GUIDELINE for Vaccination and antibody testing

Published: 01/11/2020
Last updated: 30/11/2023
Last reviewed:

The Vaccination and antibody testing in cats guidelines were published by Herman Egberink et al. in Viruses 2022, 14, 1602; this update has been drafted by Herman Egberink and Katrin Hartmann.

Introduction

Vaccination guidelines, as published by the ABCD, aim to support the practitioner in making an informed decision regarding vaccination schedules for an individual animal and/or group of animals. After primary vaccination, revaccinations are recommended every 1-3 years with the core vaccines for feline calicivirus (FCV) and feline herpesvirus (FHV), depending on the risk of infection, and every 3 years with the core vaccine for feline panleukopenia virus (FPV). Vaccination intervals are based on the minimal duration of immunity (DOI) as determined in experimental vaccination-challenge studies performed by the vaccine industry. However, individual differences exist at which age kittens can be vaccinated successfully (because maternally derived antibodies, to different viruses, vary in duration) and how long vaccine-induced DOI lasts. In addition, vaccine-induced immunity in adult cats can be much longer than those DOI determined by industry challenge experiments. Moreover, cats might have undergone subclinical infection and might be protected lifelong even without having received any vaccination. Additionally, individual immune reaction and subsequent DOI might vary and depend on many different factors, such as age, nutritional status, concurrent subclinical infections, and breed (Bergmann et al., 2018). To achieve an optimal vaccination schedule for the individual animal and to avoid unnecessary vaccinations, antibody testing can be helpful (Egberink et al., 2022). However, it is important to differentiate between actively or passively acquired antibodies during the interpretation of antibody testing, including titre testing. While titres of passively acquired antibodies, which generally only persist for weeks, allow a quantitative interpretation about the level of protection (“protective titre”), this is not the case for actively acquired antibodies. Following infection or vaccination, antibodies and cellular immune responses are induced through the activation of T- and B-cells and the formation of memory cells. The presence of antibodies indicates that an immune response has been induced, irrespective of the antibody titre.

Live vaccines induce a humoral immune response (antibodies) as well as a cell-mediated immune response (CMI). The CMI plays an important role in the control of intracellular pathogens, such as viruses. However, vaccination-challenge experiments have provided excellent data to show that there is also a good correlation between the vaccine-induced antibody titre and protection against certain diseases (Roth and Spickler, 2010). Antibody titre as a measure of immunity has been shown to be useful for the core vaccines against canine distemper virus (CDV), canine adenovirus (CAV)-1, canine parvovirus (CPV)-2, and rabies (Frymus et al., 2009; Killey et al., 2018) in dogs and against feline panleukopenia virus (FPV), and also rabies in cats. For rabies virus vaccination, however, national, and regional legislation will determine recommendations for primary vaccinations and revaccinations, and thus, antibody testing is not performed routinely to determine the need for vaccination.

For some other vaccine-preventable diseases, such as those caused by FCV and FHV, a correlation between antibody titres and protection does not exist, and the role of antibodies is less clear. Protection against FCV has been shown to correlate to humoral virus-neutralising antibodies (VNAs) and CMI (Tham and Studdert, 1987). The important role of CMI in FCV is supported by cats being protected against infection despite an absence of detectable VNAs (Knowles et al., 1991; Poulet et al., 2008; Lesbros et al., 2013). For FCV, the level of mucosal IgA is a stronger correlate of protection than blood antibody levels, but levels of mucosal antibodies cannot be measured easily (Sato et al., 2017). Also because of FCV strain variation in the field, the value of antibody testing in predicting protection is limited (Bergmann et al., 2019; Hofmann-Lehmann et al., 2022). Neutralising antibody titres detected against laboratory strains of FCV might not correlate with neutralisation (protection) against field strains, due to the absence of, or insufficient, antibody cross-neutralisation.

For FHV infection, as in other alphaherpesvirus infections, CMI is more important than humoral immunity for protection (Thiry et al., 2009). However, CMI can only be measured by sophisticated laboratory methods (Thiry et al., 2009). It has been shown that serum antibody testing is also not useful to predict protection against FHV infection (Bergmann et al., 2020) . The absence of serum antibody in vaccinated cats does not mean that cats will develop disease following infection. Also, being a pathogen of the respiratory tract, mucosal cellular and humoral responses are important in protecting cats against FHV infection (Thiry et al., 2009).

