What we need to know

Professor Michael J. Day

BSc BVMS(Hons) PhD DSc DiplECVP FASM FRCPath FRCVS

School of Veterinary Sciences, University of Bristol

INTRODUCTION: Vaccination and Titer Testing in Dogs and Cats

Vaccination delivery to dogs and cats has changed in the past decade. In response to questions over vaccine safety, guidelines groups have introduced new vaccination schedules that vaccine manufacturers have accommodated by introducing products with
extended duration of immunity (DOI) and products with fewer antigenic components. The latest advance in vaccinology is the availability of simple in-practice test kits that demonstrate whether an individual animal has serological evidence of protection. These test kits can now inform decision-making about vaccination in practice. This presentation briefly reviews recommended vaccination schedules and focuses on the potential applications for in-house serological testing.

VACCINATION GUIDELINES: Vaccination and Titer Testing in Dogs and Cats

The WSAVA Vaccination Guidelines, as endorsed by BSAVA, consider vaccines as CORE or NON-CORE. CORE vaccines are those that every dog or cat should receive as they confer protection against diseases that are life-threatening or of significant morbidity. Even in developed countries, these
diseases have not been eliminated, and the occurrence of regional outbreaks indicates the importance of maintaining herd immunity through vaccination. The CORE vaccine-preventable diseases of the dog are those induced by canine distemper virus (CDV), canine parvovirus-2 (CPV), and canine adenovirus-1 (CAV). The CORE vaccine-preventable diseases of the cat are those induced by feline parvovirus (FPV), feline calicivirus (FCV), and feline herpesvirus-1 (FHV). WSAVA guidelines encourage the use of NON-CORE vaccines after making an appropriate benefit-risk analysis tailored to the individual pet's lifestyle and risk of exposure. For that reason, in the UK, practitioners generally include vaccines to prevent leptospirosis as a CORE canine vaccine. They may include feline leukemia virus (FeLV) vaccines in the CORE feline vaccination schedule.

Modified live virus (MLV) or ‘infectious’ CORE vaccines should be administered to puppies and kittens from 8 – 9 weeks of age, with a second dose given 3 – 4 weeks later and a final dose between 14 – 16 weeks. This change ensures that all pups and kittens can mount at least a primary immune response to vaccination – even when maternally-derived antibody (MDA) persists to 12 weeks of age. Pups and kittens should also receive a booster CORE vaccine either 12 months after the final of the early life series or at 12 months of age.

Adult dogs and cats should receive MLV CORE vaccines no more frequently than every three years, and WSAVA recommendations also call for triennial revaccination with FeLV. In the UK, most canine CORE vaccines on the market now carry a licensed 3-year DOI, and at least some FPV vaccines are licensed for triennial use. NON-CORE or ‘non-infectious’ vaccines other than FeLV must still be given annually to adult animals where these products are incorporated into an individualized vaccination program.

The over-arching new concept in vaccination is that vaccines be delivered as one component of an ‘annual health check’ consultation that addresses all aspects of the health and well-being of the individual animal. Vaccination programs should be tailored to the requirements of the specific pet (individualized medicine) based on a thorough assessment of that animal's lifestyle and risk factors. Most UK practitioners will currently administer triennial MLV CORE vaccines to the dog with an annual leptospirosis vaccine. Practitioners will also use a triennial program for feline CORE vaccines. Still, where an individual cat is perceived as having a higher risk for the feline infectious respiratory disease complex, that cat may receive annual FCV and FHV components, which are available as a separate combination product.

CORRELATES OF PROTECTION: Vaccination and Titer Testing in Dogs and Cats

For licensing studies, it is still necessary for animals to be used in experimental challenge studies in which vaccinated animals are challenged with the virulent form of a pathogen some time (e.g., 3 or 4 years) after vaccination to demonstrate either sterilizing immunity (failure of the pathogen to infect) or amelioration of clinical disease (infection occurs but the effects are limited). Studies over the decades have shown strong correlates of protection in such challenging studies.

For CDV, CPV, CAV, and FPV, the presence of serum antibodies that neutralize infectious viruses and prevent infection and disease provides a robust correlate of protection. This correlation is so strong that it is possible to state that the presence of serum antibodies to one of those viruses equates definitively with protective immunity. Some regulatory authorities are now beginning to accept seroprotection rather than an experimental challenge in modulating license claims. The presence of serum antibodies does not, however, provide a correlate of immunity for FCV and FHV protection. For respiratory pathogens such as FCV, mucosal secretory IgA provides a correlate of protection, but it is impossible to measure these antibodies routinely. For FHV, there is a stronger correlation between safety and cell-mediated immunity (CMI), but it is challenging to measure CMI on a routine basis.

