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Dr. Jean Dodds, Kris Christine & Friends -- on recent Vaccination Legislation
WY's Pet Euthanasia Rule
From: Peter & Kris Christine [mailto:LedgeSpring at lincoln.midcoast.com]
Sent: Thursday, May 11, 2006
Subject: WY's Pet Euthanasia RuleGreetings:Wyoming has a new rule which would require cats and dogs deemed "unvaccinated" to be immediately euthanized if they SCRATCH or bite a human, see news story http://www.wyomingnews.com/news/more.asp?StoryID=107547 and my letter to the state veterinarian below. Bear in mind, vaccinated animals overdue for boosters are considered to be "unvaccinated" despite the science showing durations of immunity for the rabies vaccine well beyond 3 years.Permission granted to post and cross-post. Pet owners living in or traveling through Wyoming with their animals would be well-advised to take action on this matter.Kris L. ChristineCo-Trustee, Co-FounderThe Rabies Challenge Fund Charitable Trust----- Original Message -----From: Peter & Kris ChristineCc: firstname.lastname@example.org ; email@example.com ; firstname.lastname@example.org ; email@example.com ; firstname.lastname@example.org ; email@example.com ; firstname.lastname@example.org ; email@example.com ; firstname.lastname@example.orgSent: Thursday, May 11, 2006 8:19 AMSubject: WY's Euthanasia Rule
May 10, 2006
Dr. Jamie Snow
Wyoming Department of Public Health
Re: Wyoming’s New Euthanasia Rule
Greetings Dr. Snow:
In light of current scientific research, Wyoming’s rabies rule requiring mandatory euthanasia for cats and dogs deemed “unvaccinated” is unjustified. While I recognize that you have a mandate to protect public health, your new rule will needlessly take the lives of countless pets which are actually immune to rabies despite being overdue for or medically exempt from rabies boosters.
Under the new rule, it appears companion animals overdue for a rabies booster by a week or month (according to vaccine manufacturer’s recommendations) would be deemed “unvaccinated,” disregarding the fact that study results published in 1992 by Michel Aubert’s research team demonstrated that dogs were immune to a rabies challenge 5 years post-vaccination (see text of that study below). Serological studies done by Dr. Ronald Schultz of the University of Wisconsin School of Veterinary Medicine show that dogs have antibody titer counts at levels known to confer immunity to rabies 7 years post-vaccination. Based on the scientific research, any dog euthanized in Wyoming under this rule within 5 to 7 years of rabies vaccination will die needlessly.
On Page 13 of the The American Animal Hospital Association's (AAHA’s) 2003 Canine Vaccine Guidelines (accessible online at Leeburg Training http://www.leerburg.com/special_report.htm), it states: “The minimum DOI for killed rabies vaccine based on challenge studies is 3 years; based on antibody titers, it is considered to be up to 7 years [Table 2].” Vaccine manufacturers are not required by FDA to demonstrate long-term duration of immunity in order to license their products. According to the American Veterinary Medical Association’s Principles of Vaccination (http://www.avma.org/issues/vaccination/vaccination.asp), “..revaccination frequency recommendations found on many vaccine labels …does not resolve the question about average or maximum duration of immunity [Page 2] and.may fail to adequately inform practitioners about optimal use of the product…[Page 4] .” This holds true for rabies vaccines, as Aubert’s and Schultz’s studies demonstrate that rabies vaccination confers minimum duration of immunity in dogs far beyond vaccine manufacturers’ 3 year booster recommendation.
In conversations with rabies vaccine researchers, I have been told that the Center for Disease Control has no recorded cases of any dog contracting rabies after receiving 2 rabies vaccinations. The implication is that 2 rabies vaccinations confer a lifetime of immunity. As a veterinarian, your own experience being vaccinated against rabies indicates the same. The Populations at Risk for Rabies sheet from Chiron Corporation, manufacturers of the RabAvert rabies vaccines for humans is accessible at http://www.rabavert.com/risk.html. Their pre-exposure vaccination recommendation for veterinarians, who are at greater risk than the general population for contracting rabies because their profession brings them into physical contact with potentially rabid animals, is for a “Primary course. No serologic testing or booster vaccination.” In other words, after the initial series of rabies vaccinations, it is not recommended that veterinarians receive further boosters or serological testing, they are considered to be immune to a challenge. AAHA’s 2003 Canine Vaccine Guidelines (accessible online at Leeburg Training http://www.leerburg.com/special_report.htm) states on Page 18 that “There is no indication that the immune system of canine patients functions in any way different from the human immune system. In humans, the epidemiological vigilance associated with vaccination is extremely well-developed and far exceeds similar efforts in animals whether companion or agricultural. This vigilance in humans indicates that immunity induced by vaccination in humans is extremely long lasting and, in most cases, life-long.” This strongly suggests that, like the human rabies vaccine, the canine rabies vaccine also provides life-long immunity.
The science reflects that Wyoming will be euthanizing immune dogs under the new euthanasia rule. I strongly urge you to re-examine this policy and consult with the world’s leading authorities on veterinary vaccines -- Dr. W. Jean Dodds of Hemopet and Dr. Ronald Schultz of the University of Wisconsin School of Veterinary Medicine -- about modifying this scientifically unwarranted euthanasia rule.