For FeLV infection, high levels of VNAs can be detected in most cats that had overcome viraemia after field infection (but usually not after vaccination), indicating a role of VNAs in protection, although cytotoxic T-lymphocytes also are very important (Flynn et al., 2002). Results of a recent study on the humoral immune response in cats with regressive or progressive FeLV infection also support a role for VNAs in protection. VNAs were only detected in the cats with regressive infections, and these cats also showed higher FeLV-antibody responses against the FeLV surface protein (gp70) compared to cats with progressive infections (Parr et al., 2021). Assays for VNAs are helpful, as VNA positive cats are immune and do not require vaccination. However, tests to measure VNAs are not widely available (except in the UK). A new FeLV antibody test became available in some European countries. It is a point-of-care (POC) test (i.e. an in-practice test) that measures antibodies to FeLV p15E (envelope transmembrane protein). This new test is based on the results of a study that assessed the diagnostic utility of the detection of antibodies against different FeLV antigens. The recombinant preparation of FeLV p15E  was identified as a potentially useful antigen for detecting antibodies that could correlate with protection (Boenzli et al., 2014). The value of this POC test to predict protection against new infection, however, has still  to be evaluated and  it remains unknown how well the presence of anti-p15E antibodies correlates with protection from FeLV infection in the field (Westman et al., 2023).

Since good correlation between antibody titer and protection against disease has been observed for FPV in particular, the focus of this Guideline will be on testing for antibodies against FPV in the context of vaccination.

Testing for FPV antibodies

Results of experimental and field studies in FPV vaccination indicate a much longer period of persistence of antibodies than 3 years; in some animals even lifelong antibody persistence was demonstrated (Scott and Geissinger, 1999; Mouzin et al., 2004). Because of the long DOI raised by the FPV vaccines, cats with sufficient antibodies will not respond to the 3-yearly booster vaccination and, therefore, these animals will be vaccinated unnecessarily. Only cats with no or very low antibody titres will benefit from vaccination. In this regard the 3-yearly booster interval, as recommended in current guidelines for FPV, is merely intended to sustain herd immunity by ensuring that all unprotected cats are vaccinated. For the individual animal, either gold standard tests or POC test kits can be used to decide whether a (re)-vaccination is needed. To measure the antibody titre, a serum sample can be sent to an external diagnostic laboratory, or by the use of a POC test that can be used in the veterinary practice (i.e. “patient side” or ïn-clinic” testing). Gold standard tests provide precise antibody titres, whereas POC test kits provide only qualitative (present or absent) or semi-quantitative (high, medium, low or no antibodies) results.

Gold standard tests

The antibodies that protect against infection are directed against the viral surface proteins and can prevent the infection of cells. These antibodies are particularly important for the prevention of systemic infections like FPV. The level of protective antibodies against FPV can be determined in diagnostic laboratories with virus neutralization (VN) or haemagglutination inhibition (HI) assays. These tests are considered gold standard methods of determining the titre of protective antibodies in serum.

The antibody titre is determined by making serial dilutions of the serum sample which are then added to a standard amount of virus. After specific incubation times, the virus-antibody mixture is inoculated onto cell cultures or added to red blood cells. The titre is defined as the reciprocal value of the highest dilution that prevents the infection of cells (VN assay) or the agglutination of red blood cells (HI assay).

