Choice of Method

Some microorganisms are easily cultivated from easily accessible sites of the animal, while others can only be cultivated if a range of specific conditions are fulfilled. A few rodent organisms, e.g., Spirillum minus, the cause of the Japanese variant of rat bite fever, sodoku, cannot even be grown in vitro. Therefore, a health program necessarily consists of different types of assays.

Enteric helminths and protozoans may be diagnosed by direct microscopy after flotation test, on smears from the caecum and ileum, or on tapes used for sampling around the anus. Inspection under a stereomicroscope may reveal ectoparasites.

A range of organs should be inspected macroscopically to decide whether further investigation by histopathology is needed, while histopathological examination is seldom the method of choice for current screening and is more efficient as a tool for a final diagnosis and validation of the impacts of a certain microorganism.

Cultivation is the diagnostic method of choice for microorganisms readily grown on artificial media, e.g., bacteria and fungi. Samples for bacteriological cultivation are typically taken from organs such as the nose, trachea, genitals, liver, and cecum inoculating on selective and nonselective media. Further procedures are then designed to grow pure cultures and to identify exactly those organisms searched for.5

For microorganisms not easily cultivated serology, such as immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and western immunoblotting is widely used, although the latter is rather laborious and, therefore, not recommended for routine use in the FELASA guidelines.48 Serology is applied for all viruses, but lactate dehydrogenate (LDH) elevating virus, which is simply monitored by testing for LDH activity. Also a few bacteriae, such as Clostridium piliforme,26 and protozoans, such as Encephalitozoon cuniculi175 are screened by serological methods. Some organisms like Mycoplasma spp. may be diagnosed by both serological and cultural methods.238 Parvoviruses are typically diagnosed by a combination of both solid-phase assays, such as ELISA or IFA, to detect common parvoviral antigens and hemagglutination inhibition assays to differentiate between the different types.250 Serolog-ical results are historical and do not give any evidence whether the animal actually harbors that particular microorganism. The main pitfalls are the lack of sensitivity due to a pure antibody response to the infection or a low specificity due to cross-reactions, which mostly occurs with bacteria, as these contain more than 2000 antibody-inducing proteins, while viruses are far more simple.

Molecular biological techniques represent another attractive choice for noncultivable microorganisms. These methods are generally divided into two: those based upon the DNA (occasionally, RNA) already being present in detectable amounts, e.g., in situ hybridization and southern blotting, and those in which the DNA or RNA must be amplified before detection, e.g., the polymerase chain reaction (PCR). To be able to apply molecular biological methods for detection or identification of an infectious agent one or several specific sequences from that organism must be known. Helicobacter hepaticus is an example of an infection for which PCR may be the method of choice.251 Figure 11.13 shows a typical range of methods applied for health monitoring of rodents.

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