Ensuring that Wyoming’s beloved companion animals are not needlessly euthanized justifies closer scrutiny of this rule.
Kris L. Christine
The Rabies Challenge Fund Charitable Trust
Alna, ME 04535
... cc: Dr. W. Jean Dodds
Dr. Ronald Schultz
The French rabies challenge study below was published in Scientific Technical Review (Rev. sci.tech. Off. int. Epiz.) 1992, 11 (3), 735-760. Anyone wishing to obtain an original copy from the publisher can contact:Magdalena Banaszak
OIE, 12, rue de Prony, 75017 PARIS
tel. + 33 (0)1 44 15 19 64
fax + 33 (0)1 42 67 09 87
PRACTICAL SIGNIFICANCE OF RABIES ANTIBODIES IN CATS AND DOGS*
RESULTS OF A SURVEY ON RABIES VACCINATION AND QUARANTINE
FOR DOMESTIC CARNIVORA IN WESTERN EUROPE
Centre national d'études vétérinaires et alimentaires, Laboratoire d'études sur la rage et la pathologie des animaux sauvages, B.P. 9, 54220 Malzéville, France
Note : Text figures have been omitted (SG) Original: French
Summary: Doubt has sometimes been cast upon the protective effect of rabies antibodies in serum. Animals and humans suffering from fatal rabies often produce high antibody titres, while rabies cases are also observed in vaccinated animals. Cellular immunity is also largely involved in protection. Nevertheless, a large number of laboratory experiments and field observations clearly demonstrate that cats and dogs which develop antibodies after vaccination and before challenge have a very high probability of surviving any challenge, no matter how strong the dose and which virus strain is used.
Rabies antibody titration can, therefore, afford a strong additional guarantee to the vaccination certificates accompanying domestic carnivores during transportation between countries. Quarantine rules should also be adapted to the epidemiological features in the exporting country, e.g. statistics of vaccination failure in cats and dogs and host-virus adaptation of the rabies strains circulating in these countries.
Cats and dogs can introduce rabies into disease-free countries if they are incubating the disease and are transported during the pre-symptomatic phase. To prevent such introduction, vaccination is recommended. The present article reviews publications dealing with rabies protection afforded to cats and dogs by vaccination. In addition, the Appendix presents the results of a survey of the current practices of OIE Member Countries in Western Europe with respect to rabies vaccination and quarantine of dogs and cats.
Only the parenteral route of vaccination will be considered, as the oral route is employed only for wandering and non-restrained carnivores; extensive results for individual cats and dogs are unavailable. Also, since oral vaccination could mobilise immunity pathways other than those obtained parenterally, the results with one procedure may not be transposable to the other.
Furthermore, no consideration will be given here to the results of vaccination after exposure, which does little, if anything, to alter disease (20).
Emphasis will be given to the most common method for measuring rabies immunisation: assays for rabies virus neutralising antibodies in serum (henceforth referred to as "neutralising antibodies"). The practical significance and consequences of rabies virus neutralising antibodies in cats and dogs are considered; namely, to what extent do neutralising antibody titres confer protection against subsequent challenge?
No consideration will be given to the question of whether real protection against challenge is provided by neutralising antibodies and/or other immunity factors. Titres of neutralising antibodies in serum are simply viewed as the easiest means of evaluating the likelihood that a cat or dog will not contract rabies following exposure.
2. STUDIES IN DOGS
2.1. Neutralising antibodies after vaccination
The kinetics obtained for neutralising antibodies after vaccination have been thoroughly described in the literature. The curve of neutralising antibodies after vaccination and boosters follows the pattern generally observed with other antigens: seroconversion and rapid rise of the level of neutralising antibodies after first vaccination, followed by a slow decrease, a new rise after booster to reach a higher level than previously observed, then a new decrease leading to a stabilised higher level (Fig. 1) (8, 51, 63). The decrease of neutralising antibody levels has been evaluated in domestic populations of owned dogs in several countries: in Canada, titres of neutralising antibodies in the sera of dogs showed a clear division between, on the one hand, dogs vaccinated or revaccinated one year before and, on the other hand, dogs revaccinated three weeks before (33). Data from Thailand and Java show that the neutralising antibody titre decreases very rapidly after 60 to 120 days to levels 5- to 25-fold less than the highest point reached during the kinetics (Fig. 2) (40, 65). The higher level of neutralising antibodies obtained when owned dogs are vaccinated several times has been described by Sasaki and colleagues (55, 56).
Kinetics of rabies neutralising antibodies in sera of laboratory dogs vaccinated with a tissue culture vaccine
Variations according to vaccination route and antigenic value of the vaccine as measured by the NIH test
Kinetics of rabies neutralising antibodies in sera of owned dogs of various ages in Thailand after one subcutaneous vaccination with a tissue culture vaccine
The number of dogs sampled was 54 at day 0 and 31 at day 360
With regard to the production of neutralising antibodies and the relationship of these antibodies to protection against challenge, a clear distinction must be made between live virus vaccines and inactivated virus vaccines. These two types of vaccine cannot be directly compared. The best relationship between antibody production and protection has always been obtained with inactivated virus vaccines and it is, therefore, the latter which will be considered in greater detail, especially as they currently represent the only type of vaccine authorised in a great many countries.
