CVMA CONVENTION 1985

A selection ofpapers presented at the 1985Canadian Veterinary Medical Association Convention

in Penticton, British Columbia

Recent Advances in Rabies Diagnosis and Research

K.M. CHARLTON, W.A. WEBSTER, G.A. CASEY, A.J. RHODES, C.D. MACINNESAND K.F. LAWSON

In Canada, rabies is a reportabledisease under the Federal govern-ment's Animal Diseases and Protec-tion Act. Administration of this actby Agriculture Canada has led tofairly uniform procedures for fieldinvestigations, laboratory diagnosis,reporting of laboratory findings and,to a certain extent, control of thisdisease in domestic animals. Althoughthere are occasional exceptions, themain steps that occur in suspectedcases of rabies are as follows: thepracticing veterinarian or ownernotifies a District Veterinarian ofAgriculture Canada's VeterinaryInspection Directorate; the DistrictVeterinarian investigates the case and,when appropriate, submits specimensto a laboratory of the AnimalPathology Division. Diagnostic testsare done and results are reported tothe District Veterinarian who, in turn,notifies the owner, the Medical Officerof Health and others involved in thecase. The district Veterinarian alsoimposes quarantines, authorizespayment of compensation, arrangesfor vaccination clinics when war-ranted, and generally keeps the publicinformed about rabies in the area.

Various provincial and municipalagencies are involved in, and affectedby, the above activities. These agenciesalso administer provincial and munic-ipal laws concerning rabies, especiallyquarantine of animals that have bittenhumans and control of rabies inwildlife. Some provincial govern-ments, namely those of Ontario andAlberta, have extensive researchprograms on the control of rabies inwildlife.

LABORATORY DIAGNOSISOF RABIES

The primary test used for thediagnosis of rabies in most industrial-ized countries is the rabies fluorescentantibody (RFA) test(l) on braintissue. This test can be completed inapproximately 2 h and is highlyaccurate when done routinely byexperienced personnel. It is used onall suitable rabies-suspect specimenssubmitted to any of the Canadianrabies diagnostic laboratories (cur-rently the Animal Diseases ResearchInstitute, LETHBRIDGE, Albertaand the Animal Diseases ResearchInstitute, NEPEAN, Ontario). Asecondary or back-up test, the mouse

inoculation (MI) test (2), is used onhuman contact specimens that arenegative on the RFA test. Observationof mice for 30 days is required toconfirm a negative RFA test. Cur-rently less than 0.1% of RFA-negativespecimens are positive on mouseinoculation(3). This comparesfavourably with 7-15% false negativeswhen the primary test consisted of aNegri body strain on brain smears(established from the records of theAnimal Diseases Research Institute,Nepean, 1955-1964). Although theRFA test is highly accurate, the longdelay required for completion of theMI test causes considerable anxietyfor exposed persons and healthofficials.

Replacement of the MI test by viralisolation in tissue culture may soonbe feasible. Although early attemptsto isolate street virus in tissue culturegave inconsistent results, recentstudies using improved isolationtechniques suggest that this methodis at least equal to and perhaps hasgreater sensitivity than the MI test.Following further studies, we hope torecommend that the MI test bereplaced by tissue culture isolation

Can Vet J 1986; 27: 85-96.

Agriculture Canada, Animal Diseases Research Institute, NEPEAN, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9 (Charlton, Webster andCasey), The Ontario Ministry of Natural Resources, P.O. Box 50, Maple, Ontario LOJ 1 EO (Rhodes and MacInnes) and Connaught Laboratories Ltd.,1755 Steeles Avenue West, Willowdale, Ontario (Lawson).

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within one year. This will have theimportant advantages of shorteningthe observation period to three or fourdays and eliminating the need forroutine inoculation of about 30,000mice annually for rabies diagnosis inCanadian laboratories.

In addition to the above routinetests, human skin biopsies, cornealsmears or other samples are occasion-ally examined by the RFA technique.In cases where paraffin-embeddedtissues are the only specimens avail-able, we can still use the RFA test aftersections have been treated with trypsin(4). However it is not as sensitive asthe RFA test on fresh tissues and,therefore, should not be used forroutine diagnosis.

