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Evaluation of the safety of two attenuated oral rabies vaccines, SAG1 and SAG2, in six Arctic mammals
Vaccine, Vol. 14, No. 4, pp. 270-273, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/96 $15+0.00
Elsevier 0264-410X(95)00208-1
ELSEVIER
Evaluation of the safety of two attenuated oral rabies vaccines, SAG1 and SAG2, in six Arctic mammals Erich H. Follmann*l,
Donald
G. Ritter-i_ and George
M. Baer$g
The safety of two attenuated oral rabies vaccines was evaluated in mink and inJive species of rodents which occur in the Arctic. A 0.03 mI sample of liquid vaccine was installed directly into the mouth of voles and lemmings and 0.1 ml into the mouth of Arctic ground squirrels and mink. Animals were euthanized at 36 and 46 days postexposure; brain tissue was analyzed by FAT and serum by RFFIT. No rabies deaths occurred in 47 animals tested. Four animals representing three rodent species seroconverted, the highest titer being 0.5 IU ml ~ t. The absence of rabies virus in brain tissue indicates the safety of these vaccines in these species. The replacement of arginine Mlith glutamic acid at position 333 reduces the pathogenicity of these vaccines, thereby presumably preventing the deleterious efhect of viral entry into CNS neurons. Copyright (6 1996 Elsevier Science Ltd. Keywords:
Rabies:
SAGl:
SAG2
oral vaccination:
pathogenicity:
rodents;
The effectiveness of oral vaccines in rabies control has been well demonstrated in both laboratory and field studies. Wild red fox (Vulpes vulpes) populations in Europe and Canada have been successfully immunized in the field resulting in reduction and eradication of fox rabies in some areas of Europe’-‘. An important consideration in field applications is not only the effectiveness of the vaccine in vaccinating the target population but its safety for any animal that may ingest the vaccine bait. Such concerns have led to extensive safety testing of several oral vaccines in numerous rodent, insectivore, carnivore, artiodactyl and nonas new oral human primate specie?’ ’ and continue vaccines are developed. Rabies commonly occurs in Nearctic wildlife populations with the Arctic fox (Alopex lugopus) as the main vector” 15. In Alaska both Arctic and red foxes harbor the disease, and epizootics occur every 334 years”. Human cases of rabies are infrequent in the ArcticI yet public health concerns exist. In Alaska human exposure to rabies typically is not from wild animals but from domesticated animals, mainly the dog, who have had *Institute of Arctic Biology,
University of Alaska Fairbanks, Fairbanks, AK 99775-7000, USA. $State Public Health Laboratory-Fairbanks, Alaska Division of Public Health, Fairbanks, AK 99775, USA. $Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Social Services, Building 15, 1600 Clifton Road, Atlanta, GA 30333, USA. §Current address: Laboratorios Baer, S.A. de C.V., Cuautla 150, Colonia Condesa, Mexico D.F. C.P.06140, Mexico. To whom correspondence should be addressed. (Received 29 March 1995; revised 18 September 1995; accepted 18 September 1995)
270
Vaccine
1996 Volume
14 Number
4
mink
contact with foxes or other wild animals. In the past 20 years over 80% of the animals submitted for rabies testing in Alaska have been foxes and 13% domestic animals. The mean annual percentage of rabid animals over this period has been about 30%. This high incidence and fear of the disease have led Alaska and some Arctic nations with similar roblems to consider rabies control quarantine” of through vaccination P* and long-term domestic animals. Although rabies in the circumpolar Arctic has been well known for many years12, the possibility of oral vaccination of wild carnivores in these harsh and remote areas has only recently been considered”. The effectiveness of oral vaccination of red foxes is well established in Europe and Canada, and it is assumed that red foxes in the Arctic can be similarly vaccinated. Laboratory experiments have shown that the more important rabies vector in the Arctic, the Arctic fox, can also be vaccinated with SAD-BHK,, and SAG1 oral vaccines2’.“. The safety of these vaccines in nontarget, Arctic, mammalian species has, however, not been established. The purpose of the present study was to determine the inocuity of SAG1 and SAG2 oral rabies vaccines in Arctic mammalian species that occur sympatrically with Arctic and red foxes. The SAG1 vaccine is a variant derived from SAD,,,,“. It differs from SAD,,,,, its parent strain, by one mutation in the codon (at position 333)‘3 of the antigenic site III of the glycoprotein. The resulting amino acid exchange is responsible for the attenuation of this vaccine strain. Because there is only one nucleotide exchange at this site, the vaccine can theoretically revert to its parental strain with only one mutation, which could occur from passage through an animal. This occurred after first” and second” passages through suckling mice; only one of several test mice