High individual variability
In laboratory dogs bred and kept under the same conditions and in comparable health, neutralising antibody titres obtained after the same form of vaccination commonly range from zero to twenty international units per ml (IU) or more (19, 51, 53).
Influence of vaccine types and potency
The first complete study of live virus vaccines was published by Dean and colleagues (32). This study established a correlation between antibody production and resistance to challenge, which was confirmed by later studies (see below). As far as inactivated virus vaccines are concerned, besides individual variation, the level of neutralising antibodies in serum correlates positively with the antigenic value of the vaccine as determined by the American National Institutes of Health (NIH) test. This observation is common in the course of vaccine production and control on laboratory dogs (Fig. 1) (47, 63). The influence of the antigenic value of the vaccine on the level of neutralising antibodies has also been demonstrated in domestic populations of owned dogs; in Switzerland, Engels and colleagues (35) showed that in owned dogs, higher titres were generally obtained with inactivated vaccines than with live (and less potent) vaccines.
However, when inactivated virus vaccines with an antigenic value (as measured by the NIH test) equal to or greater than 1.0 IU per dose are employed, no correlation can be shown between the level of neutralising antibodies in individual dogs and the titre of the vaccine. This result was first demonstrated in owned dogs in France by Blancou and colleagues (13). In this experiment, dogs were sampled randomly from populations living under various conditions and were vaccinated with a range of commercially available vaccines. Chappuis and colleagues (30) and Lazarowicz and colleagues (45) used laboratory dogs to investigate whether the administration of vaccines from the same producer would entail a correlation between the NIH titre of vaccines and the level of neutralising antibody response. Even under standardised conditions, no correlation was found. The same conclusion can be drawn from the results of Barth and colleagues (7).
In summary, a significant variation of neutralising antibody response can be shown only under a broad range of vaccine potencies (61). When the potencies of commercial inactivated virus vaccines are fairly high, the neutralising antibody response will be related only to the immune responses of individual dogs.
Influence of the route of vaccination
Since Pasteur, the route of vaccination has been subcutaneous (s.c.). Fuenzalida in 1967 demonstrated that the intramuscular (i.m.) route resulted in higher neutralising antibody titres in sera of dogs (37). Apart from Merry (46), who found no clear advantage for the i.m. over the s.c. route, the results obtained by Fuenzalida were largely confirmed (22, 25, 63). However, the advantage of the i.m. route diminishes with high potency vaccines (Fig. 1) (63) and the use of adjuvanted vaccines renders the i.m. route excessively painful. Adjuvants confer a longer lasting immunity, which can be obtained with a smaller quantity of antigen, as first demonstrated on laboratory dogs (52), then on owned dogs (43, 68, 69). Despite the use of smaller quantities of antigen and a reduced vaccination schedule (less frequent boosters), the neutralising antibody levels reached after one, two or three years with adjuvanted vaccines were equivalent to those reached with non-adjuvanted vaccines given according to the usual schedule (two injections of vaccine the first year, with annual boosters).
The importance of the vaccination route was clearly demonstrated with intradermal injection of vaccine in dogs (68). Unfortunately, the advantages of an enhanced response obtained with a minute dose of vaccine (2 x 0.1 ml) were offset by the fact that intradermal injection must be performed on the inside of the ear and, hence, this procedure must be conducted dangerously near the mouth of the animal.
Influence of age
It has been shown that dogs 11-16 weeks of age respond better to Flury low egg passage (LEP) or high egg passage (HEP) vaccine than dogs 5-10 weeks of age (81% vs. 38% protection from challenge, respectively) (41). The relationship between the age of animals and protection from challenge was confirmed in a laboratory study by Bunn in 3- to 5-month-old pups. Three months after vaccination with Flury LEP vaccine, ten of forty pups had antibody titres below 1/5 (24, 25).
A survey on owned dogs in France showed that even beyond three months of age, older dogs produced higher titres (Fig. 3) (13).
Correlation between rabies antibody level reached after one vaccine injection and age of dogs
Study conducted on 66 owned dogs in France
The influence of age on the neutralising antibody response in dogs was also clearly demonstrated on owned dogs in Thailand by Teepsumethanon and colleagues (65). These authors described the kinetics of neutralising antibodies in three age groups: 3 weeks to 3 months, 6 to 12 months, and more than 12 months. Whenever the mean level of neutralising antibodies was evaluated after vaccination, the older dogs had the highest levels of response. Given the difficult conditions prevailing in Thailand, the superior response of older dogs could also be related to the increased life expectancy of dogs with a more powerful immune system (Fig. 2).
The presence of specific neutralising antibodies transmitted to puppies via colostrum impedes development of active immunity. The interference between passive neutralising antibodies of maternal origin and active immunisation has been studied by Précausta (52, 53). Puppies of non-immune bitches vaccinated at the age of one month respond with the same neutralising antibody level as puppies vaccinated at seven months of age. Puppies of immune bitches vaccinated at one month of age show neutralising antibody levels which decrease according to the same kinetics as unvaccinated members of the same litter.
