52       Laboratory diagnosis of viral disease

 

Many viral diseases of animals can be diagnosed on the basis of clinical signs together with postmortem findings and histopathological findings. However, confirmation of the involvement of specific viral pathogens often requires special laboratory procedures. Surveillance for particular viruses is an important aspect of the management of valuable animals such as bulls used for artificial insemination and stallions which have the potential to spread infection to many other animals. As part of international trade regulations, certification of freedom from particular viral diseases must accompany animals exported to countries in which the diseases are exotic. Moreover, rapid and accurate laboratory confirmation of exotic viral diseases, including those with zoonotic potential, is essential for the successful implementation of eradication policies and for the protection of human health. Surveillance of animal populations for new or emerging viral diseases is an important responsibility of national veterinary services.

More than 200 major viral diseases of veterinary importance affect animal species. Because of the considerable resources required for the provision of comprehensive diagnostic services in virology, national diagnostic services usually concentrate on those diseases prevalent in a country. Moreover, laboratories often provide diagnostic services for particular animal species. Special laboratory containment facilities are mandatory for some viruses which cause highly contagious diseases such as foot-and-mouth disease. The Office International des Épizooties (OIE) in Paris, also known as the World Organization for Animal Health, monitors and publishes details of significant animal disease outbreaks worldwide. This surveillance work relies on international cooperation and a network of laboratories dealing with viral diseases of international importance.

Collection, preservation and transportation of samples

Failure to provide a suitably selected specimen for the diagnostic laboratory is the single most important cause of unreliable laboratory results. Ideally, specimens for laboratory examination should be collected as early as possible from affected animals before secondary bacterial infections become established. It is advisable to collect samples from apparently normal in-contact animals because some of these animals may be actively shedding virus. The specimens selected for examination should relate to the clinical signs or to lesion distribution at postmortem.

Preservation of the infectivity or antigenicity of viruses may be required for particular tests. As many viruses are labile, specimens for virus isolation should be collected into transport medium, refrigerated and transmitted to the laboratory without delay. Samples should be frozen at –70°C if delay in delivery is anticipated. Freezing in a domestic freezer at –20°C significantly decreases the infectivity of most viruses. Transport medium consists of buffered isotonic saline containing a high concentration of protein, such as bovine albumin or foetal calf serum, which prolongs virus survival. Antibiotics and antifungal drugs are added in order to inhibit growth of contaminants. Samples for electron microscopy, in which the demonstration of virion morphology is the primary objective, require less exacting conditions for storage and transportation. Air-dried smears for fluorescent antibody (FA) staining should be fixed in either acetone or methyl alcohol for up to 10 minutes in order to preserve viral antigens. This fixation process allows penetration of FA conjugates into cells. A similar fixation procedure is required for cryostat sections of frozen tissues prior to FA staining. Formalin-fixed tissue samples embedded in paraffin wax can be stored for many years and used to demonstrate the presence of viral antigen by immunohistochemical techniques.

Guidance from clinicians regarding the possible aetiology of the disease under investigation is essential for deriving maximum benefit from laboratory tests. This requires an accurate assessment of the history and clinical signs, together with a tentative clinical diagnosis. In some instances, postmortem and histopathological examination of tissues may be sufficient for diagnostic purposes, particularly if characteristic inclusion bodies are found in infected tissues.

Detection of virus, viral antigens or nucleic acid

The presence of virus in tissues can be confirmed by isolation of live virus, by demonstration of virus particles or viral antigen and by detecting viral nucleic acid. Virus isolation using cell culture, fertile eggs or experimental animals is the standard against which other diagnostic methods are usually compared. Diagnostic laboratories usually have a limited range of cell lines, appropriate for the range of samples received. Embryonated eggs are widely used for the isolation of influenza A virus and avian viruses. Because of ethical considerations and cost, virus isolation in experimental animals is now employed infrequently.

Virus isolation is a sensitive procedure when cultural conditions are optimal for a particular virus and this method also generates a supply of virus for further studies. However, it is labour-intensive, slow and expensive. A number of blind passages may be required before a virus becomes adapted to a particular cell line and, as a consequence, a test result may not be available for some weeks. Because some viruses do not produce a cytopathic effect, additional detection procedures such as haemadsorption and FA staining may be needed to demonstrate their presence in cell cultures. Even when a virus produces a pronounced cytopathic effect, additional tests are often required for definitive identification.

The sensitivity and versatility of methods for the detection of viral nucleic acids have greatly improved in recent years and these procedures are now becoming the methods of choice for viral identification. These methods are particularly valuable when dealing with viruses which are either difficult to grow or cannot be grown in vitro. They are useful for detecting latent infections in which infectious virus is absent and also for specimens containing inactivated virus. Cloned viral DNA is available for the probing of samples and tissues by nucleic acid hybridization. This technique, however, has been largely replaced in recent years by PCR, which has the advantage of amplifying the target gene sequences. Application of this technique has been extended for detection of RNA viruses through the use of reverse transcriptase. Because of their exquisite sensitivity, PCR techniques require rigorous standardization to exclude cross-contamination and to ensure reproducibility and reliability.

Diagnostic serology

Serological procedures can be used for the retrospective diagnosis of viral diseases and for epidemiological surveys. These procedures can be automated and diagnostic reagents for many viral pathogens are available commercially. Single blood samples from animals in susceptible populations suffice for establishing the prevalence of a disease. When using serological procedures for the diagnosis of endemic disease in flocks or herds, paired serum samples taken at an interval of at least three weeks are required to demonstrate rising antibody titres. The first samples should be collected when clinical signs are first evident and the second samples during convalescence. A single blood sample may be adequate for diagnosis if reagents are available for demonstrating IgM antibodies, which are indicative of a primary immune response. Difficulties with the interpretation of serological tests may arise due to cross-reactions with antigenically related viruses. In young animals, passively acquired maternal antibodies, which may persist for several months, can lead to difficulty in interpreting results.

Interpretation of test results

Because false-positive and false-negative results can occur in many test procedures, inclusion of positive and negative controls is essential. The sensitivity and specificity of a particular diagnostic test should be established. The sensitivity of a diagnostic test, expressed as a percentage, is the number of animals identified as positive out of the total number of animals with the disease. The specificity of a test is the percentage of uninfected animals in which the result is negative. In order to detect all animals with an important viral infection, a test with high sensitivity is required. For laboratory confirmation of a viral infection in an individual animal, a test with high specificity is essential.

The isolation of virus or the demonstration of antibody to a specific virus does not necessarily confirm an aetiological link with a disease state. For the conclusive confirmation of test results, it may be necessary to demonstrate a correlation between the site of virus recovery and the nature and extent of lesions. Circumstantial evidence for the aetiological involvement of a virus in a clinically affected animal is supported by the recovery of the same virus from susceptible in-contact animals. Moreover, a rising antibody titre to the putative causal virus is of diagnostic importance. Published reports on the potential importance of a similar disease syndrome and its aetiology may point to the suitability of particular laboratory investigations for that particular viral infection.