GOLD STANDARD TESTS: Vaccination and Titer Testing in Dogs and Cats

Traditionally, two gold standard tests for CORE virus infections have measured the correlation between challenge immunity and seroprotection. These are the virus neutralization (VN) and haemagglutination inhibition (HAI) tests. A positive VN test indicates that serum from the animal contains antibodies that will neutralize infectious virus particles in vitro and prevent them from subsequently producing infection and cell damage (as assessed by the cytopathic effect on cells in vitro). A positive HAI test indicates that serum from the animal contains antibodies that will bind to infectious virus particles and neutralize the ability of that virus to cause agglutination of erythrocytes from particular animal species.

An excellent correlation exists between a positive VN test and protection against CDV, CPV, CAV, FPV, and rabies. There is also an excellent correlation between a positive HAI test and protection against CPV and FPV. For FCV, the positive VN test and protection correlation are considered only good to fair (as secretory IgA provides a better measure). For FHV, the positive VN test and protection correlation are only fair (as CMI is a better correlate of immunity).

Other serological test methods (e.g., ELISA or IFA-based) must, in turn, be correlated with the gold standard VN or HAI tests. Both of the in-house test systems that will be described below have had such correlation and validation with sera derived from animals in challenge studies tested by the
gold standards.

THE CONCEPT OF TITER: Vaccination and Titer Testing in Dogs and Cats

A titer measures the concentration of antibodies in a serum sample. Obtaining a titer involves performing an immunological test in which the serum sample is subjected to a series of doubling dilutions (that progressively reduce the antibody concentration). Each serum dilution is tested, and the highest dilution that gives an unequivocally positive reaction in the test provides the titer. The titer is the reciprocal of that dilution (e.g., a dilution of 1/10 provides a titer of 10).

Testing laboratories have traditionally provided a titer for serological tests of antibodies related to vaccine protection. The actual number may sometimes differ for the same sample tested by different laboratories. The number is relatively arbitrary as the titer is not a single defined
number but represents a range. For example, a sample with a titer of 10 indicates that the value is not less than 5 and not more than 20 (one doubling dilution above and below the titer). A sample with a titer of 1280 has a titer that is not less than 640 and not more than 2560. In
this regard, a titer of 640 from one laboratory is the same as one of 2560 from another testing laboratory.

For this reason, the WSAVA Vaccination Guidelines Group encourages practitioners to consider that any titer (above the cut-off for the gold standard test; typically 20 for HAI and 100 for VN) should be regarded as positive, and protection in challenge studies is still conferred throughout a broad spectrum of titers. For this reason, the new in-house test kits either provide a simple yes-no answer or a semiquantitative score – as antibodies above control levels correlate with protection (for those diseases defined above).

IN-HOUSE TEST KITS: Vaccination and Titer Testing in Dogs and Cats

Now, two companies produce in-house test kits for the determination of protective serum antibodies to CORE infectious diseases post-vaccination. Both test kit systems are simple to use, provide a rapid answer (protection or not) within 20 – 30 minutes, and are relatively inexpensive (costing around the same for testing as for revaccinating the animal). Several diagnostic laboratories have validated Both test kit systems and correlated them with gold-standard tests. The test kits are the TiterCHEKTM system (manufactured by Synbiotics and now owned and distributed by Pfizer) and the VacciCheckTM system (produced by Biogal Laboratories). The TiterCHEKTM system provides a yes-no (protected or not protected) answer for CDV and CPV. The VacciCheckTM system provides a semiquantitative score for serum antibody titers against CDV, CAV, and CPV. A feline VacciCheckTM system scores serum antibody titers against FPV, FCV, and FHV. The kits have excellent overall sensitivity (detecting samples with antibodies from those seropositive by gold standard) and specificity (detecting samples without antibodies from those seronegative by gold standard).

A set of excellent ‘YouTube’ videos produced by the US Charity ‘Maddies Fund’ is available on the web that provides very clear instructions on how to perform and interpret each of these test systems. Minor differences between the two systems are summarized below:

APPLICATIONS OF IN-HOUSE TESTING

To Determine Puppy Protection and Detect Genetic Non-Responders: Using in-house test kits provides a simple measure of whether a puppy (CDV, CAV, CPV) or kitten (FPV) is protected after the initial series of early life vaccinations. This has the benefit of identifying animals that may not have responded to early life vaccination (particularly where a 14 – 16-week vaccine is not given) and may remain unprotected until the time of the 12-month booster. At this stage, a seropositive and protected animal may not require the 12-month booster and could go straight to a triennial CORE vaccination program.

