1. Abstract accepted for 45th AASV Meeting, Dallas, Texas, March 1-4, 2014
MERIAL Momentum – 23rd IPVS Congress, Cancun, Mexico, June 8th-11th 2014
Detection of compliance to PCV2 vaccination using a delayed type hypersensitivity test (DTH test)
Antonio Callen1, DVM, PhD, Sonia Carceles DVM1, Albert Vidal2 DVM MSc,
Han Smits3, DVM, MSc, Olivier Merdy3, MSc, Lorenzo Fraile4, DVM, PhD
1Merial laboratorios, S.A, Barcelone, Spain, 2Vall Companys, S.A., Lerida, Spain,
3 Merial SAS, Lyon, France, 2 University of Lerida, Spain
Introduction
Delayed type hypersensitivity (DTH) type IV is a very well known immunological reaction associated to cell mediated immunity. It was first described as early as 1890 by Robert Koch, and it has been a common practice since, both in human and in the veterinary health to check for tuberculosis infection, and as a complementary surveillance tool for tuberculosis eradication campaigns in ruminants.4,7 Recent investigations have used this reaction to test the immunological response to mycoplasma and circovirus vaccines.8,9
While, the efficacy of PCV2 vaccines is out of question, occasionally results are not as expected, and one of the first questions that arise in these situations is the compliance of vaccination.5,6 Hence, the objective of this paper is to summarize some preliminary field experiences and results with a compliance test based on the DTH principle in different field conditions.
Principle of the test
The principle of the DTH type IV immunological reaction is based on a intradermal application of an antigen. If the inoculated animal has had any previous contact with the antigen, after infection or vaccination, leading to an immunological reaction, there will be a specific cellular immune response restricted to the area around the inoculation point. This reaction is characterized by a discoloration and sometimes induration or oedema, about 24 to 48 hours after inoculation. It corresponds histologically to a monocytic perivascular infiltration of the dermis, being the consequence of a temporary perivascular accumulation of lymphocytes (CD3) around the local blood vessels.,7,8,9
Material & Methods
Vaccination compliance test description: Candidate animals are preferably tested 3 to 5 weeks after the presumed date of vaccination.6,7 To carry out the test, a concentrated PCV2 antigen suspension is injected intradermally in a visible and clean part of the body. Best is the lower abdomen between the last two caudal nipples. As inoculum, 0.1 ml of the antigen suspension of CIRCOVAC® (Merial) is used. The antigen has to be injected intradermally. Proof of a good application is the emergence of a blister about 3-4 mm Ø. According to some preliminary pre-study tests, the best interval for assessing results is 20 to 24 h after the antigen inoculation. The result is visible by simple eye examination and can be recorded according to the characteristics of the reaction, i.e. discolouration, induration and oedema. It can be described qualitatively or quantitatively.
Trial I: With the aim to compare the response of vaccinated and not vaccinated animals in field conditions, 30 piglets from a commercial PRRS negative farm were randomly split in 2 groups according to the vaccination procedure at 3 weeks of age: either vaccinated with 0.5 ml of reconstituted CIRCOVAC (Group V, n=20) or controls (Group C, n=10) injected with 0.5 ml of the CIRCOVAC adjuvant without the antigen. Three weeks post-vaccination or injection, all the piglets were intradermally inoculated with the antigen (0.1 or 0.2 ml of the antigen suspension of CIRCOVAC). Group V was subdivided in two subgroups, according to the dose of antigen inoculated intradermally: 0.1 ml (Group V1, n=11) or 0.2 ml (Group V2, n=9). All piglets from the control group were inoculated only with 0.2 ml of antigen suspension. As negative control, all the piglets from all groups were injected intradermally with 0.2 ml of normal saline in the area symmetric to the point of antigen suspension inoculation, the same day.
The presence of a skin reaction and its possible diameter was blindly evaluated by a tape measure at 20h, 24h and 30h after the inoculation. Blood samples were drawn the day of vaccination, the day of antigen intradermo- injection, and 2 weeks later, and were tested for PCV2 and PRRS serological status.
Histological assessment of the type of immunological reaction was conducted by skin biopsies collected 24 h after the antigen inoculation from 5 animals showing a clear positive response. The samples were stained to differentiate the cellular subpopulations.