Evaluation of the safety of SAGf and SAG2 H-l. Follmann et al. exhibited this in each of these studies. Reversion to the parental strain could be a potential problem in a field situation because SADa,,, has induced rabies in certain skunks (Mephitis mephitis)‘” and species of rodents’,“, baboons (Papio ursinu.~)‘~.SAG1 has been shown to be safe for six wild species of murid rodents administered liquid vaccine by the oral, nasal and ocular routes]‘. SAG2 is characterized by two mutations in the codon at position 333 yielding glutamic acid at this position instead of serine as in SAG1 and arginine as in SADBern22,23. The probability of having two mutations occur in this codon as a result of animal passage is far less likely, and SAG2 is thus considered more genetically stable than its two ancestors, and is avirulent for adult immunocompetent mice as a result of replacement of arginine at position 333. SAG2 also has been demonstrated to be safe in a broad range of species” (C. Schumacher, personal communication); our study reports on its safety in six additional mammalian species found in the Nearctic as well as in certain regions of the Palearctic.
MATERIALS
AND METHODS
Five species of Arctic rodents representing four genera vison) were tested. Singing and mink (Mustela voles (Microtus miuris) were captured in box traps north of the Brooks Range, Alaska; collared lemmings (Dicrostonyx groenlandicus) originated in the same area but the animals used were bred in captivity. Tundra voles (Microtus oeconomus) and red-backed voles (Clethrionom_ys rutilis) were captured in box traps in and Arctic ground squirrels the Fairbanks area, (Spermophilus part-vii) on the north slope of the Alaska Range, all three in Interior Alaska. Mink were laboratory raised at the Oregon State University Experimental Fur Farm. These species were selected because they occur sympatrically in Arctic/subarctic areas that would be considered for bait vaccination of Arctic and red foxes. Other mammals occur there also but were unavailable for the experiments reported on here. All animals were adults as determined by size and mass; none were juveniles. The likelihood of neonates encountering a bait would be extremely low and, therefore, are not felt to be a potential problem in a rabies control program. Animals were held in separate cages in the Bio-safety Level 3 facility in the Animal Quarters at the Institute of Arctic Biology of the University of Alaska Fairbanks. Rodents were provided various combinations of carrots, lettuce, sunflower seeds and commercial feeds (Lab Chow and Rabbit Chow, Ralston Purina Co., Checkerboard Square, St. Louis, MO 63 164, USA) and mink with a commercial feed (Mink and Fox Food, Milk Specialties Co., New Holstein, WI 53061, USA). Food and water were provided ad lib. Experimental protocols for the study were approved by an independent campus-wide animal welfare committee. Two separate experiments were conducted. Singing voles, collared lemmings and mink were tested with SAG1 oral rabies vaccine, while tundra and red-backed voles and Arctic ground squirrels with SAG2 vaccine. A time delay between experiments necessitated the use of SAG2 in the second experiment because SAG1 was no longer available. Both vaccines are manufactured by
VIRBAC (13-eme Rue-L.I.D., 06517 Carros Cedex, France) and were provided to us through the Centers for Disease Control (CDC) (Atlanta, GA 30333, USA). Vaccines were titrated by intracerebral inoculation of l-3 day old CD-HalICR mice at the State Virology Laboratory prior to use. Voles and lemmings were administered 0.03 ml of vaccine and Arctic ground squirrels and mink 0.1 ml by direct installation into the mouth. This route of administration ensured exposure to the vaccine. Attempting to expose animals by providing a bait would complicate our analysis because of uncertainty whether the animals, representing six species, would be attracted to the bait and subsequently exposed by puncturing the vaccine container. The amount of vaccine administered was based on body size and was consistent with other studies. Two red-backed voles were held in the same room during the SAG2 trials but were not administered vaccine and served as controls. Animals administered SAG1 were observed for 36 days prior to euthanasia and those administered SAG2 for 46 days. At the termination of the observation period mink were anesthetized with ketamine hydrochloride (Ketaset, Aveco Co., Inc., Fort Dodge, IA 50501, USA) and rodents with methoxyflurane (Metofane, Pitman-Moore, Inc., Mundelein, IL 60060, USA). Blood samples were obtained by cardiac puncture. Mink were euthanized by overdose with pentobarbital sodium (Anthony Products Co., Arcadia, CA 91006, USA) and rodents with overdose of 2-Bromo-Chloro1,1,l -trifluoroethane (Halothane, Halocarbon Laboratories, N. Augusta, SC 29841, USA). Serum samples from the mink, singing voles and collared lemmings were tested for rabies antibodies by the rapid fluorescent focus inhibition test (RFFIT) at the CDC while sera from the tundra and red-backed voles and Arctic ground squirrels were tested at the Department of Veterinary Diagnostics. Kansas State University (Manhattan, KS 66506, USA). The presence of rabies virus neutralizing antibodies would suggest that the vaccine virus was immunogenic in these species. Brain tissue was analyzed for rabies antigen using direct immunofluorescence (FA) at the State Virology Laboratory. The presence of rabies antigen in brain tissue would suggest pathogenicity of the vaccine virus.
