beam that passes over the molecules within the liquid sample as they advance single file through the flow chamber (Pedreira et al. 2013). This technique allows for rapid, objective, and quantitative evaluation of cells, even when present in small numbers within the sample. Flow cytometry uses light scatter and absorption to measure individual cells and has the added ability to sort cells based on these characteristics into separate collection tubes for future experiments. Flow cytometry is a useful tool for the diagnosis and study of hematologic neoplasia in veterinary medicine (Meichner et al. 2020).
Secondary Binding Tests
Secondary binding tests measure antigen–antibody interaction in vitro. These tests measure the secondary effect, or consequence, of the antigen–antibody interaction. Agglutination, precipitation, neutralization (of bacteria, viruses, toxins, etc.), and complement activation are all examples of secondary binding tests (Tizard 2013). They are not as sensitive as primary binding tests, but they are much easier and faster to perform. In general, these tests are not used often in veterinary dermatology.
Tertiary Binding Tests
These are in vivo tests that measure the protective effect of antibodies within circulation and provide information about the significance of the immune response (Tizard 2013). These tests are much more complicated to perform and are not used often in veterinary dermatology.
Polymerase Chain Reaction
PCR is a molecular copying technique useful for detecting very small amounts of DNA in a sample. PCR makes millions to billions of copies of specific regions of DNA, referred to as amplicons, in order to amplify, quantify, and further study the molecule of interest (Waters and Shapter 2014). The basic steps of PCR involve denaturing the DNA of interest using heat, annealing oligonucleotide pairs (primers) to desired regions of the DNA, and making copies of this DNA template using DNA polymerase. Each time this reaction takes place, the DNA is doubled, so that at the end of 20 cycles there are more than one million copies of the template DNA. Once the DNA is amplified, additional laboratory techniques such as quantification of a molecule of interest, DNA sequencing, DNA cloning, and ELISA can be used to study the DNA further. PCR tests provide rapid and accurate results; however, the presence of DNA does not prove that the molecule of interest is functional or viable. Moreover, the presence of DNA does not prove causality of disease and may simply represent a contamination of the sample. In other words, finding dermatophytic fungal DNA on a host does not prove the fungal organism is alive and causing infection (Jacobson et al. 2018). Further diagnostics confirming fungal invasion of the host tissue (e.g. trichogram, cytology, or histopathology) would be required to confirm the presence of dermatophytosis.
Pulsed‐Field Gel Electrophoresis
Prior to whole‐genome sequencing made possible by PCR technology, pulsed‐field gel electrophoresis (PFGE) was the gold standard for fingerprinting DNA. This technique uses enzymes to digest whole DNA from microbes or mammalian cells into large fragments that are separated according to their size using a fluctuating electric field (Herschleb et al. 2007). “Fingerprinting” comes from the unique pattern the DNA fragments create for each organism. PFGE can separate very large fragments of DNA, unlike other types of conventional DNA electrophoresis. The unique DNA fingerprint is a form of genotyping that can be used to discriminate among different strains of an organism (e.g. for identifying different strains of Staphylococcus pseudintermedius grown on aerobic culture). DNA fragments are separated and extracted for further evaluation using other laboratory techniques. This technique is mainly used for research at this time.
Transmission Electron Microscopy
Transmission electron microscopy () creates much higher‐resolution images than is possible with standard light microscopy. Transmission electron microscopes shine a high‐energy particle beam of electrons through a thin sample, such as a 1 μm thick sample of skin, to visualize the finest details of cellular structures. The wavelength of electrons is much smaller than that of light, and these electrons interact with atoms in tissue to create an image with extraordinary detail. For example, TEM was used to visualize the intercellular lipids of the stratum corneum to demonstrate differences in quality and distribution of lipids in atopic dogs (Dillard et al. 2018; Inman et al. 2001). A newer technique called cryo‐electron microscopy uses cold temperatures and vitreous ice to create three‐dimensional (3D) images of macromolecular structures (Dillard et al. 2018). This technique allows scientists to study the physical alterations of normal and diseased tissues. The technique is used mostly for research at this time.
Whole‐Genome Sequencing
Whole‐genome sequencing is the process of determining the entire DNA sequence of an organism at one time. It provides detailed information regarding an organism's genetic makeup, including mutations and functional variations in DNA. Also known as “next‐generation (nex‐gen) sequencing,” this research technique is growing in popularity and may represent a new gold standard in DNA analysis. The technique provides such detailed information about an individual organism that the information can be used to create targeted drug therapy. Because exons provide the information for making proteins, whole‐exome sequencing provides information on variations in the protein‐coding regions of many genes at one time and is an efficient way to study disease‐causing mutations. Whole‐genome sequencing provides a way to study those diseases caused by mutations occurring outside of exons (Abdelbary et al. 2017; Mardis 2013). It is being used to study mutations in microbes that promote drug resistance and survival within the host (Abdelbary et al. 2017). This may represent a way to identify and develop targeted antimicrobial therapy in the future.
Matrix‐Assisted Laser Desorption Ionization‐Time of Flight Mass Spectrometry
Another newly popular research modality within microbiology is matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS), which identifies microorganisms based on their unique mass spectrum. MALDI‐TOF MS is a “soft” ionization technique causing minimal to no fragmentation of the molecules, which allows for the study of larger proteins. In this technique, the microorganism is mixed with a matrix (matrix‐assisted) and crystalized onto a target plate. The purpose of the matrix is to protect the microorganism from decomposition during exposure to the laser‐emitted heat. The plate is placed into the plate chamber and laser energy is used to apply heat to the sample, causing it to desorb (laser desorption) into singly charged ions. The ions are passed through a flight tube and separated based on their mass‐to‐charge ratio, with the smaller, lighter ions passing through first (ionization‐time of flight). The mass spectrum generated from this ionization step is detected and compared to a library of known mass spectra and the microorganism is identified (mass spectrometry).
This technique provides rapid, accurate, and cost‐effective identification of microorganisms, and it represents a major innovation for identification of clinically relevant bacteria, fungi, viruses, and parasites (Benagli et al. 2011; Bourassa and Butler‐Wu 2015; Kostrzewa et al. 2019; Pavlovic et al. 2015; Tartor et al. 2019). After as little as two days of colony growth on culture, dermatophyte genus identification is achieved using MALDI‐TOF MS, which allows for expedited antifungal treatment (Welker et al. 2019). MALDI‐TOF MS is also a sensitive, specific, and inexpensive way to study antimicrobial resistance and cellular biomarkers of infection, and can identify and characterize newly emerging microorganisms faster and more completely than culture techniques. This diagnostic modality is not without limitations, however, including nonstandardized protocols, limited database quality and diversity, and unexpected results (Van Belkum et al. 2017).
Conclusion
As scientific technology advances,
