Background

Caprine tuberculosis (TB), caused either by Mycobacterium bovis or M. caprae, and paratuberculosis (PTB), caused by M. avium subsp. paratuberculosis (Map), are endemic diseases in goat herds of the Iberian Peninsula [1–3]. Both infections may have an impact in terms of economic loss. Moreover, M. caprae and Map can be transmitted between domestic hosts and wildlife species [2, 4]. In addition, M. caprae is a zoonotic agent [5–7] and Map has been associated with Crohn’s disease [8, 9].

Control of PTB in small ruminants can be facilitated by vaccination because the bacterial burden is greatly reduced, containing the spread of the disease and preventing clinical expression [10]. Furthermore, vaccination against PTB with Map-killed formulations has been demonstrated to be an economically efficient strategy to achieve control of the disease in small ruminants [11–13] and cattle [14].

On the other hand, it has been shown that exposure to M. avium can interfere with the diagnosis of M. tuberculosis complex organisms such as M. bovis or M. caprae[15–17]. Similarly, there are some reports showing that natural infection with Map compromises TB diagnosis in cattle [18, 19] and goats [20]. Therefore, PTB could also affect the specificity of diagnostic tests used in TB control programs. The Spanish eradication program of bovine TB expressly forbids the use of Map-based vaccines in cattle because of this potential interference with TB diagnostics. In contrast, vaccination against PTB in goats is permitted with the exception of a minority of herds that are subjected to TB control in some regions. To date, the potential effect of Map-based vaccines on the diagnosis of TB has only been investigated post hoc under field conditions [21, 22].

It has been reported that TB diagnostic tests currently used in cattle, such as the skin test or the interferon-gamma (IFN-γ) assay, can be used for diagnosis of TB in goats [23–25]. Moreover, in recent years, new IFN-γ assays based on antigens secreted by active growing bacilli, such as the peptide cocktail ESAT-6/CFP-10 (E/C) or Rv3615c [26, 27], have been developed as alternatives to bovine and avian tuberculins. These antigens are not present in either M. bovis BCG or Map and can be viewed as novel DIVA (differentiation of infected and vaccinated animals) regents able to distinguish TB-infected from TB- or PTB-vaccinated animals.

The aim of the present work was to assess how vaccination against Map affects standard and novel diagnostic tests in goats. Thus, we evaluated the interference of a commercial Map-killed vaccine on TB diagnosis in goats vaccinated and subsequently challenged with M. caprae. The objectives of the present work were: (1) to investigate the possible interference of vaccination before and after M. caprae infection on TB diagnostic tests (single and comparative skin tests and IFN-γ assay); (2) to assess the usefulness of DIVA-peptide candidates E/C and Rv3615c; and (3) to evaluate immunological and post-mortem indicators of the effects of PTB vaccination on M. caprae infection.

Results

Assessment of diagnostic tests

The effect of Map vaccination and subsequent infection with M. caprae on TB diagnostic assays based on cell-mediated immunity was assessed during this study. Four vaccinated goats were subjected to the tuberculin cervical comparative intradermal test (CIT) at week 14, and all were negative for TB and correctly classified as avian reactors. However, the four goats were classified as positive for TB if only the bovine tuberculin (PPD-B) result (as a single intradermal tuberculin test [SIT]) was considered (data not shown), which indicated that the specificity of the SIT was severely compromised by PTB vaccination. The skin test was repeated in all goats at week 26 (12 weeks post-infection [wpi] with M. caprae). In the vaccinated group, 9 of 10 goats were bovine reactors (positive for TB) using the CIT (1 goat was an avian reactor), but all goats were classified as TB reactors by applying the SIT interpretation. All unvaccinated goats were positive for TB with both tests (Table 1). Differences between groups in the mean specific thickness increase for each tuberculin were statistically significant only for ΔPPD-A (avian tuberculin), with a mean increase of 14.4 mm (11.9–17, 95% CI) in the vaccinated group versus a mean increase of 10.8 mm (8.8–12.9, 95% CI) in the control group (p < 0.05) (Table 1). Differences were also found in the mean value of ΔPPD-B minus ΔPPD-A. This value was significantly lower in the vaccinated group (5.2 mm, 2.9–7.5, 95% CI) than in the unvaccinated group (10.3 mm, 8.8–11.9, 95% CI) (p < 0.005) (Table 1).

