INTRODUCTION

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Canine parvovirus type 2 (CPV2) emerged in 1978, almost simultaneously in Europe and North America, as a new pathogen of dogs that was responsible for myocarditis and hemorrhagic gastroenteritis in pups (2, 7, 11, 12). The close antigenic and genomic relationships that exist between CPV2, feline panleukopenia virus, and mink enteritis virus (18) suggest that CPV2 may have originated by genetic mutation in a wild host receptive to one of the feline panleukopenia virus-like parvoviruses that infected carnivores (19).

By use of monoclonal antibodies, restriction enzyme analysis, and DNA sequencing, Parrish et al. demonstrated that the original antigenic type (CPV2) has been replaced, over the period from 1979 to 1981, by an antigenic variant or biotype (CPV2a) that differs from the original strain in three coding regions of the gene for the VP2 capsid protein (13, 14). A second biotype (CPV2b) appeared around 1984, and the only significant difference from CPV2a was the substitution of one amino acid (AsnAsp) in the VP2 protein (13, 14). Both of these biotypes have now replaced the original strain CPV2 throughout the canine population worldwide. In particular, in the United Kingdom, Australia, and Italy the CPV2a biotype is more common than the CPV2b biotype; in Germany and Spain the two biotypes appear to be distributed about equally; and, in contrast, CPV2b appears to be more common in the United States (6, 8, 10).

An important question concerns the clinical and immunological significance of the antigenic variation of CPV2. Previously, experiments have not demonstrated any significant relevance of the antigenic changes with respect to the ability of CPV2 vaccines to protect dogs from the infection (1, 9). Furthermore, a preliminary study showed a one-way cross-reactivity (CPV2bCPV2) of sera from pups inoculated with CPV2 or CPV2b modified live virus vaccines (17).

The aim of this study was to compare the neutralizing antibody titers of two groups of dogs inoculated, respectively, with a CPV2 or CPV2b modified live virus vaccine. Our results pose questions regarding the interpretation of serological data, especially those obtained by hemagglutination inhibition (HI) tests, with respect to the immune status of pups.

	MATERIALS AND METHODS

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Vaccines. (i) CPV2 vaccine. A modified live CPV2 vaccine (17/80 ISS strain) (3) with a titer of 10^5.50 tissue culture infectious doses (TCID₅₀)/ml was used. The virus was cultivated on the canine A-72 cell line grown in Dulbecco minimal essential medium (DMEM) supplemented with 10% fetal calf serum.

(ii) CPV2b vaccine. A modified live CPV2b vaccine (29/97-40 strain) (5) with a titer of 10^4.50 TCID₅₀ was used. The virus was cultivated on the Crandell feline kidney (CrFK) cell line grown in DMEM supplemented with 10% fetal calf serum.

(iii) Virus titrations. The virus titration test was performed in microtiter plates. Tenfold dilutions of each virus were prepared in quadruplicate in DMEM and mixed with 50 µl of a suspension containing 200,000 A-72 cells for CPV2 vaccine and 200,000 CrFK cells for CPV2b vaccine. The plates were incubated at 37°C for 5 days in a humidified CO₂ atmosphere. The plates were then frozen and thawed three times, and the supernatant of each well was tested for CPV hemagglutination (HA) activity using 1% pig erythrocytes. Fifty percent end points were calculated using the Kärber formula.

Experimental procedures. Thirty-six pups, 9 to 10 weeks old, from seven litters were randomly assigned to two groups (A and B) and housed in two separate and isolated facilities. The pups in each group were handled by different workers. All pups were serologically negative to CPV at the time of vaccination, as determined by HI and neutralization (Nt) tests. Group A pups (n = 18) were inoculated subcutaneously with 1 ml of the CPV2 vaccine, and group B pups (n = 18) received 1 ml of the CPV2b vaccine. Thirty days after vaccination, the antibody titer of each pup was evaluated by HI and Nt tests using both CPV2 and CPV2b viruses. No illness was observed in any pup throughout the study.

Serological assays. (i) HI test. HI tests were carried out at 4°C using 1% pig erythrocytes and 8 HA units of either CPV2 (17/80 ISS strain) or CPV2b (29/97-40 strain). Serial twofold serum dilutions were made in phosphate-buffered saline (pH 7.2), starting from a 1:10 dilution. Titers were expressed as the reciprocal of the highest serum dilution that completely inhibited the HA, and the geometric means were also calculated.

(ii) Nt test. Serial twofold dilutions (starting from 1:10) of each serum in DMEM were mixed with 50 µl of a virus suspension containing 100 to 300 TCID₅₀ of either CPV2 (17/80 ISS strain) or CPV2b (29/97-40 strain). After 1 h of incubation at room temperature, 100 µl of a suspension containing 200,000 A-72 cells for CPV2 and 200,000 CrFK cells for CPV2b was added to each well. The plates were incubated at 37°C in a humidified CO₂ atmosphere for 5 days, and virus was then detected using the HA test as described above for virus titration. Neutralizing antibody titers were calculated as the reciprocal of the highest serum dilution that completely neutralized the virus (absence of HA activity). The geometric means of each group (A and B) also were calculated.