RESULTS AND DISCUSSION The titer of SAG1 oral vaccine was MLD,, mice: 107.5/0.01 ml and of SAG2 MLD,, mice: 106.68/0.01 ml. These are vaccines that would be used in the field in a fox vaccination program although the volume contained in each bait (l-2 ml) would be greater than that employed in these experiments. It is highly unlikely, however, that these animals would ingest such large volumes. None of the 47 animals given either SAG1 or SAG2 vaccine and maintained through the observation periods developed encephalitic signs, ataxia, lethargy or irritability, nor was rabies antigen detected in brain tissue (Table I). Four animals (1 each of singing, tundra and red-backed voles and 1 collared lemming) died during the observation period, but none were rabid. The results indicate that the SAG1 and SAG2 vaccines do not induce rabies in several Arctic species when
Vaccine 1996 Volume 14 Number 4
271
Evaluation of the safety of SAG1
Table 1 Safety trials of SAG1 and SAG2 mink and five species of wild Arctic rodents
Species
No. Deathsa
Microtus miuris Dicrostonyx groenlandicus Mustela vison
7 9 7
1 1 0
Clefhrionomys rutilis Microtus oeconomus Spermophilus parryi Clethrionomys rutilis (control)
10
1
SAGP: E.H. Follmann et al.
and
oral rabies vaccines in
Rabiespositive
Seroconversion rate
016
116
018
o/a
on
o/7
o/9
:
:,
o/9
o/76 216” l/9
2
0
012
-d
o/a
“During the observation period; none due to rabies. %era from only 7 animals were available for analysis. CSera from only six animals were available for analysis. dNo serum available for analysis
administered orally at titers of 107-‘/O.Ol ml and 106.6”/ 0.01 ml, respectively, including the Arctic fox (in which the safety and effectiveness of SAG1 were previously demonstrated”). Similar results were reported for several rodent species in Europe’“.‘6, for the red fox” and for a variety of other mammals representing carnivores, artiodactyls, rodents, insectivores and a nonhuman primate, and in four species of birds (E. Masson, unpublished results). Of 43 animals exposed to either SAG1 or SAG2 rabies vaccine, 1 singing vole, 2 tundra voles and 1 Arctic ground squirrel developed antibodies in response to oral exposure (Table I ). Due to procedural and shipping problems insufficient serum was available from six animals (4 red-backed voles, including 2 controls; 2 tundra voles) to test for antibodies. Titers were 0.11 IU ml ~ ’ for the singing vole, 0.4 and 0.5 IU ml ~ ’ for the tundra voles, and 0.4 IU ml _ ’ for the Arctic ground squirrel. These low response rates are consistent with the results obtained in another study evaluating SAG1 in Sera in that study were analyzed European rodents”. using an ELISA test with reduced sensitivity to rodent antibodies. Nevertheless, some responses were noted in two specimens of Apoderuus sp. Only a few animals seroconverted in our study, probably due to individual variation in the immune response and to differences in the amount of vaccine absorbed in the buccal mucosa prior to swallowing. The results of the present study indicate the safety of both SAG1 and SAG2, two closely related oral rabies vaccines, in six species of mammals found in Arctic and subarctic areas. The absence of rabies antigen in brain tissue after an extended observation period indicate the safety of these vaccines in nontarget species. Because rabies virus is highly neurotropic and its pathogenicity results from entry into CNS neural tissue, the absence of viral antigen in brain tissue of these 6 species exposed orally to SAG1 and SAG2 rabies vaccine, suggests that the virus is highly attenuated, presumably due to the amino acid exchange at site III, specifically replacement of arginine with glutamic acid at position 333. This exchange reduces the pathogenicity of the attenuated viruses present in SAG1 and SAG2 vaccines by preventing the deleterious effect of viral entry into CNS neurons. SAG2 is the more advanced of the two vaccines, with greater genetic stability, and it is reasonable to assume that it, too, would be safe in the three species in which we evaluated SAGl.