Table 1 Increases in skin-fold thickness and corresponding results of tuberculin skin tests
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The tuberculin-based IFN-γ test was performed every 2 weeks throughout the experiment. The kinetics of IFN-γ responses of the vaccinated group showed a PPD-A-biased response, which switched to a PPD-B-biased mean response 4 weeks after M. caprae infection (at week 18 of the experiment) (Figure 1). In the unvaccinated control group, a PPD-B-biased response was obtained 4 weeks after M. caprae infection, whereas no response was observed before this time point in any of the goats (data not shown). When the results were analyzed individually (Table 2), Map-vaccinated goats began showing responses to PPD-A (“avian reactors”) at week 2 after vaccination (2 of 10 goats), and all of them subsequently became avian reactors between weeks 4 and 10. However, 1 of 10 goats became a “bovine reactor” (positive to PPD-B) at week 12 of the experiment (12 weeks post-vaccination [wpv]), and this figure rose to 6 of 10 at week 14, in the blood samples taken just before infection with M. caprae. Interestingly, in the next blood sampling at 2 wpi (week 16), again 8 of 10 goats showed a stronger reaction to PPD-A than to PPD-B, and the 2 other vaccinated goats were negative to both tuberculins. By contrast, at week 18 (4 weeks after M. caprae challenge), 9 of 10 (90%) of the vaccinated goats were bovine reactors. At week 20, all goats were positive to PPD-B (bovine reactors), and these results were maintained during the rest of the trial with the exception of one goat that tested bovine-negative at week 24 and another that was an avian reactor at week 28 (see Table 2). In the unvaccinated group, all goats were bovine reactors at week 18 and remained so until the end of the experiment, with the exception of two goats that were negative at week 24 but returned to positivity at weeks 26 and 28 (data not shown).

Figure 1
figure 1

Kinetics of IFN-γ released after stimulation of whole blood with tuberculins in the vaccinated group. Avian tuberculin (PPD-A, ▲), bovine tuberculin (PPD-B, ■). Results are expressed as mean ΔOD450 (mean of OD450 of antigen-stimulated values with unstimulated values subtracted) ± 95% confidence interval.

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Table 2 Results of the standard IFN-γ assay (Bovigam™)
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Next, we assessed the interference of Map vaccination with the IFN-γ assay by employing the defined antigen reagents E/C and Rv3615c. Responses were assessed at the same five time points described in the Materials and Methods section. At weeks 14 (0 wpi) and 16, all goats were defined as TB-negative (ΔOD450 ≤ 0.05) according to the test results. At weeks 20, 24, and 28, 90%, 100%, and 70% of vaccinated goats were positive with the E/C cocktail and 100%, 90%, and 80% were positive with Rv3615c, respectively (Table 3). However, these proportions increased to 100%, 100%, and 90% at weeks 20, 24, and 28, respectively, when the E/C and Rv3615c results were considered together. Very similar results were obtained in the unvaccinated group, in which 100%, 80%, and 90% of goats were positive to E/C at weeks 20, 24, and 28, respectively; 100%, 80%, and 80% of goats were positive to Rv3615c; and 100%, 80%, and 90% of goats were positive again when combining the results for both antigens at the same time points (data not shown).

Table 3 Results of the IFN-γ assay using two DIVA reagents: ESAT-6/CFP10 (E/C) and Rv3615c
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Seropositivity to Map was assessed using the Paratub.Serum-S ELISA kit (Institut Pourquier, Montpellier, France) applying the cut-off defined above. All goats (n = 20) were negative at week 0. Unvaccinated goats also remained negative during the experiment (data not shown). In contrast, 1 of 10 (10%) and 5 of 10 (50%) vaccinated goats were positive or doubtful at weeks 14 (0 wpi) and 28 (14 wpi), respectively, with different degrees of intensity (Table 4). The mean intensity of antibody response (%S/P) increased moderately, but was statistically significant (p < 0.05), from 20% (4%–36%, 95% CI) at week 14 to 55% (19%–91%, 95% CI) at week 28 (2 weeks after CIT).