Trial II: In order to evaluate the test response to different vaccination approaches, a total of 102 weaned piglets were selected from one weaning batch of one sow farm. The experimental animals were placed in 24 pens (8 per group) along with coetaneous piglets not included in the trial, and each pen was randomly assigned to one of the following vaccination groups at weaning (3 weeks of age): 0.5 ml of CIRCOVAC, IM route (n=32) or 0.5 ml of

2. Abstract accepted for 45th AASV Meeting, Dallas, Texas, March 1-4, 2014
MERIAL Momentum – 23rd IPVS Congress, Cancun, Mexico, June 8th-11th 2014
CIRCOVAC injected with a needle free injection device (PULSE® 250, MS Schippers) at 2.5 psi (n=38) or 2 ml of an extemporaneously reconstituted vaccine combination against PCV2 (subunit vaccine) and mycoplasma IM (Vaccine B, n=32). All the animals were vaccinated in the neck. Three weeks after vaccination, two piglets per pen were administered intradermally 0.1 ml of CIRCOVAC antigen in the abdomen skin, as in trial I, with a precision syringe with needle, and another two between the shoulders with a needle free device (DermoJet®). Three pens of the PULSE group coincidentally included two additional piglets tested. The presence of a skin reaction and its possible diameter were blindly evaluated 24h later, and blood samples were collected to check for the absence of previous infection through PCV2 IgG/IgM antibody detection.
Serological assays: Serum samples were assayed for PCV2 IgG/IgM antibodies in both trials (INGEZIM PCV 11.PCV.K2, Ingenasa), whereas in the first trial the analysis included additional detection of total PCV2 antibodies in an indirect ELISA (BIOCHEK PCV2 antibody test kit), and PRRS (European strain) antibodies in a blocking ELISA (INGEZIM PRRS COMPAC, Ingenasa).
Statistical analysis: The proportion of visible skin reaction was compared between groups by Fisher’s exact test, whereas comparison of the diameter of reaction between groups was evaluated by Mann-Whitney test. In trial I, each reading period was evaluated independently. Spearman’s rank correlation coefficient r was calculated between DTH results observed at 20 h and 24 h in vaccinated animals.
Results
DTH reaction (trial I): the results of the local reaction to the antigen observed at 20, 24 and 30h are summarized in Tables 1a, 1b & 1c, respectively. In this preliminary trial, a significant reaction (characterized by a red discolouration and oedema) was observed around the point of inoculation of the antigen in 100% of the vaccinated animals. Two vaccinated animals, one of group V1 and another of group V2, were only weakly positive at 24h and another V2 piglet with a clear positive response at 20h was negative at 24h. At 30 h, both the number of animals with a positive response and the surface of the reaction of the positive animals clearly decreased (Table 1c). The maximal diameter of the skin reaction was observed in the 20h to 24h post-inoculation interval with a very strong correlation (r = 0.80) between these two first measurements timepoints. No reaction to the saline placebo was observed in these pigs. Fifty percent (5/10) of the controls reacted positively. However, 2/5 positives were classified as positive only at 20h and the remaining 3/5 control pigs displayed a long-lasting skin reaction of very limited diameter.
The percentage of vaccinated piglets responding positively to the DTH test was significantly higher than the controls both 20h following inoculation (p=0.03) and 24h following inoculation (p<0.001) but returned to be not significant at 30 post-inoculation (p=0.21) when the number of vaccinated animals had been reduced for skin biopsies.
The average diameter of the reaction was similar for both vaccinated groups, V1 and V2 (p>0.65), but differed significantly between vaccinated and not vaccinated animals both 20h and 24h following inoculation (p≤0.001) (see Tables 1a, 1b & 1c).
DTH reaction (trial II): in this trial, the reaction to the abdominal intradermal injection of the antigen was qualitatively and quantitatively similar to Trial I, i.e. red discolouration, but with no oedema. However, the transdermal application of the antigen with DermoJet yielded a response characterized by red discolouration and /or subcutaneous induration. The results of this trial are summarized in Table 2.
All the animals vaccinated IM with CIRCOVAC displayed a visible skin reaction following the DTH test, independently of the method of antigen inoculation (32/32). Almost all the pigs (35/38) vaccinated with CIRCOVAC with the needle-free device PULSE displayed a visible skin reaction to the antigen inoculation as well. In contrast, 12/32 piglets vaccinated with Vaccine B did not show any reaction in this test with no influence of the ID injection device and location on the positivity ratio (11/16 with DERMOJET, 9/16 with standard equipment).
