J. Vet. Med. B 51, 99–103 (2004) 2004 Blackwell Verlag, Berlin ISSN 0931–1793
Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland
Antibody Titres Against Canine Distemper Virus in Vaccinated and Unvaccinated Dogs A. Jo´z´wik1,3, T. Frymus1, B. Mizak2 and A. RzeZ_ utka2 Addresses of authors: 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland; 2Department of Carnivores and Fur Animal Diseases, National Veterinary Research Institute, Pulawy, Poland; 3 Corresponding author: Tel.: +48 22 8475818; fax: +48 22 8475818; E-mail:
[email protected] With 3 figures and 1 table
Received for publication October 27, 2003
Summary In 248 urban dogs the serum antibody titre against canine distemper virus (CDV) was determined by the immunoperoxidase monolayer assay. Among them 72 animals had been routinely vaccinated at least once against distemper, 20 dogs had never been immunized and the vaccination history of 156 was unknown. The mean antibody titre in vaccinated dogs (114 ± 8.2) indicated immunity (‡100) and was significantly higher when compared with unvaccinated animals (29 ± 8.8, P < 0.05) or with dogs with unknown vaccination history (29 ± 5.0, P < 0.001). Among 156 dogs with unknown vaccination history only 26 (19%) had antibody titres indicating immunity against CDV (‡100). Vaccinated dogs were divided into five groups according to the time since the most recent distemper vaccination: group A: up to 1 month (n ¼ 13); group B: 1–2 months (n ¼ 6); group C: 2–12 months (n ¼ 20); group D: 12–24 months (n ¼ 4); group E: 25–36 months (n ¼ 9) and group F: 36 or more months (n ¼ 18). The mean titre in group A was 490 ± 4.5, in B 359 ± 3.8, in C 204 ± 4.6, in D 269 ± 1.9 and the differences between these groups were not significant. However, dogs in groups E and F had significantly lower mean antibody titres (21 ± 8.3, P < 0.01 and 39 ± 7.6, P < 0.001, respectively) when compared with group A. Nine dogs suffering from distemper confirmed by the immunofluorescence test or nested RT-PCR were also included in this study. In five of them high titres of anti-CDV antibody were detected (160–1280). All these animals recovered (in two localized muscle twitching persisted). On the contrary, the remaining four patients with low antibody titre to CDV (5–40) died.
Introduction Outbreaks of canine distemper have been recently documented in several countries, such as Denmark (Blixenkrone-Møller et al., 1993), the US (Patronek et al., 1995), Japan (Gemma et al., 1996), Finland (Ek-Kommonen et al., 1997) and Germany (Haas et al., 1997). In a previous report we demonstrated that distemper is still an important infection also in Poland and that vaccination significantly reduces the risk of this disease (Jo´z´wik and Frymus, 2002). However, the duration of the post-vaccinal immunity against the canine distemper virus (CDV) and also the required frequency of revaccinations of adult dogs remain controversial aspects of the immunoprophylaxis of distemper. The first studies by Baker in 1962 showed that 1 year after vaccination the antibody titre U. S. Copyright Clearance Center Code Statement:
decreased to low levels in 33% of dogs, and after 2 years in the remaining animals (cit. after Carmichael, 1999). Probably according to these data, all vaccine manufacturers recommended annual revaccinations against distemper, what has been established as a common practice in many countries including Poland. However, some recent data indicate that the postvaccinal immunity against CDV persists much longer than 1 year. Olson et al. (1997) examined serologically 30 dogs exported after the vaccination to Iceland, which is considered to be free from CDV and where vaccination against distemper is not permitted. They demonstrated in 73.3% of these animals neutralizing titres ‡16 of anti-CDV antibody after a mean time of 5 years and 5 months since the most recent immunization (range from 4 years and 2 months to 9 years and 10 months). Similarly Carmichael (1999) in nine vaccinated beagles kept in strict isolation found even after 6 years neutralizing antibody titre ‡80 against distemper. Also the studies of Schultz (1999) revealed that after a vaccination against distemper 95% of dogs were protected from clinical disease for at least 5 years. It has been also