurate genome sequencing of pathogens. The complete genomic sequence of individual pathogens can be completed within a matter of hours to days using next-generation sequencing technologies.

For diagnostic purposes genetic sequencing may be paired with PCR amplification of specific genetic markers or regions of known pathogens, or it may be used in de novo applications that determine the genetic sequence and identity of an unknown or previously uncharacterized pathogen.^20,21 Sequence analysis, using an approach known as Sanger sequencing, is most com­monly used in diagnostic medicine to confirm the identity of a pathogen testing positive in a PCR assay, to detect genetic variants or diagnostically important mutations in a pathogen's genetic makeup, and for forensic investigation into the source of an agent or disease outbreak based on specific genetic markers in the recovered pathogens. The target for Sanger sequencing is the amplified DNA (or cDNA) generated from routine standard PCR or real-time PCR testing of diagnostic samples. Sanger sequencing is currently used by the majority of veterinary diagnostic laboratories as a supplementary diagnostic tool. Next-generation sequencing is a rapidly developing tool that, unlike Sanger sequencing, which targets small pieces of DNA such as a specific part of a single gene, targets entire genes or genomes, making it an ideal tool for discovery of novel pathogens; identification of genetic disorders, which is often done by looking for the same genetic markers in animals previously linked to human genetic diseases¹; and genomic and phylogenetic research of both pathogens and host animals.^33,34 Next-generation sequencing is not yet a routine diagnostic tool, as the sensitivity of next-generation sequence analysis is highly dependent on the quality of the sample, the precision needed in sample processing, and the relatively complex data analysis. However, the field of study is rapidly evolving. A number of platforms including devices for field applications³⁵ are currently available, each of them operating with slightly different methods resulting in both advantages and limitations.³⁶ Sampling bias, enzymatic reaction with associated error rate and bias, and computing database bias all must be addressed before reliable results can be communicated to the client, the public, and those making animal health policy decisions.³⁷ Multiple guidelines addressing the importance of a rigorous data analysis have been published for human medicine and are equally applicable to veterinary medicine for ensuring the reliability of results.³⁸ Relevant examples of new and emerging viruses identified using next-generation sequencing approaches include the Schmallenberg virus that spread rapidly in European cattle, resulting in reproductive inefficiency and fetal abnormalities,³⁹ a new Bluetongue serotypes,⁴⁰ as well as a tickborne “looping-ill”-related virus in goats and novel astroviruses in both cattle and sheep associated with encephalitis and gastrointestinal diseases.³⁹ Importantly, next-generation sequencing continues to expand the clinically important molecular characterization of uncultured bacteria.⁴¹ In the future, metagenomics and next-generation sequencing data are expected to allow monitoring of treatment interactions with the host's microbiome, as has already been demonstrated with poultry data.⁴²

Genetic Diseases

Diseases that are linked to genetic mutations in specific breeds— such as bovine leukocyte deficiency and complex vertebral malformation in Holstein cattle, β-mannosidosis in cattle or goats, and spider-lamb syndrome in sheep—can be controlled by selective breeding using PCR-based testing to identify carriers of