used to convert the viral RNA to DNA prior to the PCR process. Real‐time PCR allows for the quantification of DNA through the use of fluorescent labeling of the DNA (or a DNA probe), which can be tracked and quantified as the PCR progresses, allowing for the determination of initial DNA quantity, less susceptibility to contamination, and greater sensitivity of detection. Standard PCR techniques analyze DNA bands at the completion of a variable number of amplification cycles and rely on size discrimination for identification, which can result in comparatively lower precision and sensitivity of detection (Applied Biosystems n.d.; Tizard 2013). Real‐time PCR can also be completed on RNA viruses through reverse transcriptase real‐time PCR.
For the detection of common causes of both respiratory and GI disease, diagnostic laboratories offer real‐time PCR analysis of a variety of pathogens common to either dogs or cats. Such services carry the benefit of screening for a variety of pathogens with a single sample (and single fee), fast turnaround time (one to three days), and ready identification of potential co‐infections. Pathogen detection may be inhibited in aged samples or those from patients receiving antimicrobial therapy and, in the case of respiratory PCR panels, recent modified live‐virus vaccination can interfere with the interpretation of results. Samples for respiratory PCR analysis should include a conjunctival and deep pharyngeal swab collected on a sterile, plastic‐stemmed swab and placed in a sterile tube. Samples obtained via trans‐tracheal wash or bronchoalveolar lavage may also be accepted and would be most appropriate to collect from animals with suspected lower airway disease. Samples for GI PCR analysis should include fresh feces (minimum 1 gram) placed in a sterile container. Both respiratory and GI samples can be refrigerated for up to 10 days (IDEXX Laboratories 2017).
4.3.4.2 Microbiological Tests
Microbiological culture is a commonly used test that, with the exception of dermatophytosis cultures, generally requires the use of diagnostic laboratory services (see Chapter 20 on Dermatophytosis for more information about the performance and interpretation of ringworm cultures). Almost any bodily fluid, excretion, or tissue can be submitted for culture to determine whether or not infectious organisms are present (most commonly bacterial or fungal agents). In contrast to molecular assays, a culture can identify live pathogens, the species of organism(s) present and, when antimicrobial sensitivity is evaluated, which pharmaceutical treatments are likely to be effective and at what dosages. Repeat cultures can be used to evaluate a patient's response to a treatment course and to determine when an infection has been resolved. Unless sampling from a contaminated area such as a wound or a sample of skin or hair (e.g. ringworm cultures), samples must be collected and handled using aseptic techniques in order to provide meaningful results. Fecal samples submitted for culture should include specific differential diagnoses to direct the microbiologist. Similarly, when fastidious microorganisms not easily cultured on standard media are suspected (e.g. Mycoplasma spp., anaerobes, spirochetes, Mycobacterium spp.), the laboratory should be consulted for recommendations on sample collection, preparation, storage, and transportation. Additionally, if the animal is receiving antimicrobial therapy at the time of sample collection, this must be taken into account for proper interpretation of results. Common indications for culturing include UTIs, non‐healing or contaminated wounds, and severe, chronic, or non‐responsive diseases of the respiratory, GI, and dermatologic systems.
Virus isolation is a specialized type of microbiological culture that relies on the need for viruses to replicate in living tissue. Most commonly, laboratory cell cultures are inoculated with test samples, incubated, and evaluated for structural changes due to viral infection (i.e. cytopathic effect). Once an active virus is cultivated, identification can be confirmed through the use of the serological methods described above. Common samples for virus isolation include tissue (belonging to viral target organs), anticoagulated whole blood, respiratory secretions, and urine. Samples should be kept in sterile containers, refrigerated, and arrive at the diagnostic laboratory within 24 hours. In addition to its utility for the identification of novel organisms and to monitor the microevolution of certain viruses (e.g. influenza), virus isolation is commonly used for confirmation of infection with adenoviruses, FeLV, and parvoviruses. Virus isolation of these pathogens may take between one and 4 weeks, limiting the usefulness of this modality in urgent clinical cases.
4.4 Indications for Diagnostic Testing
In the animal shelter setting, diagnostic testing can be employed on both an individual animal and population level basis. Before developing a testing strategy for either of these scenarios, the shelter practitioner should consider the costs and benefits of diagnostic testing as it relates to shelter operations, testing methodology, animal and human health, and the specific disease of interest (Box 4.2). Performing diagnostic tests is generally indicated when the disease in question is common in the shelter population, is likely to be acquired or transmitted in the shelter, presents a risk for spread into the community, requires immediate treatment and/or could be life‐threatening, or poses a zoonotic risk (Hurley and Pesavento 2013).
4.4.1 Individual Animal Testing
There are two high‐level indications for diagnostic testing of individual animals, including (i) screening for common diseases found in a population and (ii) diagnosis of existing disease states. Screening tests are those that are conducted on all members of a defined population for the detection of diseases that are commonly present but have not yet become clinical. Screening tests provide for early detection of disease, thus allowing for earlier management and, in most cases, an improved prognosis for the individual animal while also having potentially (widespread) implications for the health and well‐being of the larger population in the case of contagious diseases. For these reasons, they are a key component of a comprehensive preventive care strategy. Such testing is most useful for diseases that have a long preclinical phase and for which the benefits of early treatment outweigh the expense of conducting the test, the cost of disease progression in the individual (both in terms of money and welfare), and the cost of disease transmission within the shelter population. In most cases, if performed, screening should be conducted at the time of shelter intake to realize these benefits; however, screening at other times may be considered for diseases that do not pose immediate threats in order to improve animal flow through the shelter system and promote more efficient utilization of resources. Diseases for which screening tests are commonly conducted include canine heartworm disease, feline retroviruses, and dermatophytosis.
Box 4.2 Diagnostic Testing Considerations
Shelter Operations
Does testing fall within the shelter's operational mission?
Are there enough resources to devote to testing?
Will test results alter current or future operations?
Do the costs of testing impact other services?
Testing Methodology
What tests are available for the disease in question?
How does disease prevalence impact test accuracy?
Can samples be collected, handled, prepared, and stored appropriately?
Do staff have the time, knowledge, skill, and training to conduct point‐of‐care tests accurately?
Animal and Human Health
Will test results alter the management of animals?
Will test results impact human health?
Disease Characteristics
Is the disease common?
Is infection or transmission within the shelter or community likely?
Is