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Vaccine 1996 Volume 14 Number 4
ACKNOWLEDGEMENTS Support for this program was provided by the University of Alaska Foundation President’s Special Projects Fund, the Institute of Arctic Biology, the Alaska Division of Public Health, and the Centers for Disease Control (CDC). VIRBAC kindly gave permission to use SAG1 and SAG2 vaccines and CDC provided them to us for use in Alaska. We thank B. Barnes, B. Boyer and S. Sharbaugh for assistance with trapping rodents. D. Hartbauer and the IAB Animal Quarters staff are thanked for care and maintenance of the animals before and during the study. We gratefully acknowledge C. Schumacher of VIRBAC for providing information on the SAG2 vaccine and for reviewing an early draft of this manuscript.
REFERENCES 1
2 3
4
5
6
7
a
9
10
11
12
13 14 15
16
17
Wandeler, A.I. Oral immunization of wildlife. In: The Natural History of Rabies (Ed. Baer, G.M.), 2nd Edition. CRC Press, Boca Raton, FL, 1991, pp. 485-503 Bogel, K., Meslin, F.-X. and Kaplan, M. (Eds) Wildlife Rabies Control. Wells Medical Ltd, Kent, UK, 1992 Winkler, W.G., Shaddock, J.H. and Williams, L.W. Oral rabies vaccine: evaluation of its infectivity in three species of rodents. Am. J. Epidemiol. 1976, 104, 294 Wachendotfer, G., Kiefert, C. and Frost, J.W. Safety tests with Flury HEP Strain 675 in wild-living European mammals. Comp. Immunol. Microbial. Infect. Dis. 1982, 5, 177 Pepin, M., Blancou, J., Aubert, M.F.A., Barrat, J., Coulon, P. and Flamand, A. Oral immunization against rabies with an avirulent mutant of the CVS strain: evaluation of its efficacy in fox ( Wpes vulpes) and its infectivity in seven other species. Ann. Inst. PasteurNirool. 1985, 136E, 65 Kalpers, J., Brochier, B., Lejeune, E. et al. First vaccination campaign of foxes against rabies by oral route in Belgium. lnnocuity controls in rodents and insectivores. Ann. Med. Vet. i987,131,473 Lawson, K.F., Black, J.G., Charlton, K.M., Johnston, D.H. and Rhodes, A.J. Safety and immunogenicity of a vaccine bait containing ERA strain of attenuated rabies virus. Can. J. Vet. Res. 1987, 51, 460 Paquot, A., Brochier, B., Thomas, I. and Pastoret, P.-P. Vaccination campaigns of the red fox (Vu$es vu$es) against rabies in Belgium: ingestion of vaccine baits by the red deer (Cervus elaphus), the roe deer (Capreolus capreolus) and the wild boar (Sus scrofa). Ann. Med. Vet. 1988, 132, 697 LeBlois, H., Tuffereau, C., Blancou, J., Arlois, M., Aubert, A. and Flamand, A. Oral immunization of foxes with avirulent rabies virus mutants. Vet. Microbial. 1990, 23, 259 Artois, M., Guittre, C., Thomas, I., LeBlois, H., Brochier, B. and Barrat, J. Potential pathogenicity for rodents of vaccines intended for oral vaccination. Vaccine 1992, 10, 524 Coulon, P., Lafay, F., Leblois, H. et al. The SAG: a new attenuated oral rabies vaccine. In: wildlife Rabies Control (Eds Bogel, K., Meslin, F.-X. and Kaplan, M.). Wells Medical Ltd, Kent, UK, 1992, pp. 105-111 Crandell, R.A. Arctic fox rabies. In: The Natural History of Rabies (Ed. Baer, G.M.), 2nd Edition. CRC Press, Boca Raton, FL, 1991, pp. 291-306 Rausch, R. Some observations on rabies in Alaska, with special reference to wild Canidae. J. Wildlife Manaue. 1958, 22, 246 Rausch, R. Observations on some natural-focal zoo&es in Alaska. Arch. Environ. Hlth 1972, 25, 246 Secord, D.C., Bradley, J.A., Eaton, R.D. and Mitchell, D. Prevalence of rabies virus in foxes trapped in the Canadian arctic. Can. Vet. J. 1980, 21, 297 Ritter, D.G. Rabies. In: Alaskan Wildlife Diseases. (Ed. Dieterich, R.A.). University of Alaska, Fairbanks, AK, 1981, pp. 6-12 Follmann, E.H., Ritter, D.G. and Belier, M. Survey of fox trappers in northern Alaska for rabies antibody. Epidemiol. Infect. 1994, 113, 137