RABIES MONOCLONALANTI BODI ES

Since the initial production ofrabies monoclonal antibodies byWiktor and Koprowski (5), additionalpanels (of antibodies) have beenproduced in the United States (6),Germany (7), France (8) and Canada(personal communication, R.B. Ste-wart, Queen's University, Kingston,Ontario). These antibodies may beproduced in tissue culture or mouseascitic fluids and can be used in manycommon laboratory tests devised forpolyclonal antibodies.

Prior to the development of monoc-lonal antibodies most rabies streetvirus isolates were considered to beantigenically homogeneous. However,testing with monoclonal antibodies(Supplied by Dr. T. Wiktor, WistarInstitute, Philadelphia and J. Smith,CDC, Atlanta, Georgia) revealedmany different antigenic profiles ofstreet viruses and vaccine strains."Typing" street virus isolates isproving to be valuable in epizootiolog-ical studies since in some cases theprogression of particular strain(s) ofvirus can be traced through animalpopulations and geographic regions.Vaccine-induced rabies can be diag-nosed by testing virus from a sus-pected case with monoclonal antibo-dies. In a field trial of an oral vaccinefor wildlife in Ontario, we aremonitoring virus isolates from anim-als in the target area.Canadian street virus isolates have

been examined with a panel ofantinucleocapsid antibodies of ADRI,

NEPEAN (9). Further studies haverevealed four antigenic groups interrestrial mammals: one major groupin eastern Canada (infecting all speciesof terrestrial mammals and which is asouthward extension of the rabiesfound in the Canadian Arctic); asecond smaller group in a limitedgeographical area in southern Onta-rio; a major group in the southernportions of Manitoba, Saskatchewanand Alberta (represents the northernextension of "skunk rabies" from themid-central U.S.A.); and a fourth verysmall antigenic group found in skunksin the Brooks, Alberta area. Fourdifferent major antigenic groups arefound in bats in Canada and these arebased both on host species and geo-graphical areas.

Other uses of monoclonal antibo-dies include selection of rabies virusmutants, and studies of the pathogene-sis including virulence factors andimmunogenicity related to specificantigenic determinants. Some of themutants selected in this manner areavirulent and are being considered ascandidate vaccines for vaccination ofwildlife (10). Mutants may be selectednot only in vitro but in vivo suggestinga mechanism of antigenic variation innature (11). The role of monoclonalantibodies in studies of the pathogene-sis will be discussed later.

RESEARCH ON RABIESCONTROL IN WILDLIFE

In Canada, the United States andEurope, the main source of rabies indomestic animals continues to becertain wildlife species that supportenzootic rabies. Although vaccinationand other control methods for domes-tic animals reduces economic lossesand the number of human exposures,substantial control or eradicationeventually must depend on what we doabout rabies in wildlife. During thepast 20 years, research on control ofwildlife rabies has centered ondevelopment of oral rabies vaccines(12,13,14). Countries that have beenespecially active in this field areCanada, the United States, Switzer-land, West Germany and France.Switzerland, West Germany andCanada currently have field trials of alive vaccine in progress. The vaccineappears to be successful in areas wherethe fox is the target species (14).

In Canada, research on oral vac-cines for wildlife has been fundedalmost entirely by the government ofOntario. During the past 17 years, aresearch group of the Ontario Minis-try of Natural Resources has workedon development of baits and baitdistribution, and the ecology ofwildlife vectors. From 1968 to 1973 theOntario Ministry of Health fundedresearch at Connaught Laboratorieson oral rabies vaccines. Recently theOntario Ministry of Natural Resour-ces has funded an intensified researchprogram on oral rabies vaccines. TheRabies Advisory Committee (RAC)was established in 1979 by an Order-in-Council of the government ofOntario. Since then, the committeehas been directing research to developa suitable rabies vaccine, and aneffective delivery system for vaccinat-ing wild animal populations. Grantswere awarded to scientists at severalinstitutions to conduct research onvarious facets of oral vaccine develop-ment. The institutions and the mainactivities were as follows: Queen'sUniversity (computer modelling,production of a pathogenic strains ofrabies virus and monoclonal antibo-dies); University of Toronto (antibodytesting, preparation and testing ofvaccine-containing capsules,genetically- engineered vaccines,testing of adjuvants); University ofGuelph (immune response in foxes);University of Saskatchewan (testingvaccines and immune response inskunks); Connaught Laboratories(development and production ofvaccines and baits, experimentationwith adjuvants, potency and safetytesting); Ontario Ministry of NaturalResources (computer modelling,wildlife ecology and development andtesting of baits); Agriculture Canada(challenge trials, testing experimentalvaccines in skunks and foxes).