Point-of-care test kits

Different POC tests are available for use in veterinary clinics. The tests are ELISA- or immuno-migration-based, and results of some of the tests have been validated against the gold standard assay (Mende et al., 2014). In one POC test (based on a solid phase dot ELISA), antibodies against FPV, as well as FCV and FHV, are detected, and with one kit a maximum of 12 feline samples can be investigated at once. The result can be read in 21 minutes by comparing the colour tone of the test spots with the control spot, which gives a semi-quantitative result. Data on sensitivity and specificity of this test were provided by the commercial company that produced the test and additionally were published in two other independent studies (DiGangi et al., 2011; Mende et al., 2014). The test kit was reported to have 99% specificity and 49% sensitivity in a study in shelter cats (DiGangi et al., 2011). Mende et al. (2014) reported the results of a study after the test had been slightly modified to improve  the sensitivity. In this study, the test showed 89% specificity and 79% sensitivity when compared with a HI titre at a cut-off value of 20; this cut-off titre was chosen as a titre of ≥20 in adult cats that have been vaccinated, or have overcome an active infection, is considered protective (Lappin et al., 2002). In another study, cats were considered to be protected against FPV if they had a HI titer of ≥40 (Mouzin et al., 2004). At a titre cut-off point of 40, the specificity of the assay was determined as 86% and sensitivity 83%. To identify cats that have insufficient antibody levels and therefore require vaccination, specificity is the more important parameter since it determines the percentage of negative tests that are correctly identified as negative, and thus the number of false positive results that can be expected. It is important to minimise the number of false positive results that can be expected because cats with a false positive test result will not receive a booster vaccination and will potentially remain unprotected. The specificity is considered acceptable assuming a titer of ≥20 is protective. Especially if the test is used in cats belonging to a population with an expected high prevalence of antibody-positive animals, the positive predictive value is high, and the test can be considered suitable for use in veterinary practice (Mende et al., 2014), for example in adult cats with a known vaccination or infection history.

New POC tests that have recently become available that detect antibodies against only FPV (and not FCV and FHV), are based on an immunochromatographic principle, and generally deliver qualitative (i.e. giving a result of protected or not protected, rather than a specific antibody titre) results in a shorter period of time. These tests have not been evaluated in independent studies so far.

Applications of POC antibody testing against FPV

To measure the antibody response in kittens following vaccination

After the initial series of vaccinations in the first months of life of the kitten, vaccine-induced protection can be determined by POC tests. As the last kitten vaccination is usually given around the age of 12-16 weeks, a positive test result in an antibody test obtained at the age of 20 weeks indicates that the animal has made an active immune response. At this age maternally derived antibodies (MDA) are expected to have waned to very low or undetectable titres in the majority of animals (Addie et al., 1998). If the last vaccine was given at an age of 16 weeks and protection was shown at 20 weeks, the WSAVA vaccination guidelines state that the 12-month booster might not be required and that animals could go straight to a triennial FPV vaccination program (Day et al., 2016). There are not much data about the age at which the immune system matures and if the quality of the induced immune response at 16 weeks is as good as in adult animals. Therefore, in the opinion of ABCD, it seems valid to advise yearly titre testing in these animals rather than going straight to a triennial vaccination programme, particularly if the last vaccine was given before the age of 16 weeks. A kitten that is negative for FPV antibodies at the age of 20 weeks should be revaccinated and tested again 3-4 weeks later to determine if antibodies have developed. If the animal is still negative, the kitten is most likely a non-responder to the particular FPV antigen and might be susceptible to infection and disease for life (Day et al., 2016).

To test whether (re)vaccination for FPV is necessary

The triennial vaccination for FPV is based on the minimal DOI. Since many vaccinated animals will maintain protective antibody titres for longer than a 3-year period, sometimes even lifelong, triennial antibody testing can be performed as an alternative for routine booster vaccination during regular health checks. Revaccinations were shown not to be beneficial especially in cats with high titres and are therefore unnecessary (Bergmann et al., 2018). For adult cats with an unknown vaccination history, or elapsed vaccination, an antibody test could be offered to owners as an alternative to revaccination for FPV. However, this may require that monovalent FPV vaccines are available to allow differential administration of specific vaccines, which is not the case in all countries.

In animals that have previously experienced a serious adverse reaction, the need for revaccination should be carefully evaluated. This holds true for Core and Non-Core vaccines. For FPV vaccines this decision can be made based on the results of a positive FPV POC antibody test. Another situation in which the requirement for vaccination can be determined by antibody measurement is in immunocompromised cats (see ABCD guidelines for Vaccination of immunocompromised cats; Hartmann et al., 2022).

To manage FPV infection and disease outbreaks in shelters

If possible, animals could also be tested before admission into a shelter to determine if they are protected against FPV. If they are not protected, the animals should be vaccinated and kept in strict isolation or preferably sent to foster homes to develop active immunity before entering the shelter. However, it is recognised that in shelters, routine antibody screening might not be appropriate because of the extra costs; therefore, often the preference is to elect for vaccination as soon as possible after entry.