Evaluation of the safety of SAG1 and SAG2: E.H. Follmann et al. 18 19 20
21
22
23
Middaugh, J. and Ritter, D. A comprehensive rabies control program in Alaska. Am. J. Public Hlth 1982, 72, 384 World Health Organization. Report of a WHO/NV1 Workshop on Arctic Rabies, WHO/Rab. Res./90.35, Uppsala, Sweden, 1990 Follmann, E.H., Ritter, D.G. and Baer, G.M. Immunization of arctic foxes (Alopex lagopus) with oral rabies vaccine. J. Wildlife Dis. 1988, 24, 477 Follmann, E.H., Ritter, D.G. and Baer, G.M. Oral rabies vaccination of Arctic foxes (Alopex lagopus) with an attenuated vaccine. Vaccine 1992, 10, 305 Flamand, A., Coulon, P., Lafay, F. and Tuffereau, C. Avirulent mutants of rabies virus and their use as live vaccine. Trends Microbial. 1993, 1, 317 Lafay, F., Benejean, J., Tuffereau, C., Flamand, A. and Coulon, P. Vaccination against rabies: construction and characterization
24
25
26
of SAGP, a double avirulent derivative of SADBern. Vaccine 1994, 12,317 Wandeler, A.I. Safety aspects for man, domestic animals and other non-target species: attenuated rabies vaccines. In: Wildlife Rabies Control (Eds Boael. K.. Meslin, F.-X. and Kaplan, M.). Wells Medical Ltd, Kent, UK,‘1992, pp. 123-124 Bingham, J., Foggin, C.M., Gerber, H. et al. Pathogenicity of SAD rabies vaccine given orally in chacma baboons (Papio ursinus). Vet. Rec. 1992, 131, 55 Flamand, A., Blancou, J., Coulon, P. et a/. The antigenic structure of the rabies glycoprotein, application of basic research to oral vaccination of foxes. In: Progress in Rabies Control (Eds Thraenhart, O., Koprowski, H., Bogel, K. and Sureau, P.). Wells Medical Ltd, Kent, UK, 1989, pp. 72-77
Vaccine 1996 Volume 14 Number 4
273
Elsevier 0264-410X(95)00208-1
ELSEVIER
Evaluation of the safety of two attenuated oral rabies vaccines, SAG1 and SAG2, in six Arctic mammals Erich H. Follmann*l,
Donald
G. Ritter-i_ and George
M. Baer$g
The safety of two attenuated oral rabies vaccines was evaluated in mink and inJive species of rodents which occur in the Arctic. A 0.03 mI sample of liquid vaccine was installed directly into the mouth of voles and lemmings and 0.1 ml into the mouth of Arctic ground squirrels and mink. Animals were euthanized at 36 and 46 days postexposure; brain tissue was analyzed by FAT and serum by RFFIT. No rabies deaths occurred in 47 animals tested. Four animals representing three rodent species seroconverted, the highest titer being 0.5 IU ml ~ t. The absence of rabies virus in brain tissue indicates the safety of these vaccines in these species. The replacement of arginine Mlith glutamic acid at position 333 reduces the pathogenicity of these vaccines, thereby presumably preventing the deleterious efhect of viral entry into CNS neurons. Copyright (6 1996 Elsevier Science Ltd. Keywords:
Rabies:
SAGl:
SAG2
oral vaccination:
pathogenicity:
rodents;
The effectiveness of oral vaccines in rabies control has been well demonstrated in both laboratory and field studies. Wild red fox (Vulpes vulpes) populations in Europe and Canada have been successfully immunized in the field resulting in reduction and eradication of fox rabies in some areas of Europe’-‘. An important consideration in field applications is not only the effectiveness of the vaccine in vaccinating the target population but its safety for any animal that may ingest the vaccine bait. Such concerns have led to extensive safety testing of several oral vaccines in numerous rodent, insectivore, carnivore, artiodactyl and nonas new oral human primate specie?’ ’ and continue vaccines are developed. Rabies commonly occurs in Nearctic wildlife populations with the Arctic fox (Alopex lugopus) as the main vector” 15. In Alaska both Arctic and red foxes harbor the disease, and epizootics occur every 334 years”. Human cases of rabies are infrequent in the ArcticI yet public health concerns exist. In Alaska human exposure to rabies typically is not from wild animals but from domesticated animals, mainly the dog, who have had *Institute of Arctic Biology,