Both live and inactivated vaccineswere tested in foxes and skunks.Although researchers made someprogress with inactivated vaccinesgiven directly into the intestine,generally the proportion of vaccinatedanimals that became immune was toosmall to warrant use in the field (15).This method of vaccination poses theadditional challenge of developing aneffective method of encapsulation toallow the vaccine to pass through the

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stomach with no loss in immunogenic-ity. In foxes, some live vaccinesincluding commercial ERA® wereeffective when given for absorptionthrough mucous membranes of themouth or intestine (15,16,17). To date,similar standard live rabies vaccineshave not been consistently successfulin skunks. On the other hand, resultsof preliminary studies at the AnimalDiseases Research Institute,NEPEAN, of a vaccinia virus recom-binant containing the rabies virusglycoprotein gene (18) are encourag-ing. Moderate to high titers of serumneutralizing antibodies were produ-cedin skunks given the recombinant inbaits, by endoscope, by intramuscularinoculation and by scarification.Work continues to develop a safevaccine that is efficacious in bothfoxes and skunks.

In other areas of the program,major achievements have been madein developing a bait that is acceptableto a high proportion of foxes andskunks, a bait distribution system (19)and a computer model of rabies inwildlife (20,21,22). The computermodel will be useful in predictingoutbreaks of rabies, in estimatinglevels of immunity to control rabies,and in evaluating the results of fieldtrials.

In summary, the progress beingmade in development of vaccines thatare immunogenic by the oral route andthe marked improvements in auxiliarysystems are encouraging signs thatvaccination of wildlife in their naturalhabitats will eventually be successful.A field trial is in progress in HuronCounty Ontario. This trial is intendedto test several features concerning thebait, the bait distribution system,acceptance of bait by wild animals,and the efficacy of ERA® in wild free-ranging foxes.

P A T H O G E N E S I S

Our knowledge of the pathogenesisof rabies comes mainly from workdone since the late 19th century. Thefollowing is a brief description of someof the important features of thisprocess. Early studies established theneurotropism of the agent and theinfectious nature of saliva of rabidanimals (23,24). Subsequent studiesestablished the following general stepsin the movement of virus through the

animal body: 1) introduction of virusinto a bite wound or laceration;2) migration via peripheral nerves tothe central nervous system (CNS);3) spread through the CNS;4) centrifugal neural transport ofvirus; and 5) infection of nonnervoustissues (25,26,27).One of the concerns with the

inoculation site has been to determinewhether virus enters peripheral nervesdirectly without preliminary replica-tion in nonnervous tissue, or indirectlyafter replication in nonnervous tissue.In some experiments, nerve resectionor limb amputation proximal to theinoculation site was lifesaving for onlya short period after inoculation(28,29), indicating that virus couldenter peripheral nerves directlywithout preliminary replication innonnervous tissue. Similarly, studiesusing a street virus with a longincubation period suggested that, insome cases, virus could be retained forprolonged periods at the inoculationsite (30). These latter findings. werecompatible with a period of replica-tion in nonnervous tissues. Immuno-fluorescence and electron microscopicstudies demonstrated that musclefibers at the inoculation site inhamsters and skunks could be infecteddirectly by rabies virus in the inoculum(31,32) and, thus muscle could be a sitefor preneural replication of virus. Therelative importance of these twomechanisms (direct and indirect entryinto peripheral nerve) has not beenestablished. The neural route to theCNS was demonstrated by experi-ments using nerve resection and fromevidence that antigen occurred in theCNS first at sites connected by nervesto areas of inoculation of virus(25,28,29). Other evidence indicatedthat this centripetal movement inperipheral nerves was in axons viaretrograde axoplasmic flow (33,34).Electron microscopical studies dem-onstrated that viral replication in theCNS occurred almost entirely inneurons (35) and that cell to celltransfer of virus (transneuronaldendroaxonal transfer of virus)occurred by a process of budding onperikaryal and dendritic plasmamembranes with simultaneous viro-pexis by adjacent axon terminals(32,36).