After ten weeks (44) to twelve weeks (52), no traces of maternal neutralising antibodies remain. Surveys in pet dog populations where systematic vaccination of adult dogs is practised (in France and elsewhere) have confirmed that no further interference between active and passive immunity occurs beyond this age (53).
Influence of the health and breeding status of dogs
Blancou and colleagues (19) compared the proportion of individuals developing neutralising antibodies in 64 dogs after the administration of adjuvanted or non-adjuvanted vaccines. This rate may vary considerably depending on the category of dog (bred for laboratories, belonging to individuals in France or uncontrolled in Tunisia). The rate drops from 100% to 59% in the case of semi-stray dogs as compared to laboratory dogs (Fig. 4). Urban dogs in Lima (Peru) exhibited better rates than in Tunisia, but the rates were still lower than in dogs kept under laboratory conditions (31). Although the health status of these populations had not been measured in the previous studies, this status is probably responsible for the differences observed by Teepsumethanon and colleagues (65) in Thailand: Thai pet dogs which had received one s.c. dose of rabies vaccine exhibited a better neutralising antibody response when they did not suffer from anaemia (Fig. 5). In 440 pets under quarantine in Hawaii, Sasaki and colleagues (56) demonstrated that those with internal parasites had significantly lower levels of neutralising antibodies than those without parasites.
Influence of the breeding standards of dogs on the level of rabies antibody reached one year after vaccination
Comparison of laboratory dogs, pet dogs in France and stray dogs in Tunisia
Influence of the health status of Thai dogs on the level of rabies antibody reached after one vaccination
Comparison of dogs with or without anaemia
2.2. Level of neutralising antibodies in sera and results of challenge
Challenge under laboratory conditions
In view of the serious problem posed by rabies, challenge of previously vaccinated dogs has often been performed even when a large proportion of the dogs under experiment exhibited a seroconversion. Moreover, such challenges are performed in response to doubts which have sometimes been cast on the significance of neutralising antibodies to rabies, due to the fact that high titres have been measured in human beings and animals dying of rabies. In fact, very few diseases show so clear a correlation as in rabies between seroconversion before challenge and protection from challenge.
In the context of movement of dogs between countries, it is possible to check the efficiency of previous vaccinations. A large number of reports can be summarised by the simple comparison of the proportion of dogs surviving challenge vs. the proportion of dogs with detectable neutralising antibodies in serum just before challenge (i.e. theoretically when the neutralising antibody level is lowest). These summaries are given in Tables 1 to 8.
Laboratory dogs: one intramuscular vaccination with various vaccines, challenge with rabies virus NYC-Ga strain, one year after vaccination
Vaccine Dogs with antibodies Dogs surviving challenge just before challenge (%) Experiment 1 LEP tissue culture 88 9/10 LEP tissue culture 73 10/10 ERA tissue culture 73 10/10 LEP chicken embryo 70 10/10 HEP tissue culture 63 10/10 CVS adjuvanted 13 7/10 None 0 0/10 Experiment 2 Suckling mouse brain 95 10/10 Suckling mouse brain 67 10/10 None 0 0/10
LEP: low egg passage
HEP: high egg passage
ERA: Elizabeth (Gaynor) Rokitniki Abelseth
CVS: challenge virus strain
Laboratory dogs: one intramuscular vaccination with various vaccines, challenge with rabies virus NYC-Ga strain, three years after vaccination
Vaccine Dogs with antibodies Dogs surviving challenge just before challenge (%) LEP tissue culture 87 29/30 LEP tissue culture 69 26/29 ERA tissue culture 57 27/30 LEP chicken embryo 54 28/30 Suckling mouse brain 48 27/27 HEP tissue culture 42 27/29 Suckling mouse brain 28 23/29 CVS adjuvanted 0 17/29 None 0 3/30 Results of challenge Antibodies before challenge yes no Rabid 3* 26 Surviving 157 47
* titres of 1/2, 1/3 and 1/5 (end point neutralising dilution of the serum)
Laboratory dogs: one vaccination with HEP vaccine, challenge with rabies virus NYC-Ga strain three years after vaccination
Vaccination Dogs with detectable Dogs surviving antibodies challenge just before challenge Intramuscular injection Undiluted 29/30 30/30 Diluted 1/10 6/10 10/10 Diluted 1/100 4/10 9/10 Subcutaneous injection Undiluted 4/29 17/29 Diluted 1/10 0/9 2/9 Diluted 1/100 0/8 2/8 None 0/30 0/30
Laboratory dogs: subcutaneous vaccination with tissue culture vaccine, challenge with rabies virus NYC-Ga strain, twenty-seven months after vaccine
Antigenic value Dogs with detectable antibodies Dogs surviving challenge of vaccine 12 months after vaccination Vaccinated Controls 0.6 8/8 8/8 0/7 1.7 19/20 17/18** 3/12 2.3 6/9 8/9*** 4/12 4.6 10/10 9/9 -
* measured by the NIH test, expressed in IU/dose
** The dog which died of rabies had always had the lowest antibody titre in the group
*** The dog which died of rabies had never seroconverted
Laboratory dogs: subcutaneous vaccination with tissue culture vaccine, challenge with rabies virus NYC-Ga strain, three years after vaccination
Vaccination Dogs with serum antibodies Dogs surviving challenge > 0.5IU/ml just before challenge Vaccinated Controls One injection of 29/30 29/30 0/20 adjuvanted vaccine
Laboratory dogs: intramuscular vaccination with tissue culture adjuvanted vaccine, challenge with rabies virus NYC-Ga strain, three years after vaccination
