Rabies is a major zoonosis for which diagnostic techniques have been standardised inter-nationally. As there is no gross pathognomonic lesion for rabies, diagnosis can only be made in the laboratory. Laboratory techniques are preferably conducted on central nervous system (CNS) tissue removed from the cranium. A composite of CNS samples should be tested and the brain stem is the most important component of the sample.
Identification of the agent: Agent identification is preferably done using the fluorescent antibody test (FAT). A drop of purified immunoglobulin previously conjugated with fluorescein isothiocyanate is added to an acetone-fixed brain tissue smear, preferably made from several parts of the brain, including the hippocampus, cerebellum and medulla oblongata. For a large number of samples, as in an epidemiological survey, the immunoenzyme technique can provide rapid results (the rapid rabies enzyme immunodiagnosis [RREID]). FAT provides a reliable diagnosis in 98-100% of cases for all genotypes if a potent conjugate is used, while RREID detects only genotype 1 virus.
Infected neuronal cells have been demonstrated by histological tests and these procedures will reveal aggregates of viral material (the Negri bodies) in the cytoplasm of neurones. However, the sensitivity of histological techniques is much less than that of immunological methods, especially if there has been some autolysis of the specimen. Consequently, histological techniques can no longer be recommended.
As a single negative test on fresh material does not rule out the possibility of infection, inoculation tests, or other tests, should be carried out simultaneously. Newborn or 3-4-week-old mice are inoculated intracerebrally with a pool of several CNS tissues, including the brain stem, and then kept under observation for 28 days. For any mouse that dies between 5 and 28 days, the cause of death should be confirmed by FAT. Alternatively, a monolayer culture of susceptible cells is inoculated with the same material as used for mice. FAT carried out after appropriate incubation will demonstrate the presence or absence of viral antigen. Wherever possible, virus isolation in cell culture should replace mouse inoculation tests.
The identification of the agent can be supplemented in specialised laboratories by identifying any variant virus strains through the use of monoclonal antibodies, specific nucleic acid probes, or the polymerase chain reaction followed by DNA sequencing of genomic areas. Such techniques can distinguish between field and vaccine strains, and possibly identify the geographical origin of the field strains. These very sensitive tests should be used by well trained personnel in specialised laboratories.
Serological tests: Virus neutralisation (VN) assays in cell cultures are the prescribed tests for international trade. Alternatively, use may be made of a test that is known to correlate with these, notably an enzyme-linked immunosorbent assay using antibody to the G protein or the neutralisation test in mice. Results are expressed in International Units or equivalent units relative to an international standard antiserum.
Requirements for vaccines and diagnostic biologicals: Rabies vaccines for use in animals contain either live virus attenuated for the target species (such as Flury low egg passage, Flury high egg passage, Street-Alabama-Dufferin or Kelev), or virus inactivated by chemical or physical means, or recombinant vaccines. The virus is cultivated in the CNS tissue of newborn animals, in embryonated eggs, or in cell cultures.
Rabies vaccines are usually lyophilised, but inactivated virus vaccines, preferably with an adjuvant, may be stored in liquid form.
Before newly developed vaccines can be licensed, the duration of immunity resulting from their use should be determined in vaccinated animals of the target species.
For live virus vaccines, the minimum virus content that will elicit an adequate immune response must be established.
The potency of inactivated virus vaccines is established and controlled by mouse vaccination followed by intracerebral challenge using tests formulated by the United States Department of Agriculture in the United States of America or the European Pharmacopoeia elsewhere. The final products of both types of vaccine are subjected to tests for innocuity and absence of toxicity.
For live vaccines that are prepared for oral vaccination of wild (or domestic) animals, safety and efficacy in target animals and safety in nontarget species must be demonstrated.
A. INTRODUCTION
Rabies is caused by a neurotropic virus of the genus Lyssavirus of the family Rhabdoviridae, and is transmissible to all mammals. As it is transmissible to humans by inoculation or inhalation of infectious virus, all suspected infected material must be handled under the appropriate safety conditions specified by the World Health Organisation (WHO) (37).
Seven distinct genetic lineages can be distinguished within the genus Lyssavirus by cross-protect
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