Following dispersal from the CNS,

virus replicates in some nonnervoustissues. In the salivary glands, replica-tion of virus in epithelial cells wasaccompanied by abundant budding onapical plasma membranes - thusaccounting for release of virions intoglandular ducts and saliva (37). Innaturally infected skunks, severalsalivary glands and nasal glandssupport growth of rabies virus (38).The mechanism of infection of sali-vary gland epithelial cells involvesprimarily neural-epithelial celltransfer of virus rather than cell to celltransfer among epithelial cells (39).There is evidence that in skunks, viraltiters in the submandibular salivarygland may be markedly influenced bythe immune response (40,41). Unpub-lished studies in our laboratory sug-gest that the immune responseimpeded neural- epithelial cell transferof virus.

Recent studies have focussed on theroles of cellular receptors and viralantigenic determinants in the patho-genesis of the disease. It has beensuggested that acetylcholine receptorsare important in the initial neuraluptake of rabies virus at the inocula-tion site (42,43,44,45,46,47). Othershave demonstrated that acetylcholinereceptors are not essential for infec-tion of rat myocytes and other cells inculture (48). Tsiang (49) suggests thatthe cellular receptor for rabies virus isnot a unique specific molecule but ismore likely a complex structureinvolving many cellular components.Monoclonal antibodies are proving

useful in studies of the pathogenesisand, possibly, in the development ofvaccines. Antiglycoprotein monoclo-nal antibodies were used to selectmutants of challenge virus standard(CVS) virus that no longer reactedwith the antibodies used for selection.Some of these mutants were apatho-genic when given to mice by theintracerebral route (50,51,52). Theloss of antigenic sites on the glycopro-tein molecule and associated virulencehas been traced further to substitu-tions of specific amino acids (53,54).As stated previously, tests are under-way to determine the efficacy of suchmutants when used as vaccines (10).The above findings complement thoseof Wunner and coworkers (55) whohave identified regions on the glyco-protein molecule that are responsible

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for different types of immuneresponses. These areas of investigationshould eventually lead to much greaterprecision in the design of vaccines,increased understanding of theimmune response to rabies and,possibly, to improved treatment ofclinical cases.

Recently we reported spongiformlesions in brains of infected animals(56). These lesions were detected firstin experimental rabies in skunks andfoxes, and later in naturally occurringrabies in the following species: fox,skunk, cow, horse and sheep.Vacuoles, 1-60 ,um occur in theneuropil of the grey matter, only rarelyin neuronal perikarya. This "spongi-form change", as defined by Mastersand Richardson (57), is a cardinalfeature of the traditional subacutespongiform encephalopathies: scra-pie, transmissible mink encephalo-pathy, kuru, Creutzfeldt- Jakobdisease, and wasting disease of muledeer. The lesions in rabies areconsidered to be etiologically distinctfrom the subacute spongiform ence-phalopathies, since the incubationperiod in rabies is much shorter andthe lesions occur only in rabies-posi-tive animals. They were not detected incontrol skunks or foxes or in animalsinoculated with rabies virus that failedto develop clinical signs. Also, inpreliminary studies, we have notdetected spongiform change in mice orrats experimentally infected with CVSrabies virus or street virus. Theexperimental disease in foxes andskunks may be useful to study thecomparative pathogenesis of spongi-form change since the rabies virus isfairly well characterized and thelesions occur in a high proportion ofanimals that have rabies encephalitis.

C O N C L U S IO N

Many of the recent advances inrabies diagnosis and research havebeen made in the fields of advancedbiotechnology, especially geneticengineering, biochemistry, immuno-cytochemistry, and monoclonal antib-ody production and testing. In thefuture it may be possible, through acombination of mutant selection andgenetic engineering, to design vaccinesthat are totally apathogenic and thatmeet requirements for specific types ofimmune responses and selected routes

of administration. Studies of thepathogenesis are likely to lead todetermination of the cellular recep-tor(s) for rabies virus, the nature of thelong incubation periods, the mecha-nisms of salivary gland infection, andfactors that are necessary for recoveryfrom CNS infection. All this shouldcontribute to the development ofbetter vaccines, postexposure treat-ment and even treatment of clinicalcases of rabies. Such achievements willbe attainable mainly through ablending of several fields of advancedbiotechnology with classical methodsof investigation. Many facets of rabieswork such as field investigations,border control, laboratory diagnosisand basic and applied research stillrequire use of proven procedures andwell established technology. Thisrequirement for the "old" methods islikely to continue, so that in manyinstances adoption of the products ofgenetic engineering and other techno-logical advances will require expan-sion rather than a change in work fromone technological field to another.

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