The average diameter of the skin reaction was high following CIRCOVAC vaccination whatever the vaccination procedure and the DTH test conditions tested but tended to depend on the method of vaccination and method of antigen inoculation. The skin reaction was nevertheless significantly smaller following Vaccine B vaccination (p≤0.001).
Serology: In trial I, the PRRS ELISA results confirmed the negative status of the farm (data not shown). Both the IgG/IgM test and the PCV2 indirect test indicated the absence of seroconversion in the control group. About half of the vaccinated pigs (8/19) showed an IgM response at 21 days post-vaccination, and also 9 out of 19 vaccinated animals showed PCV2 seroconversion as detected in the indirect test at 3 and /or 5 weeks after vaccination.
In trial II, all 48 sampled piglets (two per pen) showed a negative response for PCV2 IgG and only 4 animals, 3 vaccinated with CIRCOVAC and one vaccinated with Vaccine B, showed a PCV2 IgM positive result.
Histology: All five biopsies revealed a reaction characteristic of type IV DTH, although the intensity of it varied among samples5. The mononuclear cells were characterized as CD3 lymphocytes.

3. Abstract accepted for 45th AASV Meeting, Dallas, Texas, March 1-4, 2014
MERIAL Momentum – 23rd IPVS Congress, Cancun, Mexico, June 8th-11th 2014
Discussion - Conclusion
The intradermal application of the PCV2 antigen of CIRCOVAC elicited a visible local response in most of the CIRCOVAC vaccinated piglets and a limited number of placebo inoculated pigs. The most obvious reaction was detectable at 24h post-inoculation. The chronological appearance of this reaction along with the histological picture are clearly indicative for a delayed type hypersensitivity (DTH) type IV reaction. Our results confirm previous studies done with different methodology.8 According to these preliminary trials, 0.1 ml of the suspension of PCV2 antigen of CIRCOVAC vaccine is able to induce a clear reaction. The results are comparable with the injection of a greater antigenic mass in 0.2 ml. This test, easily applicable in the field, can be used as a practical alternative to check the compliance of PCV2 vaccination with CIRCOVAC in daily practice. Indeed, data obtained in our second trial provide further evidence of the potential of such a test in animals vaccinated with CIRCOVAC by intramuscular injection or with a needle free device. However, the first results obtained in animals vaccinated with a PCV2 subunit vaccine showed a lower DTH reaction than with the full virus antigen. The reduced response observed with this vaccine, both for the percentage of positive pigs and the average diameter might be related to a lower level of cellular mediated immunity (CMI) induced by this vaccine, related to the presence of only one antigenic component, i.e. ORF2.1,2 However, we cannot rule out a closer antigenic nature between the vaccine and the test material to explain a more intense response to the test in the homologous system.
These preliminary results are indicative of a high sensitivity of the test implemented 3 weeks post-vaccination for the piglets vaccinated with CIRCOVAC. However, the results obtained in placebo vaccinated controls are suggestive of some problems of specificity, at least for the use of the test for vaccination compliance purposes. One explanation for these positive results might be the presence of a residual maternally derived CMI in 6 weeks old piglets. We likely did not have an early infection as our serology results confirmed, and in fact the DTH results were independent of the seroconversion pattern in the vaccinated animals (results not shown). Indeed, OH et al. (2012) reported the presence of CMI and a positive DTH response in not vaccinated piglets produced from CIRCOVAC vaccinated sows.4 While, in our trials, the sows had not been vaccinated and the piglets are a few weeks older than the ones included in the previous study, we should not reject this possibility because of the high seroprevalence of PCV2 in sows and gilts observed in the field. Our results are clearly indicative of the need to check non vaccinated and non-infected animals of the same age and older to get a further insight into the evolution and potential impact of cell mediated maternal derived immunity (MDI).
In conclusion, the intradermal application of PCV2 antigen is a feasible and convenient alternative to elicit a DTH type IV response that can be useful for CIRCOVAC vaccination compliance. These preliminary trials might be the first steps to a wider use of this technique, related to CMI, in multiple applications in the veterinary field. Further experimental and field studies are needed to evaluate the sensitivity and specificity of this test in different field conditions and for a better standardization of the technique.