University of Alaska Fairbanks, Fairbanks, AK 99775-7000, USA. $State Public Health Laboratory-Fairbanks, Alaska Division of Public Health, Fairbanks, AK 99775, USA. $Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Social Services, Building 15, 1600 Clifton Road, Atlanta, GA 30333, USA. §Current address: Laboratorios Baer, S.A. de C.V., Cuautla 150, Colonia Condesa, Mexico D.F. C.P.06140, Mexico. To whom correspondence should be addressed. (Received 29 March 1995; revised 18 September 1995; accepted 18 September 1995)
270
Vaccine
1996 Volume
14 Number
4
mink
contact with foxes or other wild animals. In the past 20 years over 80% of the animals submitted for rabies testing in Alaska have been foxes and 13% domestic animals. The mean annual percentage of rabid animals over this period has been about 30%. This high incidence and fear of the disease have led Alaska and some Arctic nations with similar roblems to consider rabies control quarantine” of through vaccination P* and long-term domestic animals. Although rabies in the circumpolar Arctic has been well known for many years12, the possibility of oral vaccination of wild carnivores in these harsh and remote areas has only recently been considered”. The effectiveness of oral vaccination of red foxes is well established in Europe and Canada, and it is assumed that red foxes in the Arctic can be similarly vaccinated. Laboratory experiments have shown that the more important rabies vector in the Arctic, the Arctic fox, can also be vaccinated with SAD-BHK,, and SAG1 oral vaccines2’.“. The safety of these vaccines in nontarget, Arctic, mammalian species has, however, not been established. The purpose of the present study was to determine the inocuity of SAG1 and SAG2 oral rabies vaccines in Arctic mammalian species that occur sympatrically with Arctic and red foxes. The SAG1 vaccine is a variant derived from SAD,,,,“. It differs from SAD,,,,, its parent strain, by one mutation in the codon (at position 333)‘3 of the antigenic site III of the glycoprotein. The resulting amino acid exchange is responsible for the attenuation of this vaccine strain. Because there is only one nucleotide exchange at this site, the vaccine can theoretically revert to its parental strain with only one mutation, which could occur from passage through an animal. This occurred after first” and second” passages through suckling mice; only one of several test mice
Evaluation of the safety of SAGf and SAG2 H-l. Follmann et al. exhibited this in each of these studies. Reversion to the parental strain could be a potential problem in a field situation because SADa,,, has induced rabies in certain skunks (Mephitis mephitis)‘” and species of rodents’,“, baboons (Papio ursinu.~)‘~.SAG1 has been shown to be safe for six wild species of murid rodents administered liquid vaccine by the oral, nasal and ocular routes]‘. SAG2 is characterized by two mutations in the codon at position 333 yielding glutamic acid at this position instead of serine as in SAG1 and arginine as in SADBern22,23. The probability of having two mutations occur in this codon as a result of animal passage is far less likely, and SAG2 is thus considered more genetically stable than its two ancestors, and is avirulent for adult immunocompetent mice as a result of replacement of arginine at position 333. SAG2 also has been demonstrated to be safe in a broad range of species” (C. Schumacher, personal communication); our study reports on its safety in six additional mammalian species found in the Nearctic as well as in certain regions of the Palearctic.
