Diagnostic Testing in Exotic Animals: What Test to Run and What Does It Mean?
Mark A. Mitchell, DVM, MS, PhD
Many of the exotic species we deal with have evolved to mask their illness to avoid predation. Although this method of self-preservation is valuable to these animals in the wild, it can make disease diagnosis in captive animals a challenge for the veterinarian. Historically, mammalian diagnostic methods have been used to characterize pathogens in exotic species. Unfortunately, some of these diagnostic assays may not have been sensitive or specific enough to characterize non-mammalian pathogens. Veterinarians have also been limited by the amount of research specifically focused on the identification of pathogens from these animals. Historically, bacterial diseases were considered the primary/only pathogen associated with exotic species because microbiological culture was the only diagnostic test available to veterinarians. In the past two decades, there has been a movement in the scientific community to develop diagnostic assays specifically tailored to diagnosing diseases in exotic animal patients. The purpose of this presentation is to introduce veterinarians to the diagnostic assays that are currently available for exotic animal patients and discuss the advantages and disadvantages associated with these assays.
Historically, microbiological culture has been used to detect bacterial organisms from various tissues and excretions. Although culture has been considered the gold standard for detecting microbes, test characteristics, including sensitivity and specificity, are unknown. In general, culture is considered to have a moderate sensitivity and high specificity. There are a number of parameters that can affect the reliability of culture, including method of specimen collection, quantity or type of sample collected, temporal or seasonal variation in shedding, and method of culture. An additional limitation of microbiologic culture is the time required to confirm a diagnosis, which may exceed 48 h to isolate an organism and 96 h to confirm the organism with biochemical tests. The delay imposed by culture techniques may defer initiation of appropriate antimicrobial therapy and control procedures. Culture of certain microbes can be difficult, if not impossible, based on our current knowledge of growth requirements. The inability to confirm the presence of a fastidious organism can influence the management of a clinical case.
Necropsy is generally regarded as the gold standard for disease diagnosis in veterinary medicine. However, this diagnostic method is subject to misclassification. Sample collection during the necropsy may be subject to sampling bias. Macroscopic lesions are generally sampled without error, whereas microscopic lesions may be overlooked. The number of samples collected at the necropsy can also affect the outcome. The author has observed two sampling methods among pathologists, those that collect a series of samples from a single organ and those that collect only one sample from an organ. Microscopic interpretation of the histologic samples may also be subject to misclassification. One pathologist's interpretation of a histologic lesion may be vastly different from another pathologist. Although necropsy provides important insight into the disease processes in exotic animal patients, veterinarians should consider that it is subject to misclassification.
Hemagglutination inhibition assays are used as screening tests for a variety of different pathogenic organisms. In brief, gander red blood cells are used to measure the ability of serum to protect against agglutination when challenged with a pathogen. The absence of red blood cell agglutination is considered a positive test result. Hemagglutination inhibition assays are susceptible to cross-reactions with related organisms. This can make it more difficult to characterize a specific virus within a group of viruses. Because the HI assay is susceptible to cross-reactions between similar viruses, it may be more useful as a screening test. Other tests, such as polymerase chain reaction assay, may be more useful for characterizing a specific pathogen among similarly related pathogens.
The impact of infectious diseases on exotic animal health has created a need for rapid and accurate detection methods for pathogens from both animal and environmental samples. Enzyme-linked immunosorbent assays combine a specific anti-immunoglobulin with an enzyme to detect a specific microbial antigen or antibody. Benefits of the procedure include speed and low labor requirements compared to culture. The sensitivity of the ELISA is generally considered to be higher than the sensitivity for culture. Because the ELISA detects antigen or antibody, it is not necessary for a sample to contain live pathogen. ELISA generally requires fewer overall organisms for detection than culture. Further research on the diagnostic value of the ELISA as a method to detect pathogens from exotic animals should be pursued.
Polymerase chain reaction, once considered only a research technique, is being used more frequently in the clinical diagnosis of disease and for epidemiological investigations. Polymerase chain reaction assay is an enzyme-mediated process used to replicate DNA from an organism with specific oligonucleotide primers that are complimentary to specific nucleotide sequences of the subject organism. Because the PCR technique can be used to create logarithmic copies of microbial DNA from a limited amount of sample, the technique can identify organisms in clinical or environmental samples at levels too low to detect with culture. Several techniques can be used to detect microbial DNA with PCR, including specific PCR, broad-range PCR, multiplexing PCR, nested PCR, and reverse-transcriptase PCR. The PCR assay amplifies DNA with a thermostable DNA polymerase in combination with a buffer, magnesium, deoxyribonucleoside triphosphates, and oligonucleotide primers. The primers anneal to complimentary regions on the coding and noncoding strand of DNA. The DNA polymerase attaches to DNA primer complexes and extends the DNA. The copy made in the first cycle serves as a template for further amplification. Multiple cycles at various temperatures are repeated and the process of disrupting the double-stranded DNA, annealing the primers to the DNA, and extending the DNA, produces a logarithmic increase in the template. There are a number of different techniques used to confirm the presence of target DNA, including gel electrophoresis, DNA sequencing, oligonucleotide probes, and restriction fragment length polymorphism.
The PCR assay is considered to be more sensitive than culture because it can amplify DNA from a single organism or part of an organism under suitable conditions. Specificity of the PCR assay can also be very high depending on the primers used. PCR assay may be prone to false positive reactions if processing of the samples is not performed under sterile conditions. To prevent amplification of contaminant DNA, processing should be conducted in separate pre-and post-PCR rooms. A number of biological inhibitors affect the results of a PCR assay, including blood, blood culture media, urine, sputum, and vitreous humor. Sample processing can also influence the PCR assay results, yielding false negatives if the DNA extraction technique does not release microbial DNA, or if the quantity of DNA available for the reaction is low.
Recent advances in exotic animal medicine have generated new diagnostic assays to assist the veterinarian with disease diagnosis. Veterinarians should become familiar with the advantages and disadvantages associated with specific diagnostic assays to improve their understanding of these testing methods. It is important that we remain vigilant and continue searching for new methods to detect pathogens that have not yet been characterized.