In the face of an outbreak of FPV disease, susceptible cats without FPV antibodies can be identified using the POC antibody test and can then be immediately vaccinated or receive hyperimmune serum (passive immunization). The advantage of such an approach is that protected antibody-positive animals can then be separated from the cats without antibodies. Antibody-positive animals do not need to be vaccinated. The antibody-negative animals, following vaccination, should be isolated, at least until the incubation period of the disease has passed (on average 2-7 days). These animals should be retested before adopting out. Passive immunization of these unprotected cats also might be a short-term option. In countries where they are available, commercial immunoglobulin preparations containing antibodies against FPV can be used. Also, homologous immune serum from blood of cats with high antibody levels can be prepared and administered. Blood donors must be screened for insidious infectious agents (e.g., FIV, FeLV, Bartonella , Haemoplasma infection) and attention must be paid to sterility, storage, and administration. Also, the blood type of a donor and recipient should match (see also ABCD Guideline for Feline Panleukopenia).

To determine the optimal age of vaccination in kittens

During the first period of their lives, kittens are protected by MDAs, which for the most part are obtained on the first day of life from the mother via the colostrum (see also ABCD Guideline for maternally derived immunity and vaccination). It is generally believed that only up to 5–10% of the MDAs are transferred during pregnancy from an immune queen to the foetuses (Scott et al., 1970; Schultz et al., 1974; Claus et al., 2006). These MDAs protect animals from infection but also interfere with immunization after vaccination (Addie et al., 1998). Very young kittens can generate an effective antibody response to vaccination only when the levels of MDA against the respective antigen have decreased below the inhibitory threshold. The level of MDAs will differ between litters and individual animals within litters, depending on the antibody levels in the colostrum of the queens and the amount of colostrum ingested. Therefore, it is common practice to perform the first core vaccination at 8–9 weeks of age (or earlier in kittens at special risk or in rescue shelters), and to give additional doses at 2- to 4-week intervals until the age of 12–16 weeks or older  (Hosie et al., 2015) with the expectation that one of these vaccinations will occur after the blocking effect of the MDA has waned, and before exposure to virulent agents. With this strategy, some vaccine doses might be given that are of no benefit if the kitten still has interfering levels of MDAs or if the kitten has already responded to an earlier vaccine (or infection) and thus is already immune. To avoid this problem, in an ideal world, the optimal age for the first core vaccination would be determined by establishing the antibody titre of each kitten to determine when interfering maternal antibodies have waned. However, the use of antibody testing in this situation has not yet been critically evaluated. If using a POC test that provides no titres but only semi-quantitative results, kittens would likely need to be re-tested every 2-3 weeks since the optimal time point for vaccination might not be determined by a single blood sample at an age of 6-8 weeks. It is recognized that repeated blood sampling of young kittens every 2-3 weeks is difficult and potentially stressful for the kittens, as well as costly, precluding routine adoption of this procedure in practice.

Data defining the levels of MDAs at which vaccination will lead to active immunization are lacking. Also, differences in the performance of available vaccines in the presence of MDAs can be expected. Where antibody testing is being used to decide upon the optimal age of first vaccination, a titre, as produced by diagnostic laboratories, is preferable to the semi-quantitative results of a POC test, given the increased precision of the result. However, antibody testing is still expensive and often impractical in this situation.

An estimate of the optimal age for first vaccination of kittens can also be calculated based on the antibody titre of the queen, and the average half-life of MDAs (9.5 days for FPV MDA), bearing in mind that individual kittens in a litter will suckle different amounts of colostrum (Sykes, 2022; Greene and Levy, 2012).

In conclusion

Determining the optimal age of immunization of kittens by antibody testing with POC tests can be problematic in the face of decreasing MDA titres for reasons described in this guideline. In contrast, antibody testing, especially against FPV, can be a useful and reliable tool to determine if a cat has developed antibodies after vaccination, and to determine whether the individual animal needs revaccination at the time proposed in the general vaccination guidelines. In vaccinated adult cats, testing for antibodies can be conducted as optional part of the annual health check appointment which could also include a complete blood count, serum biochemistry and urinalysis at least in mature animals. Since data on the role of ageing of the immune system on the persistence of levels of antibodies are lacking, yearly testing in older cats (> 15 years) is strongly advised.

Acknowledgement

ABCD Europe gratefully acknowledges the support of Boehringer Ingelheim (the founding sponsor of the ABCD), Virbac, IDEXX GmbH and MSD Animal Health.

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