MATERIALS
AND METHODS
Five species of Arctic rodents representing four genera vison) were tested. Singing and mink (Mustela voles (Microtus miuris) were captured in box traps north of the Brooks Range, Alaska; collared lemmings (Dicrostonyx groenlandicus) originated in the same area but the animals used were bred in captivity. Tundra voles (Microtus oeconomus) and red-backed voles (Clethrionom_ys rutilis) were captured in box traps in and Arctic ground squirrels the Fairbanks area, (Spermophilus part-vii) on the north slope of the Alaska Range, all three in Interior Alaska. Mink were laboratory raised at the Oregon State University Experimental Fur Farm. These species were selected because they occur sympatrically in Arctic/subarctic areas that would be considered for bait vaccination of Arctic and red foxes. Other mammals occur there also but were unavailable for the experiments reported on here. All animals were adults as determined by size and mass; none were juveniles. The likelihood of neonates encountering a bait would be extremely low and, therefore, are not felt to be a potential problem in a rabies control program. Animals were held in separate cages in the Bio-safety Level 3 facility in the Animal Quarters at the Institute of Arctic Biology of the University of Alaska Fairbanks. Rodents were provided various combinations of carrots, lettuce, sunflower seeds and commercial feeds (Lab Chow and Rabbit Chow, Ralston Purina Co., Checkerboard Square, St. Louis, MO 63 164, USA) and mink with a commercial feed (Mink and Fox Food, Milk Specialties Co., New Holstein, WI 53061, USA). Food and water were provided ad lib. Experimental protocols for the study were approved by an independent campus-wide animal welfare committee. Two separate experiments were conducted. Singing voles, collared lemmings and mink were tested with SAG1 oral rabies vaccine, while tundra and red-backed voles and Arctic ground squirrels with SAG2 vaccine. A time delay between experiments necessitated the use of SAG2 in the second experiment because SAG1 was no longer available. Both vaccines are manufactured by
VIRBAC (13-eme Rue-L.I.D., 06517 Carros Cedex, France) and were provided to us through the Centers for Disease Control (CDC) (Atlanta, GA 30333, USA). Vaccines were titrated by intracerebral inoculation of l-3 day old CD-HalICR mice at the State Virology Laboratory prior to use. Voles and lemmings were administered 0.03 ml of vaccine and Arctic ground squirrels and mink 0.1 ml by direct installation into the mouth. This route of administration ensured exposure to the vaccine. Attempting to expose animals by providing a bait would complicate our analysis because of uncertainty whether the animals, representing six species, would be attracted to the bait and subsequently exposed by puncturing the vaccine container. The amount of vaccine administered was based on body size and was consistent with other studies. Two red-backed voles were held in the same room during the SAG2 trials but were not administered vaccine and served as controls. Animals administered SAG1 were observed for 36 days prior to euthanasia and those administered SAG2 for 46 days. At the termination of the observation period mink were anesthetized with ketamine hydrochloride (Ketaset, Aveco Co., Inc., Fort Dodge, IA 50501, USA) and rodents with methoxyflurane (Metofane, Pitman-Moore, Inc., Mundelein, IL 60060, USA). Blood samples were obtained by cardiac puncture. Mink were euthanized by overdose with pentobarbital sodium (Anthony Products Co., Arcadia, CA 91006, USA) and rodents with overdose of 2-Bromo-Chloro1,1,l -trifluoroethane (Halothane, Halocarbon Laboratories, N. Augusta, SC 29841, USA). Serum samples from the mink, singing voles and collared lemmings were tested for rabies antibodies by the rapid fluorescent focus inhibition test (RFFIT) at the CDC while sera from the tundra and red-backed voles and Arctic ground squirrels were tested at the Department of Veterinary Diagnostics. Kansas State University (Manhattan, KS 66506, USA). The presence of rabies virus neutralizing antibodies would suggest that the vaccine virus was immunogenic in these species. Brain tissue was analyzed for rabies antigen using direct immunofluorescence (FA) at the State Virology Laboratory. The presence of rabies antigen in brain tissue would suggest pathogenicity of the vaccine virus.