Vaccination Dogs with detectable antibodies Dogs surviving just before challenge challenge Yes 14/25 23/25* No 0/10 2/10
* One of the two dogs which died following challenge was seronegative, the other had a titre of 1/4 (endpoint neutralising dilution of the serum)
Laboratory dogs: intramuscular vaccination with ERA vaccine, challenge with rabies virus fox strain four or five years after vaccination
Vaccination Dogs with detectable Dogs surviving challenge antibodies just before challenge Four years before challenge Yes 5/10 7/10 No 0/9 0/9 Five years before challenge Yes 7/14 13/14 No 0/14 5/14
Laboratory dogs: subcutaneous vaccination with adjuvanted tissue culture vaccine, challenge with rabies virus fox strain, two years after booster vaccination
Antigenic value Dogs with detectable Dogs surviving challenge of vaccine* antibodies just before challenge Vaccinated Controls 4.2 9/10** 10/10 0/5
* measured by the NIH test, expressed in IU/dose
** two dogs with an antibody titre <0.5 IU/ml
Sikes and colleagues (62) employed several types of vaccine on dogs and challenged them one or three years after vaccination (Tables 1 and 2). Sikes (61) commented on the three-year experiment as follows: "In this study, as in many others, presence of neutralising antibodies to rabies at the time of challenge did not indicate protection for all of the animals. Likewise, absence of neutralising antibodies in serum at the time of challenge did not mean the animals were unprotected. However, there was strong statistical significance (P < 0.1) that animals with neutralising antibodies at the time of challenge were better protected than those with no detectable neutralising antibodies."
Sikes employed the NYC-Ga (New York City-Georgia) dog salivary gland strain of rabies virus. The same strain has also been used for challenge in other experiments (Tables 3 to 6) and the results confirm each point of the statements made by Sikes (60) regarding vaccination of dogs:
a) generally, groups of dogs with a high percentage of seroconversion will have the highest probability of surviving challenge;
b) on an individual basis:
- a dog with neutralising antibodies just before challenge will have the best chance of surviving a severe challenge;
- a dog with no detectable neutralising antibodies just before challenge will have a high chance of surviving a severe challenge if it seroconverted after vaccination;
- some dogs will not survive a severe challenge even if they have detectable neutralising antibody titres before challenge; generally these titres are the lowest of the group.
In studies of fox strains of rabies virus (Tables 7 and 8), the possibility of procuring a strong immunity as long as four to five years after vaccination, and of enhancing protection by the use of adjuvanted vaccines, has been demonstrated. These studies also confirmed the correlation between neutralising antibodies and protection against a fox strain.
Bunn and colleagues (28, 29) gathered pre-challenge neutralising antibody titres and challenge results obtained on dogs by the United States National Veterinary Services Laboratories and by vaccine manufacturers. Most of the dogs were challenged with the NYC-Ga strain, but results obtained with fox or skunk strains were also added. Sera were titrated either by the virus neutralisation test in mice (MNT) (5) or the rapid fluorescent focus inhibition test (RFFIT) (62). Data on neutralising antibodies originally expressed in arithmetical dilutions by Bunn (26, 27) have been converted into IU in Figure 6. Beyond 0.03 IU/ml with the MNT or 0.05 IU/ml with the RFFIT, the expected survival to challenge by a dog strain reaches 95%. With 288 dogs having RFFIT titres above 0.1 IU/ml, a 100% survival rate was obtained. The maximum survival rate observed among animals with the highest neutralising antibody titres measured by MNT was 99.5%.
Survival rate after challenge of laboratory dogs correlated with the level of rabies antibody reached before challenge
Dogs were vaccinated with various vaccines and challenged one year after vaccination with NYC-Ga, fox or skunk strains; the number of dogs in each class is written at the top of the bars
Given the higher susceptibility of dogs to dog strains (e.g. NYC-Ga), which was proven by cross challenge of dogs with homologous and heterologous (fox) strains (15, 17), the challenge with fox strains could be expected to be less severe. Unfortunately, the data are too scarce to permit a definitive conclusion.
Natural infection of vaccinated dogs
The number of vaccinated dogs which become naturally infected is related to several factors other than vaccine potency, such as probability of encountering an infected animal, severity of bites, health status and immune efficiency of the vaccinated dogs, and host-virus adaptation. Such considerations could explain why vaccinated dogs suffer rabies more often in the course of dog rabies enzootics than during fox rabies enzootics. In Thailand, 9% of the dogs found positive upon laboratory diagnosis had been vaccinated within the previous two years (39). In Nigeria, a survey of 2,500 dogs vaccinated over two years, showed that at least four died of rabies three to eight months after vaccination (1, 2).
The following reasons (16) for the failure of immunity may be suggested:
- inappropriate vaccination with inadequately stored or improperly injected vaccine
- vaccination during the incubation of rabies or before the onset of an immunological response
- a heavy challenge overwhelming host defences
- intrinsic incapacity in the host.