References
1. Fort et al. (2009) Vaccine, 27 : 4031-4037.
2. Fort et al. (2012) Vet. Immunology Immunopathology, 150 : 128-132.
3. Oh et al. (2012) Journal Gen. Virol. 93: 1556-1562.
4. Monaghan et al.(1994) Vet. Microbiol.40 : 111-124.
5. Opriessnig et al. (2013) Proc.44th AASV, San Diego, 37-38.
6. Pittman et al. (2010) Proc. 21st IPVS Congress, Vancouver, O183, 217
7. Razzaque et al. (1983) Arch. Dermatol. :119: 934-945.
8. Seo et al. (2012) BMC Vet. Research, 8: 194.
9. Seo et al. (2013) J. Vet. Med. Sci. 75 (2): 245-247.
® CIRCOVAC is a registered trademark of Merial.
® PULSE needle free is a registered trademark of MS SCHIPPERS.
® DermoJet is a registered trademark of AKRA DERMOJET.
® INGEZIM PCV 11.PCV.K2 and INGEZIM PRRS COMPAC are registered trademarks of INGENASA
® BIOCHEK is a trademark of BIOCHEK smart veterinary diagnosis

4. Abstract accepted for 45th AASV Meeting, Dallas, Texas, March 1-4, 2014
MERIAL Momentum – 23rd IPVS Congress, Cancun, Mexico, June 8th-11th 2014
Table 1.- DTH response (skin reaction presence and diameter-SRD-, in mm) in piglets vaccinated with CIRCOVAC or placebo (Control) 3 weeks before (Trial I).
a) 20h post-inoculation of PCV2 antigen
CIRCOVAC
Control
Group
V1
V2
V1+V2
C
Antigen dose
0.1 ml
0.2 ml
0.1/0.2
0.2 ml
N
11
8*
19
10
No response
1 (9.1%)
1 (12.5%)
2 (10.5%)
5 (50%)
Response
10 (90.9%)
7 (87.5%)
17 (89.5%)
5 (50%)
SRD (Mean +/-SD)
29.8±14.1
29.5±16.8
29.7±14.8
9.3±10.4
SRD (p-value)
V1 vs V2:p=0.94
V1+V2 vs C:p=0.001
b) 24h post-inoculation of PCV2 antigen.
CIRCOVAC
Control
Group
V1
V2
V1+V2
C
Antigen dose
0.1ml
0.2 ml
0.1/0.2
0.2 ml
N
11
8
19
10
No response
0 (0%)
1 (12.5%)
1 (5.3%)
7 (70%)
Response
11(100%)
7 (87.5%)
18 (94.7%)
3 (30%)
SRD (Mean +/-SD)
27.7±16.3
27.1±20.0
27.5±17.4
4.2±8.2
SRD (p-value)
V1 vs V2: p=0.80
V1+V2 vs C:p<0.001
c) 30h post-inoculation of PCV2 antigen.
CIRCOVAC
Control
Group
V1
V2
V1+V2
C
Antigen dose
0.1ml
0.2 ml
0.1/0.2
0.2 ml
N
8
6
14
10
No response
3 (37.5%)
2 (33.3%)
5 (35.7%)
7 (70%)
Response
5 (62.5%)
4 (66.7%)
9 (64.3%)
3 (30%)
SRD (Mean +/-SD)
10.0±15.9
14.8±19.3
12.1±17.1
3.0±5.6
SRD (p-value)
V1 vs V2: p=0.65
V1+V2 vs C: p=0.20
*one animal died before antigen administration; SD: Standard deviation

5. Abstract accepted for 45th AASV Meeting, Dallas, Texas, March 1-4, 2014
MERIAL Momentum – 23rd IPVS Congress, Cancun, Mexico, June 8th-11th 2014
Table 2.- DTH results (skin reaction presence and diameter-SRD-, in mm) in piglets according to the method of inoculation of the antigen (DERMOJET vs Needle), the vaccine used and the vaccination route (Trial II).
Vaccine history
CIRCOVAC
Control
IM
MS Pulse
IM
Material used for the DTH test
DermoJet
Needle
DermoJet
Needle
DermoJet
Needle
No response
0/16
0/16
1/19
2/19
5/16
7/16
(0%)
(0%)
(5.3%)
(10.5%)
(31.3%)
(43.8%)
Response
16/16
16/16
18 /19
17/19
11/16
9/16
(100%)
(100%)
(94.7%)
(89.5%)
(68.7%)
(56.3%)
SRD
(Mean +/-SD)
26.5±17.9
39.7±21.2
20.9±12.2
23.2±19.2
7.6±7.0
7.9±12.2
Difference between vaccines in skin reaction diameter* (p-value)
p=0.001
p<0.001
SD: Standard deviation
*using identical injection equipment