RESULTS AND DISCUSSION The titer of SAG1 oral vaccine was MLD,, mice: 107.5/0.01 ml and of SAG2 MLD,, mice: 106.68/0.01 ml. These are vaccines that would be used in the field in a fox vaccination program although the volume contained in each bait (l-2 ml) would be greater than that employed in these experiments. It is highly unlikely, however, that these animals would ingest such large volumes. None of the 47 animals given either SAG1 or SAG2 vaccine and maintained through the observation periods developed encephalitic signs, ataxia, lethargy or irritability, nor was rabies antigen detected in brain tissue (Table I). Four animals (1 each of singing, tundra and red-backed voles and 1 collared lemming) died during the observation period, but none were rabid. The results indicate that the SAG1 and SAG2 vaccines do not induce rabies in several Arctic species when
Vaccine 1996 Volume 14 Number 4
271
Evaluation of the safety of SAG1
Table 1 Safety trials of SAG1 and SAG2 mink and five species of wild Arctic rodents
Species
No. Deathsa
Microtus miuris Dicrostonyx groenlandicus Mustela vison
7 9 7
1 1 0
Clefhrionomys rutilis Microtus oeconomus Spermophilus parryi Clethrionomys rutilis (control)
10
1
SAGP: E.H. Follmann et al.
and
oral rabies vaccines in
Rabiespositive
Seroconversion rate
016
116
018
o/a
on
o/7
o/9
:
:,
o/9
o/76 216” l/9
2
0
012
-d
o/a
“During the observation period; none due to rabies. %era from only 7 animals were available for analysis. CSera from only six animals were available for analysis. dNo serum available for analysis
administered orally at titers of 107-‘/O.Ol ml and 106.6”/ 0.01 ml, respectively, including the Arctic fox (in which the safety and effectiveness of SAG1 were previously demonstrated”). Similar results were reported for several rodent species in Europe’“.‘6, for the red fox” and for a variety of other mammals representing carnivores, artiodactyls, rodents, insectivores and a nonhuman primate, and in four species of birds (E. Masson, unpublished results). Of 43 animals exposed to either SAG1 or SAG2 rabies vaccine, 1 singing vole, 2 tundra voles and 1 Arctic ground squirrel developed antibodies in response to oral exposure (Table I ). Due to procedural and shipping problems insufficient serum was available from six animals (4 red-backed voles, including 2 controls; 2 tundra voles) to test for antibodies. Titers were 0.11 IU ml ~ ’ for the singing vole, 0.4 and 0.5 IU ml ~ ’ for the tundra voles, and 0.4 IU ml _ ’ for the Arctic ground squirrel. These low response rates are consistent with the results obtained in another study evaluating SAG1 in Sera in that study were analyzed European rodents”. using an ELISA test with reduced sensitivity to rodent antibodies. Nevertheless, some responses were noted in two specimens of Apoderuus sp. Only a few animals seroconverted in our study, probably due to individual variation in the immune response and to differences in the amount of vaccine absorbed in the buccal mucosa prior to swallowing. The results of the present study indicate the safety of both SAG1 and SAG2, two closely related oral rabies vaccines, in six species of mammals found in Arctic and subarctic areas. The absence of rabies antigen in brain tissue after an extended observation period indicate the safety of these vaccines in nontarget species. Because rabies virus is highly neurotropic and its pathogenicity results from entry into CNS neural tissue, the absence of viral antigen in brain tissue of these 6 species exposed orally to SAG1 and SAG2 rabies vaccine, suggests that the virus is highly attenuated, presumably due to the amino acid exchange at site III, specifically replacement of arginine with glutamic acid at position 333. This exchange reduces the pathogenicity of the attenuated viruses present in SAG1 and SAG2 vaccines by preventing the deleterious effect of viral entry into CNS neurons. SAG2 is the more advanced of the two vaccines, with greater genetic stability, and it is reasonable to assume that it, too, would be safe in the three species in which we evaluated SAGl.
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ACKNOWLEDGEMENTS Support for this program was provided by the University of Alaska Foundation President’s Special Projects Fund, the Institute of Arctic Biology, the Alaska Division of Public Health, and the Centers for Disease Control (CDC). VIRBAC kindly gave permission to use SAG1 and SAG2 vaccines and CDC provided them to us for use in Alaska. We thank B. Barnes, B. Boyer and S. Sharbaugh for assistance with trapping rodents. D. Hartbauer and the IAB Animal Quarters staff are thanked for care and maintenance of the animals before and during the study. We gratefully acknowledge C. Schumacher of VIRBAC for providing information on the SAG2 vaccine and for reviewing an early draft of this manuscript.
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