Whatever the origins of rabies cases recorded in vaccinated dogs, their number seems relatively low in areas contaminated with fox rabies (e.g. in Europe) (20). In France, only ten cases of so-called vaccination failures in dogs (and four among cats) have been registered over a period of twenty-three years (6). This number should be compared with the 4,250,000 cats and dogs vaccinated annually in France (this figure is based on the annual number of vaccine doses sold for domestic carnivores). The probability of a cat or dog becoming rabid if vaccinated can be estimated as 14/(23 x 4,250,000), which is less than 1/6,980,000. In France, dogs in contact with a rabid animal in an enzootic area are not sacrificed and can be kept alive if, prior to contamination, they have been properly vaccinated (with certificate and identification). In such cases, the animals are immediately revaccinated. A study of more than 3,500 dogs which had close contacts (bites in 36% of cases) with foxes (mainly) or other carnivores which were diagnosed as rabid by laboratory examination, revealed that only three dogs developed rabies (50). The failure rate in animals which were definitely contaminated can be estimated as 3/3,500, given that injection of vaccine after contamination has been shown to provide no protection (20). It must be emphasised that these failures were recorded before 1984 and that failure is now less probable, given the generalisation of adjuvanted vaccines for dogs. In the United States of America, four rabies vaccine failures were recorded in cats and dogs in 1988 with 33,182,575 vaccinated domestic carnivores the same year (rate = 1/8,296,000) (34).
Such evaluations could be useful in comparing the risks of vaccination with those of quarantine. For even when they are strictly managed, quarantines still entail a risk. For instance, in many countries, the quarantine period is six months. However, longer incubation periods have been reported in dogs (8.5 months after challenge) (67) and in other carnivores (12 months or more for foxes) (57). According to Sasaki and colleagues (55), Beynon determined that a quarantine period of nine months would be necessary to detect all cases of incubating rabies with a 95% degree of confidence.
3. STUDIES IN CATS
3.1. Neutralising antibodies after vaccination
Although fewer studies have been conducted on vaccination of cats against rabies, several of the characteristics observed in dogs were also observed in cats:
- the kinetics of neutralising antibodies follow the same profile in the two species (8, 23, 64)
- the relationship between the potency of vaccines and the level of neutralising antibodies: Lawson and colleagues (44) have shown that the less diluted modified live vaccines induced the highest rate of seroconversion in vaccinated cats (Table 9), but Lazarowicz and colleagues (45) obtained no correlation of the antigenic value of inactivated virus vaccines as determined by the NIH test and mean neutralising antibody titres in vaccinated cats. However, as for dogs, it is necessary to take account of the fact that the production of antibodies (and protection against challenge) obtained after administration of live virus vaccine (44) and inactivated virus vaccine (45) can show great divergence and are not readily comparable.
- intramuscular vaccination provides longer lasting protection than subcutaneous vaccination (Table 10) (59).
Laboratory cats: intramuscular vaccination with various vaccines, challenge with rabies virus fox strain five weeks and four years after vaccination
Vaccination Cats with detectable antibodies Cats surviving just before challenge challenge Five weeks before challenge ERA undiluted or diluted 1/10 19/19 19/19 Inactivated virus vaccine 20/20 20/20 undiluted or diluted 1/10 ERA diluted 1/100 or 1/1000 5/20 12/20 HEP diluted 1/1000 1/40 11/40 Inactivated virus vaccine diluted 1/1000 0/5 2/5 None 0/11 3/11 Four years before challenge ERA 7/8 8/8 None 0/8 1/10
ERA: Elizabeth (Gaynor) Rokitniki Abelseth
HEP: high egg passage
Concerning the influence of age, Précausta and colleagues (53) described the good neutralising antibody response achieved by three-month-old kittens, even those born of immune queens.
To our knowledge, Blancou and colleagues (18, 19) are the only authors who have tested vaccination results on owned cat populations. Pet cats appeared to respond well to the administration of an adjuvanted vaccine. In contrast, the same authors obtained mild or zero neutralising antibody responses when vaccinating cats sampled from stray populations in France. With stray cats, the worst result was obtained with non-adjuvanted vaccines: 5 of 9 individuals did not respond (Fig. 7).
Influence of the breeding standards of cats on the level of rabies antibody reached one year after vaccination
Comparison of laboratory cats, pet cats in France and stray cats in Tunisia
3.2. Level of neutralising antibodies and result of challenge Challenge under laboratory conditions
Although there are few field studies on the immunity of pet or stray cat populations, there are numerous laboratory studies on challenge of vaccinated cats (Tables 10 to 12).