pointed out that the recommended vaccination schedules for adult dogs are contrary with those widely accepted in the human medicine (Paul et al., 2003). Welldocumented epidemiological studies indicate that immunity against many diseases induced in human beings by vaccination is extremely long lasting, even life-long. For example, vaccination against measles, a Morbillivirus infection with several similarities to distemper, is carried out only in children, without revaccination of adults (Collier and Oxford, 1993). Based on these data the need for annual revaccination of adult dogs against the most important diseases including distemper has been questioned recently by the American Animal Hospital Association Canine Vaccine Task Force (Paul et al., 2003). However, the post-vaccinal immunity developed in experimental conditions in isolated dogs may last longer than that obtained in a natural environment. It is well known that many factors can suppress the immune response to vaccination. The most important of them are the maternal immunity of a puppy, helminth infestation and stress because of other reasons (Greene, 1998; Jarrett and Ramsey, 2001). Also, improper handling and storage of the vaccine may affect its efficacy, specially in the case of immunization against distemper (Chappuis, 1995). Finally, commercially available vaccines may have different immunogenicity. A major distemper epidemic has been described in Finland in dogs vaccinated with a product of poor efficacy (Ek-Kommonen et al., 1997;
0931–1793/2004/5103–0099$15.00/0
www.blackwell-synergy.com
A. Jo´z´wik et al.
100
Two hundred and forty-eight healthy and sick dogs brought to the Clinic of Small Animals of the Warsaw Agricultural University were examined serologically to determine the antibody titre against CDV. Seventy-two animals had been vaccinated previously at least once against distemper, 20 dogs had never been immunized against this disease and in 156 dogs the vaccination history was unknown. In the group of vaccinated animals, the time of the last immunization against distemper was recorded. Seven dogs were less than 3 months old, 14 animals were between 3 and 6 months old, 15 were between 7 and 12 months old, 21 were between 1 and 3 years old, 59 were between 3 and 6 years old and 132 dogs were older than 6 years. At the time of blood sampling 22 dogs revealed clinical signs suggestive of distemper. Beside serological testing these animals were also examined by the direct immunofluorescence test or by nested RT-PCR to confirm the CDV infection as described earlier (Jo´z´wik and Frymus, 2002; Jo´z´wik and Frymus, 2004). Blood samples were collected from the cephalic vein and were centrifuged to obtain sera. Collected sera were frozen at )20C until further analysis. Serum antibody titres were determined by the immunoperoxidase monolayer assay (IPMA) as previously described (Mizak and Borowski, 1998; Borowski and Kimak, 2001). Briefly, each serum was serially diluted and added into microwells, which had been previously overlaid with Vero cells infected with the Lederle strain of CDV. Rabbit anti-dog IgG (Sigma-Aldrich, Poland) labelled with horseradish peroxidase was used as a conjugate and 3-amino-9-ethylcarbazole (Sigma) as a substrate. The plates were read by the use of the light microscope Jenaval FA (Carl Zeiss Jena, Jena, Germany) at magnification 100·. For the purpose of determining whether dogs were immune against distemper, the titre ‡100 was considered to indicate immunity ( Borowski and Kimak, 2001; A. Borowski, B. Mizak and I. Kimak, unpublished data). The animals were divided into groups depending on the vaccination status (vaccinated, unvaccinated, unknown history) and time since the most recent immunization against distemper (group A: up to 1 month; group B: 1–2 months; C: 2–12 months; D: 12–24 months; E: 25–36 months and F: 36 or more months). Antibody titres in each group were expressed in log2 and geometric mean as well as standard deviations were obtained. The significance of the differences between the groups were determined by the Student’s t-test using the computer program Statistica (StatSoft, Krako´w, Poland) and Microsoft Excel (Microsoft, Troy, NY, USA). For statistical analysis, a value of P < 0.05 was considered significant.