WSAVA guidelines recommend the final CORE vaccination at 14 – 16 weeks. The puppy can be tested from 2 weeks after this vaccination (typically at 18 weeks). Seropositivity at this stage indicates that the pup has made an endogenous immune response to the vaccine, as there can be no MDA remaining at this time. A seronegative puppy at 18 weeks should be revaccinated (perhaps with an alternative product) and then tested 2 weeks later. A positive result indicates protection. A second negative result may indicate that the pup is either a ‘low responder’ or a ‘non-responder.’ Performing a gold standard test at this stage may show the low antibody titer typical of a low responder dog. Such an animal will be protected from clinical disease but not from infection. Alternatively, the dog may lack antibodies and be a genetic non-responder incapable
of ever making an immune response to that particular antigen. Such dogs are, therefore, susceptible to infection and disease for life. Dogs of the rottweiler breed have a higher proportion of genetic non-responders to parvovirus and rabies virus vaccines. Although non-responder rottweilers are no longer recognized in the US (the gene pool has been selected against them), they are still seen in Europe. Genetic non-responders generally cannot respond to one (rather than all) CORE vaccine antigens. The estimated prevalence of non-responders (US data) for CPV is 1 in every 1000 dogs and 1 in every 5000 dogs for CDV. CAV non-responders are rare (estimated < 1 in every 100,000 dogs).

A recent Danish study has evaluated seroconversion in a population of 135 pups aged between 8 weeks and 12 months. Most of these dogs will have finished an early life protocol (unlikely to have included a 14 – 16 week vaccine) but have not yet received a 12-month booster. The prevalence of
As determined by VacciCheckTM testing, 25.3% of this population were nonresponders for CPV, 20.7% for CAV, and 12.6% for CDV.

To Decide about Vaccination of a ‘Lapsed’ Adult Dog

Much is currently made of revaccinating ‘lapsed’ adult or adult dogs adopted without a vaccination history. Most current data sheets for MLV CORE vaccines suggest that it is necessary to treat such animals as puppies and give two injections 3 – 4 weeks apart. Immunologically, an
adult dog can be primed, immunized, and boosted from a single injection of MLV CORE vaccine as there is no inhibitory MDA. However, such dogs may not require vaccination at all – either because they have been previously vaccinated or, in some instances, have acquired natural immunity from field exposure to the virus. Owners may, therefore, be offered serology rather than automatic vaccination in this circumstance. An adult dog with serum antibodies to CDV, CAV, and CPV is protected already and does not require revaccination at that time point. Similarly, a ‘lapsed’ or
adopted adult cat with serum FPV antibody is protected and does not require that component of vaccine at that time point.

To Minimize Risk in an Animal Previously Having an Adverse Reaction to Vaccine

Adverse reactions of a broad spectrum are recognized post-vaccination in dogs and cats. The prevalence of these is low, and most have mild and transient effects. However, some (e.g., canine immune-mediated disease) are potentially life-threatening. If there is a suspicion that
vaccination might have triggered a disease, then such animals should be subject to rigorous benefit-risk analysis before revaccination is considered. For CORE vaccine antigens, this decision is now made simpler by the availability of in-house serology. A dog with serum antibodies to CDV, CAV, and CPV does not require revaccination with MLV CORE vaccines, and serious consideration should be given to which NON-CORE products such an animal receives.

Serology Replacing Revaccination in an Annual Health Check

The Annual Health Check concept is gaining momentum in the US and Europe. So, too, is the adoption of triennial CORE revaccination schedules for adult animals. However, many US practices have continued in this rapidly changing arena. Instead of offering triennial CORE revaccination, these practices now provide the alternative of triennial serological testing using one of the in-house systems. Seropositive dogs (or cats seropositive for FPV) are not revaccinated with CORE vaccines as these are not required. NON-CORE vaccines may still be used annually, and FCV and FHV revaccination might be considered annually for cats at risk. Where this approach is used, the testing interval is reduced to annually for senior animals (dogs > 10 years and cats >15 years) to ensure that immunosenescence (aging of the immune system) is not an issue.

Management of Disease Outbreaks in Shelters

One of the most valuable applications of in-house serology has been managing infectious disease outbreaks in shelters – specifically for CDV, CPV, and FPV outbreaks. The ability to rapidly and cheaply test populations to identify animals that are protected or susceptible
has allowed many animals to live that might otherwise have been euthanized as they were of unknown status.

In the face of a disease outbreak, all animals currently resident within the shelter should be tested. Those who are seropositive are protected and will not become infected or die. This protected population should be separated from low or negative responder animals, which should be isolated.
The susceptible population should not be adopted out of the shelter until at least 2 weeks for CPV or FPV or until at least 6 weeks for CDV (reflecting the incubation periods of the diseases). The susceptible population might be retested after these intervals.

The second population to be considered is those animals that wish to enter the shelter. These should also be tested before considering admission. Seropositive animals may enter as they are protected from disease. Seronegative animals should be vaccinated and then ideally sent to foster homes. They should not be allowed to enter the shelter until they have seroconverted (when retested two weeks later).

This approach has proven successful in controlling infectious disease outbreaks in shelters. However, it is not applicable to outbreaks of the feline infectious respiratory disease complex as serology is not correlated with protection.