Laboratory cats: intramuscular vaccination with tissue culture adjuvanted vaccine, challenge with rabies virus NYC-Ga strain, one to three years after vaccination
Vaccination Cats with detectable Cats surviving antibodies just before challenge challenge One year before challenge Yes (subcutaneous) 5/5 5/5 No 0/4 0/4 Three years before challenge Yes (intramuscular) 25/25 24/25* No 0/10 1/10
* Prior to challenge the cat which died of rabies had an antibody titre of 1/2 (endpoint neutralising dilution)
Laboratory cats: subcutaneous vaccination with tissue culture vaccine, challenge with rabies virus NYC-Ga strain, 3.4 to 3.7 years after vaccination
Vaccination Cats with antibodies >0.5 IU/ml Cats surviving challenge 0-6 months before challenge Vaccinated Controls Non-adjuvanted 8/8 8/8 0/5 vaccine Adjuvanted vaccine 5/5 5/5 0/5 Adjuvanted vaccine 8/11 10/10 1/10
Laboratory cats: challenged with rabies virus NYC-Ga strain four to six-and-a-half months after vaccination
Vaccination Cats with antibodies >0.5 Cats surviving challenge IU/ml just before challenge Vaccinated Controls Inactivated virus in cell culture: antigenic value: 0.9* 8/8 7/8** antigenic value: 1.8 5/8 8/8 0/16 Modified live virus: ERA 2/8 3/8***
* Measured by the NIH test, expressed in IU/dose
** The cat which died of rabies had a pre-challenge titre of 5.34 IU/ml
*** The cats which died of rabies had the lowest antibody titres
ERA: Elizabeth (Gaynor) Rokitniki Abelseth
Challenge was performed with a dog strain (NYC-Ga) mimicking the situation of canine street rabies (42, 61, 64) or, in other experiments, a fox strain mimicking the situation of sylvatic fox rabies in continental Europe (38, 44). With both strains, the general conclusion was the same as for dogs: the probability of a cat surviving challenge can be predicted by the level of neutralising antibodies. Of course, unexpected deaths can occur: Kihm and colleagues (42) reported a rabies death in a cat which had a pre-challenge titre of 5.34 IU, and Blancou and colleagues (18) in another cat with a pre-challenge titre of 0.87 IU/ml.
The cumulative challenge results on cats reported by Bunn (26, 27) are described in Figure 8 and Tables 13 and 14. With a neutralising antibody level of more than 0.1 IU (measured by MNT) or more than 0.2 IU (measured by RFFIT), all of the cats survived challenge.
Survival rate after challenge of laboratory cats correlated with the level of rabies antibody reached before challenge
Cats were vaccinated with various vaccines and challenged one year after vaccination with NYC-Ga,
fox or skunk strains; the number of cats in each class is written at the top of the bars
Challenge results from rabies immonogenicity tests conducted in dogs and cats with vaccines approved for use in the United States of America
Vaccine Antibody titre* <5 5-9 10-19 20-39 >40 Flury modified live 1/50** 0/16 0/26 0/15 0/40 vaccine SAD modified live 5/55 3/36 1/41 0/35 1/188 vaccine SAD inactivated virus, 21/156 2/63 1/116 0/79 0/150 tissue culture origin Pasteur inactivated 5/44 1/45 0/38 0/32 0/76 virus, tissue culture origin Pasteur inactivated 13/133 2/62 5/73 1/34 0/164 virus, nervous tissue origin
* antibody titres expressed as 50% endpoint neutralising dilutions established by either the MNT or RFFIT
** challenge results are expressed as number of animals which died/number of animals challenged
SAD: Street Alabama Dufferin strain
Neutralising antibody titres in dogs and cats and protection from challenge with rabies virus
Animals Antibody test Antibody titre* <5 5-9 10-19 20-39 >40 Dogs MNT 56/251** 9/100 9/92 1/63 0/171 RFFIT 84/241 13/112 9/119 0/87 0/201 Total 140/492 22/212 18/211 1/150 0/372 Cats MNT 25/155 5/57 5/94 0/33 0/144 RFFIT 17/87 3/59 1/62 1/49 1/187 Total 42/242 8/116 6/156 1/82 1/331
* antibody titres expressed as 50% endpoint dilutions established by either the virus neutralisation test in mice (MNT) or the rapid fluorescent focus inhibition test (RFFIT)
** challenge results are expressed as number of animals which died/number of animals challenged
Natural infection of vaccinated cats
The safety problem associated with the receptivity of cats to live virus vaccines such as Flury LEP and HEP or Street Alabama Dufferin (SAD) strain vaccines will not be reviewed here (11). But it should be remembered that while cats are the species with the largest number of rabies cases directly induced by the inoculation of live modified virus strains, other species such as dogs and foxes are also receptive (72).
Inactivated virus vaccines are employed on cats as they are more efficient in protecting the species against natural challenge. However, considering the results of challenge experiments on vaccinated cats, natural infection among vaccinated pet cats is suspected to be as frequent as for vaccinated dogs. But investigations on rabies cases in vaccinated cats are scarce: apart from the four cases reported in France (6) there appear to be no other reports. This discrepancy is due to the fact that dogs have been studied considerably more than cats.
4. THE SIGNIFICANCE OF NEUTRALISING ANTIBODIES IN NON-VACCINATED CARNIVORES
4.1. Non-specific and specific neutralising factors
Sekine and colleagues (58) found that sera of normal rabbits and guinea-pigs contained non-specific inhibitors capable of neutralising the virus in the presence of complement. In a well-conducted seroneutralisation on mice, therefore, inactivation of sera is performed for 30 minutes at 56°C. Virus inhibition by other substances was described in infected skunks and foxes (74). Infection by mycobacteria, e.g. Bacillus Calmette-Guérin (BCG), can also induce the production of rabies neutralising antibodies in mice and provide protection against rabies in a number of animals (70). Since more specific immunological tests (such as enzyme-linked immunorescent assay: ELISA) have become widespread, non-specific neutralising factors have not generated further scientific reports.