The geometric mean titres in the groups of dogs with different vaccination history are shown in Fig 1. The anti-CDV antibody titres depending on the time of the most recent distemper vaccination are shown in Fig. 2. Among 156 dogs with unknown vaccination history only 26 (19%) had antibody titre indicating immunity (‡100) against distemper, whereas most (127) animals appeared to be susceptible to this disease (Fig. 3). Among 22 dogs suspected of having distemper in nine animals the CDV infection was confirmed by the immunofluorescence test or nested RT-PCR. Only in five of these nine infected patients high titres of anti-CDV antibody were detected. Among these five dogs with strong serologic response (‡100), three recovered completely and in two animals only localized twitching of head and limb muscles were seen till the end of the observation period (>12 weeks). On the contrary, all four patients with low antibody response ( 0.05.
600 Geometric mean titre
Rikula et al., 2000). Because of these factors and little evidence on the duration of protective immunity in dogs vaccinated under natural conditions many authors still recommend annual booster in adult animals (McCaw et al., 1998; Jarrett and Ramsey, 2001). Taking into account these discrepancies the aim of the study was to investigate in natural conditions the anti-CDV antibody titres in dogs at different time after a routine distemper vaccination and to analyse the immunity of these animals in comparison with non-vaccinated dogs.
500
490 ± 4.5
400
359 ± 3.8
300
269 ± 1.9 204 ± 4.6
200 100
21 ± 8.3
39 ± 7.6
0 A up – 1 month (n =13)
B 1–2 months (n =6)
C 2 – 12 months (n =20)
D 12 – 24 months (n =4)
E 25 – 36 months (n =9)
F more than 36 months (n =18)
Time since the last distemper vaccination
Fig. 2. Mean anti-canine distemper virus serum antibody titres in dogs at different time since the most recent distemper vaccination (by immunoperoxidase monolayer assay). AB: P > 0.05; AC: P > 0.05; AD: P > 0.05; AE: P < 0.01; AF: P < 0.001.
Death 5 + +
Death 10 + +
Death 40 Not done +
40 + +
Derived from a rural area No # Mixed 731A
5
No # St Bernardshund 815A
2,5
No #
Mastino napoletano Mixed 738A
32A
Mixed 818A
7
Contacts with dogs
Laryngotracheitis, bronchitis and gastroenteritis Localized twitching of muscles, ataxia, paresis Laryngotracheitis, bronchitis, neurological signs, hyperkeratosis of nose and footpads Purulent nasal discharge, localized twitching of muscles Recently visited a dog show, sick since 9 days Neurological signs since 1 month Yes $
Dachshund 33A
5
+ ) Pneumonia 2 weeks ago No $
Mixed Mixed 726A 759A
7
No #
Siberian husky 752A
12
Yes Yes $ $
320
Localized twitching of muscles Localized twitching of muscles Death 640 Not done +
Laryngotracheitis, bronchitis, neurological signs, hyperkeratosis of the nose and footpads Localized twitching of the muscles of limbs
Recovery Recovery 1280 160 + + Purulent ocular and nasal discharge Purulent nasal discharge
) )
1280 + +
Breed No.
5 12
Clinical signs History Sex
Vaccinated against distemper Age (m-ths)
Table 1. Epidemiological, serological and virological data of nine dogs with clinical distemper
The standard for assessment the level of immunity to distemper is to challenge the vaccinated animals with a virulent strain, but as an alternative method, measurement of serum antibody titres has been widely used (McCaw et al., 1998; Carmichael, 1999; Schultz, 1999). The importance of the humoural immune response of a dog in determining the outcome of infection by CDV has been well documented (Appel, 1969; Krakowka et al., 1975; Rima et al., 1991; Greene and Appel, 1998). However, titres considered to indicate immunity vary among laboratories and methodologies. Krakowka et al. (1975) reported that vaccinated dogs with antibody titres 16 in the serum neutralization (SN) test survived challenge with virulent CDV. Rima et al. (1991) reported that dogs with neutralizing