Statistical analysis. The statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance on ranks.

	RESULTS

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The results of HI tests carried out on the sera of pups inoculated with CPV2 (group A) or CPV2b (group B) are reported in Tables 1 and 2, respectively. For dogs given CPV2 (group A) (Table 1), the homologous geometric mean antibody titer was 1,950 and the mean heterologous titer was 1,138, and this difference is statistically significant (P = 0.036). For dogs inoculated with CPV2b (group B) (Table 2), the homologous and heterologous geometric means were, respectively, 3,475 and 2,655; the difference is not statistically significant (P = 0.108).

The Nt antibody titers of pups inoculated with CPV2 (group A) or CPV2b (group B) are shown in Tables 3 and 4, respectively. In general, higher titers were observed in pups inoculated with CPV2 than in pups inoculated with CPV2b. The homologous geometric mean antibody titer for pups in group A (Table 3), inoculated with CPV2, was 4,732, whereas the mean heterologous titer was 162, which is a statistically significant difference (P < 0.01). Pups in group B (Table 4), inoculated with CPV2b, had a homologous mean titer of 1,138 and a mean heterologous titer of 940, which is not statistically significant (P = 0.184).

	DISCUSSION

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Effective vaccines are generally available for the prevention of CPV2 infection. Both modified live and inactivated CPV2 vaccines have been demonstrated to have the ability to immunize pups efficiently. Inactivated vaccines, however, provide only a short immunity to the infection. Although dogs may be protected for several months against disease, they may have subclinical infection (15). In contrast, modified live vaccines have been shown to protect dogs for at least 2 to 3 years.

Previous studies (4, 15, 16) have demonstrated that pups with HI titers of >1:80 are immune to oronasal CPV2 challenge. In those studies, clinical illness was not observed, virus was not recovered from fecal specimens, and active serological responses were not observed after challenge. On the other hand, pups with HI titers of <1:40 become subclinically infected, with virus shedding and antibody responses indicative of active infection.

The results of this study raise puzzling questions about the clinical significance of the antigenic CPV biotypes regarding (i) the serological tests presently used for diagnosis and (ii) the actual immunity of vaccinated dogs, considering that most of the licensed CPV vaccines are presently produced from virus (CPV2) with the original antigenic structure.

The HI test using CPV2 as the antigen is presently employed by most investigators to measure antibody titers to CPV, since it has been correlated with the level of pup immunity to CPV2. However, the results presented here and our field experience indicate that the HI test, unlike the Nt test, does not always detect differences in amounts of antibody against either the homologous (CPV2) or the heterologous (CPV2b) virus. In contrast, using the Nt test, the antibody titers of pups vaccinated with CPV2 were much higher to the homologous virus (CPV2) than to the heterologous virus (CPV2b). These findings suggest that HI antibody values using a CPV2 antigen may not allow a true prediction of the earliest age at which pups would be expected to become susceptible to CPV infection or amenable to immunization. In several cases which we investigated, pups with HI of titers 1:160 to 1:320 to CPV2 were not vaccinated because they were considered still protected, yet they developed parvoviral disease caused by CPV2b shortly after serological evaluation was done (C. Buonavoglia, unpublished data). Such findings prompted this study.

Another question concerns the real immunity conferred by CPV2 vaccines against the CPV2a and CPV2b biotypes now circulating in the dog population. Our results (obtained by serological tests and without challenge trials) revealed that pups inoculated with CPV2 vaccine had significantly higher Nt antibody titers to the homologous virus (CPV2) than to the heterologous virus (CPV2b). In contrast, pups inoculated with CPV2b vaccine had similar Nt antibody titers to both viruses.

Despite the serological problem noted here, the problem may not be critical because, in the field, documented parvoviral infections due to CPV2 vaccine failures as a consequence of a low level of immunity in dogs against the heterologous viruses are rare. Pups vaccinated with CPV2 possess antibodies against the heterologous virus which probably are at levels which still provide complete or partial immunity to young pups at 2 to 4 months of age, a time when they are more susceptible to CPV infection. Nevertheless, there is evidence to suggest that optimal protection may not be provided if CPV2 vaccines are used, considering that (i) the original CPV2 has disappeared from the canine population worldwide and (ii) the CPV2 vaccine appears to confer a somewhat lower and shorter immunity against the CPV2b biotype. As suggested in 1982, "more effective vaccines that induce longer lived immunity to infection should be sought to control the spread of canine parvovirus" (15).