In endemic areas, serosurveys in wild carnivores demonstrated a high proportion of apparently healthy individuals with neutralising antibodies in serum (54, 71) and it has been suggested that these antibodies may have been produced following contact with virus from other species, which was therefore immunising but rarely fatal (12). However, the same observations have also been reported for dog populations in areas where dog rabies is endemic: in Thailand, in areas where no canine vaccination programme has ever been conducted, 15-20% of dogs had neutralising antibodies, yet remained perfectly normal when observed for prolonged periods (75); similar results had also been reported previously in other countries of Asia and in Africa (3, 36). These observations correlate with the high probability of inter-individual contamination within the reservoir species, which is not the case for pet populations in areas where rabies is endemic. The possibility of non-fatal contamination of dogs by non-canine strains (e.g. those from wild animals living in the region) has also been proposed (20). Several questions thus arise regarding:
a) the specificity of serum titrations and the threshold level for protection against rabies;
b) the possibility of rabies outbreaks in naturally seroconverted dogs, and the interval between seroconversion and the onset of clinical symptoms.
4.2. Rabies infections
The viral infection triggers the production of neutralising antibodies. When a high dose of rabies virus reaches the central nervous system, neutralising antibodies are not detectable before or at the onset of clinical signs; they are usually induced by longer incubation periods. This phenomenon has been studied mainly in laboratory rodents, which supply the chief model of rabies immunopathology (49, 73). Unfortunately (but not surprisingly, considering the difficulty of handling rabid carnivores), there appears to be no literature on the frequency and intensity of neutralising antibody production in non-vaccinated infected cats and dogs. Some data can be found in articles by Artois and colleagues (4), Blancou and colleagues (17) and Fekadu (36) regarding latent or abortive rabies.
Bell and colleagues (10) proved that dogs which recovered from rabies after intracerebral inoculation of homologous strains, had high titres of neutralising antibody in the cerebrospinal fluid as well as in serum and retained these titres for several months, whereas vaccinated dogs did not have high cerebrospinal fluid titres. Murphy and colleagues (48) demonstrated the same phenomenon in cats.
Bell and colleagues (9) were the first to apply cerebrospinal fluid titration for an epidemiological survey. Of 120 dogs sampled in an area where rabies was enzootic (Buenos Aires), none was found to be positive; thus, it cannot be concluded that non-fatal rabies is common.
Blenden and colleagues (21) have suggested that the kinetics of antibody levels in blood and cerebrospinal fluid should be compared, to determine whether specific antibodies have been produced by infection or by immunisation. Without a booster after a first blood and cerebrospinal sampling, the antibody level should remain stable in cases of immunisation, or increase in cases of infection. In fact, such procedures have never been routinely used anywhere. Indeed, given the variability of the titration test, the constancy of an antibody titre over time is difficult to verify even in a vaccinated animal.
Given the lack of easily-performed experimental methods, the only basis for considering that an individual dog or cat possessing rabies neutralising antibodies has been vaccinated is good individual identification and certification.
Laboratory conditions described in the challenge of vaccinated cats and dogs generally appear more severe than natural conditions of challenge in the field. In normal practice, experimenters use extremely long intervals between vaccination and challenge (three to five years) and high virus doses involving 100% mortality in controls. In areas contaminated by fox rabies, natural challenge is not as severe for dogs and this could compensate for the fact that the health status of pets may be lower than that of dogs bred in the laboratory. Epidemiological observation is by far the more important evidence; in continental Europe, rabies vaccination of cats and dogs is so efficient that where the annual risk of a fatal case of rabies has been evaluated for a vaccinated pet, this risk is minute (1/6,980,000). It is also noteworthy that in continental Europe, fox rabies has never been propagated by domestic animals from an enzootic area to a free one - even if administrative rules concerning compulsory confinment, leashing or vaccination have sometimes been broken either deliberately or by the simple fact that rabid pets have escaped from their owners.
If a neutralising antibody titration were required for certifying the immunological capacity of vaccinated animals, two questions would arise regarding:
a) the choice of techniques for antibody titration
b) the definition and acceptance of a minimum antibody titre considered as providing protection against rabies.
A general analysis of challenge experiments leads to the conclusion that neutralising antibody titres enable prediction of survival more often on a qualitative basis (i.e. Do the animals have detectable neutralising antibodies or not?) than on a quantitative basis. This fact becomes apparent when one tries to determine a "protective" threshold. For this purpose, either method of seroneutralisation (RFFIT or MNT) can be employed, provided a correlation between the two methods has been demonstrated in the same laboratory (14, 66).
Agreements on the international transfer of dogs and cats could be formulated, therefore, based on a designated minimum level of neutralising antibodies, and could be proposed as an alternative to quarantine measures. The designated threshold could be based on the results presented in this study. The security of the protection constituted by this threshold would be increased by the extent to which it excedes the level recognised as effective against experimental challenge in cats and dogs (0.1 IU/ml and 0.2 IU/ml, respectively, measured by RFFIT).
The author wishes to express his gratitude to Dr J. Blancou for kindly revising the manuscript of this paper.
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