antibody titres higher than 50 in the SN test recovered from distemper. Carmichael (1999) showed in vaccinated beagle dogs kept in strict isolation that neutralizing antibody titre ‡80 indicated immunity to CDV. McCaw et al. (1998) considered in vaccinated dogs SN antibody titre ‡96 as protective. Gillespie (1966) showed that pups with maternal antibody SN titres above 100 were definitely immune and that this is a serologic assurance that the dog is resistant to distemper. Besides the SN assay other methods have been used to determine the antibody level to CDV in dogs. Twark and Dodds (2000) studied the post-vaccinal immunity to CDV by the indirect immunofluorescent antibody test. We used a similar method, the immunoperoxidase monolayer assay (IPMA). The comparative studies of Borowski and Kimak (2001) of the SN test and IPMA revealed a high correlation coefficient (r ¼ 0.94) between both methods and a high repeatability of titres using IPMA. Within distemper vaccines control system they demonstrated that only these dogs survived the challenge, which developed IPMA titre at least 100 (A. Borowski, B. Mizak and I. Kimak, unpublished data). Thus we considered in our study this antibody level as indicating immunity. Also our findings in dogs suffering from distemper confirm that only patients with IPMA titre ‡100 are able to recover (Table 1). Our studies were performed on dogs routinely vaccinated with commercially available live virus products. There is one Polish distemper vaccine in the market, but to the best of our knowledge the most frequently used products in Warsaw in the last decade were manufactured by Fort Dodge, Pfizer, Intervet and Merial. We found that in dogs vaccinated at least once against distemper, the mean serum antibody titre to CDV was 114, whereas animals never immunized against
CDV detected by IF assay (imprints)
Discussion
At present not observed
Fig. 3. Percentage of dogs with serum antibody titres indicating immunity to distemper (‡ 100 in the immunoperoxidase monolayer assay) among 156 animals with unknown vaccination history.
101
An episode of laryngotracheitis 10 days ago Sick since 3 days Lives in a dog shelter, sick since 1 month Derived from a dog shelter
CDV detected by RT-PCR (blood)
81%
Yes
Anti-CDV Ab titre (IPMA)
Titre 12 weeks). On the contrary, all four patients with low antibody response to CDV died. They suffered from distemper for longer than 9 days but in none of them antibody titre achieved 100 despite administration of antiCDV immunoglobulin therapy to two of them. The results shown in Table 1 confirm that serological examination of a distemper patient may have a prognostic value. It can be concluded that dogs from an urban population vaccinated with commercial modified live virus distemper vaccines have for 2 years antibody titres indicating immunity, and then the antibody level significantly decreases. Unknown vaccination history should raise suspicion, that the dog is not protected against CDV and prompt for basic vaccination.
References Appel, M. J. G., 1969: Pathogenesis of canine distemper. Am. J. Vet. Res. 30, 1167–1182.
A. Jo´z´wik et al. Blixenkrone-Møller, M., V. Svansson, P. Have, C. O¨rvell, M. Appel, I. R., Pedersen, H. H. Dietz, and P. Henriksen, 1993: Studies on manifestations of canine distemper virus infection in an urban dog population. Vet. Microb. 37, 163–173. Borowski, A., and I. Kimak, 2001: Application of IPMA for detection of antibodies to canine distemper virus. Medycyna Wet. 57, 595–597 (in Polish). Carmichael, L. E., 1999: Canine viral vaccines at a turning point – a personal perspective. In: Schultz R.D. (ed.), Veterinary Vaccines and Diagnostics. Advances in Veterinary Medicine, 41, 289–308. Chappuis, G., 1995: Control of canine distemper. Vet. Microbiol. 