The laboratory confirmation of rinderpest can be achived by several methods which detect live virus, virus antigens, virus genetic material or antibodies directed against the virus. Characterisation of rinderpest viruses relies primarily on isolating live virus and/or examining viral RNA. Maximising the chances of a successful result and information gained requires different samples. Paired samples should be submitted, one chilled sample, in transport medium if indicated, for virus isolation or further pathogenesis studies, and another fixed in 10 per cent buffered formol saline. The latter will survive extremes of temperature and still allow molecular biological studies and strain differentiation. Every effort must be made to obtain both fresh and formalin fixed material.
The key to diagnostic success is the examination of as many samples as possible from several sick animals. The crucial factor is the selection of suitable donor animals.
Virus is first shed in the excretions and secretions of the infected Animals towards the end of the incubation period, before the onset of illness. It continues to be shed thoughout the prodromal fever, the mucosal erosion phase and the diarrhoeic phase. Shedding of virus stops in early convalescence, a few days after the fever has regressed. The infectious period, therefore, lasts at most from 10 to 16 days. The optimal period for collecting suitable diagnostic specimens, however, is much shorter because viral titres peak before or at the onset of fever and antigens peak early in the mucosal erosion phase. Dead animals are poor donors of diagnostic samples and are best avoided unless no other source of samples is available. Similary, animals late in the course of didease, distressed by mocopurulent nasal and ocular discharges, and soiled animals voiding fetid, fluid faeces are less likely to yield positive diagnostic samples. The most suitable live donors of samples for virus isolation or antigen detection are febrile, have mucosal erosions and clear lachrymal secretions.
The first rinderpest-specific humoral antibodies belong to the IgM class of immunoglobulins and emerge toward the end of the mucosal erosion phase or in the early diarrhoeic phase of the disease. A few days later, rinderpest-specific antibodies are also found in the IgA and IgG classes of animals which have been affected for the last 10 days.
The specimens required for diagnosis from the selected live animals are biopsy samples of peripheral lymph nodes, gum debris, tears, and clotted blood. All tissue samples or gum debris should be kept chilled and transport medium (phosphate buffered salin pH 7,6 (PBS) containing antibiotics and fingizone - BUT NO GLYCEROL) may be added to help preserve the specimens. For virus isolation samples should be transported as quickly as possible chilled, not frozen. If samples are to be stored for long periods, they should be frozen at -70° Cnot -20° C.
Sample as many animals and as many tissues as possible, to increase the chance of virus isolation and detection.
A peripheral lymph node should be located and grasped firmly through the skin. A wide-bore needle (18 gauge) with its stilette in place should be thrust into the parenchyma of the node. Remove the stilette and attach a 10 or 20 ml syringe, pre-wetted with a drop of Heparin Injection BP. Aspirate a plug of tissue into the syringe and eject into a suitable container containing 0,5 ml of transport medium.
Collect the necrotic debris coating eroded gums on a spatula or finger rubbed across the gums and inside the lower and upper lips. Scrape off into a convenient container.
Collect tears onto cotton buds swabs inserted into and twirled around the conjuctival sac behind the eyelids. Break off the bud or swab into a suitable container (glass or plastic universal bottle) and add 150 µl of PBS (Appendix 2).
Collect blood for serum into plain, sterile containers, preferably silicone-coated, and allowed to clot undisturbed for a period of at least 24 hours. Separate serum by centrifugation, transfer to suitable clean, sealed containers and store at 4° C or below.
Select the least two animals for slaughter and proceed as follows:
Where possible, all dead animals should be necropsied. Fresh, clean carcasses of animals that have died early in the course of the disease are worth sampling by collecting aliquots of spleen, lymph nodes and tonsils for antigen-detection tests. Slices of these tissues together with portions of effected mucosae should also be fixed in formol saline for histopathological examination. Animals that have died late in the course of the disease are soiled and fetid and are usually emaciated and dehydrated. They should be examined to supplement the clinical findings but their tissues should only be collected for histopathology. Decomposed animals are not worth examining.
Sample bottles must be labelled clearly in waterproof ink on good quality adhesive tape. Thise should be done at the time of collection, together with the writing of comprehensive field notes (see Appendix 4). Tissues placed in fixative require no further care other than ensuring that screw-caps are tightly in place and that gross overheating does not occur.
Serum samples can be stored indefinitely in clean crew-capped bottles at -20 ° C or, if a freezer is not available, for several weeks at 4 ° C.
All the samples should remain chilled on ice until they are returned to the laboratory. Transoport medium is not strictly necessary for trasporting solid tissues, as these contain sufficient protein to protect the virus and are not amenable to buffering. However, a small amount of transport medium (PBS with high levels of antibiotics and fungistat may be added to combat surface contamination and dehydration. Glycerol should not be included because it kills rinderpest virus.