44, 351–358. Collier, L., and J. Oxford, 1993: Childhood infections caused by paramyxoviruses. In: Collier, L., and J. Oxford (eds), Human Virology, pp. 113–117. Oxford University Press, Oxford. Ek-Kommonen, C., L. Sihvonen, K. Pekkanen, U. Rikula, and L. Nuotio, 1997: Outbreak of canine distemper in vaccinated dogs in Finland. Vet. Rec. 141, 380–383. Gemma, T., T. Watari, K. Akiyama, N. Miyashita, Y.-S. Shin, K. Iwatsuki, Ch. Kai, and T. Mikami, 1996: Epidemiological observations on recent outbreaks of canine distemper in Tokyo. J. Vet. Med. Sci. 58, 547–550. Gillespie, J. H., 1966: The significance of passive immunity and the biological tests used in the study of distemper. J. Am. Vet. Med. Assoc. 149, 623–628. Greene, C. E., 1998: Canine immunization recommendations. In: Greene, C. E. (ed.), Infectious Diseases of the Dog and Cat, pp. 751–752. W. B. Saunders Company, Philadelphia. Greene C. E., and M. J. Appel, 1998: Canine distemper. In: Greene, C. E. (ed.), Infectious Diseases of the Dog and Cat, pp. 9–22. W. B. Saunders Company, Philadelphia. Haas, L., T. Harder, H. Liermann, W. Martens, I. Greiser-Wilke, D. Maack, V. von Messling, and B. Liess, 1997: Zur Situation der Hundestaupe in Deutschland. Kleintierpraxis 42, 613–620. Jarrett, O., and I. Ramsey, 2001: Vaccination. In: Ramsey I., and B. Tennant (ed.), Manual of Canine and Feline Infectious Diseases, pp. 41–51. British Small Animal Veterinary Association, Gloucester. Jo´z´wik, A., and T. Frymus, 2002: Natural distemper in vaccinated and unvaccinated dogs in Warsaw. J. Vet. Med. B. 49, 413–414. Jo´z´wik, A., and T. Frymus, 2004: Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper. Vet. Res. Commun. (in press). Krakowka, S., R. Olsen, A. Confer, A. Koestner, and B. McCullough, 1975: Serologic response to canine distemper viral antigens in gnotobiotic dogs infected with canine distemper virus. J. Inf. Dis. 132, 384–392. McCaw, D. L., M. Thompson, D. Tate, A. Bonderer, and Y. J. Chen, 1998: Serum distemper virus and parvovirus antibody titres among dogs brought to a veterinary hospital for revaccination. J. Am. Vet. Med. Assoc. 213, 72–75. Mizak, B., and A. Borowski, 1998: Application of PLA test for the evaluation of canine distemper virus, adenovirus type 1 and parvovirus replication in cell cultures. Medycyna Wet. 54, 753–756 (in Polish). Olson, P., H. Finnsdo´ttir, B. Klingeborn, and A˚. Hedhammar, 1997: Duration of antibodies elicited by canine distemper virus vaccinations in dogs. Vet. Rec. 141, 654–655. Patronek, G. J., L. T. Glickman, R. Johnson, and T. J. Emerick, 1995: Canine distemper infection in pet dogs: a case control study of risk factors during a suspected outbreak in Indiana. J. Am. An. Hosp. Assoc. 31, 230–235. Paul, M. A., M. Appel, R. Barrett, L. E. Carmichael, H. Childers, S. Cotter, A. Davidson, R. Ford, D. Keil, M. Lappin, R. Schultz, E. Thacker, J. L. Trumpeter, and L. Welborn, 2003: Report of the American Animal Hospital Association (AAHA) Canine Vaccine Task Force: executive summary and 2003 canine vaccine guidelines and recommendations. J. Am. An. Hosp. Assoc. 39, 119–131.
Antibody Titres Against Canine Distemper Virus in Vaccinated and Unvaccinated Dogs Rikula, U., L. Nuotio, and L. Sihvonen, 2000: Canine distemper virus neutralising antibodies in vaccinated dogs. Vet. Rec. 147, 598–603. Rima, B. K., N. Duffy, W. J. Mitchell, B. A. Summers, and M. J. G. Appel, 1991: Correlation between humoral immune responses and presence of virus in the CNS in dogs experimentally infected with canine distemper virus. Arch. Virol. 121, 1–8. Schultz, R. D., 1999: Duration of immunity to canine vaccines: what we know and don’t know. In: Carmichael L. (ed.), Canine Infectious
103
Diseases: From Clinics to Molecular Pathogenesis. International Veterinary Information Service, Ithaca. (http://www.ivis.org/ proceedings/Baker_Can_Inf_Dis/bpd/d21_frm.asp). Twark, L., and W. J. Dodds, 2000: Clinical use of serum parvocvirus and distemper virus antibody titres for determining revaccination strategies in healthy dogs. J. Am. Vet. Med. Assoc. 217, 1021–1024.