Table 4 Seroreactivity to Map (Paratub.Serum-S™ ELISA)
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Pathology and bacteriology

At necropsy on week 28 (14 wpi), visible pathology typical of TB was observed in the lungs and pulmonary lymph nodes (LN) of all animals irrespective of their vaccination status. Of the seven lung lobes evaluated, the vaccinated group presented an average of three lobes with gross lesions (2–4, 95% CI), slightly lower than the mean of four lobes (3–5, 95% CI) found in the unvaccinated group, but this difference was not statistically significant (p = 0.324).

The mean log10-transformed volume of gross lesions in lungs was similar in the unvaccinated (1.3 log10 cm3; 1–1.7, 95% CI) and vaccinated (1.1 log10 cm3; 0.5–1.6, 95% CI) groups (p = 0.470), but values were spread over a wider range in the vaccinated group. By contrast, statistically significant differences between vaccinated and control goats were found after assessing the gross pathology in LN: The unvaccinated animals presented with a mean log10-transformed volume of visibly affected tissue of 3.9 log10 mm3 (3.6–4.3, 95% CI) compared with 3.3 log10 mm3 (2.7–3.8, 95% CI) obtained in the vaccinated group (p < 0.05). The individual pathological parameters are represented in Figure 2A and B.

Figure 2
figure 2

Post-mortem analysis as measured by gross pathology and bacterial burden. Results are plotted for individual goats. (A) Total volume of visible lesions (VL) in lungs as log10 cm3. (B) Total volume of VL in respiratory lymph nodes (LN) as log10 mm3. (C) Bacterial load as log10 cfu/g. (●) vaccinated goats, (■) unvaccinated goats. Horizontal lines indicate median values. Significance determined by unpaired t-test: *p < 0.05.

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In addition, none of the vaccinated goats showed extrapulmonary gross lesions, whereas dissemination was observed in 4 of 10 unvaccinated goats that presented with gross lesions beyond the thoracic area (2 goats with lesions in retropharyngeal LN, 2 goats with lesions in mesenteric LN, and 1 goat with lesions in the spleen). These lesions were histopathologically confirmed to be tuberculous.

In three animals, focal lesions were also found in the LN draining the Map vaccine inoculation point (axillary, prescapular, or subcutaneous LN). These lesions were attributed to the vaccine inoculation because of their localization and histological characteristics: lesions predominantly necrotizing rather than granulomatous, without Langerhans cells, but surrounded by dispersed polymorphonuclear cells and abundant fibrosis.

Finally, the mycobacterial load in LN was calculated as log10 cfu/g. The bacterial burden in the pulmonary and retropharyngeal LN of all animals (n = 20) ranged from 2 to 4 log10 cfu/g (Figure 2C). The total bacterial load in the unvaccinated group was 3.5 log10 cfu/g (3.2–3.7, 95% CI), slightly higher than that in the vaccinated group (3.1 log10 cfu/g, 2.7–3.6, 95% CI). However, this difference was not statistically significant (p = 0.203).

Cross-sectional analysis

To evaluate the vaccine effect in terms of heterologous protection, post-mortem parameters were also assessed in combination. A mild positive correlation between bacteriological and pathological findings was found by correlating individual logarithmic values of bacterial load and total volume of visible lesions (r = 0.386, p < 0.05). However, a higher dispersion of individual values was observed in the vaccinated group compared with the unvaccinated group (Figure 3A), and a stronger positive correlation was found when only vaccinated goats were assessed (r = 0.523, p < 0.05), whereas results obtained from unvaccinated goats did not show significant correlations (r = -0.274, p > 0.05).

Figure 3
figure 3

Protective efficacy measured by post-mortem results, and cross-sectional comparison of IFN-γ results with vaccination outcome. (A) Correlation between pathological and bacteriological parameters of all goats (n = 20). Dashed line (y = −x + 4.16) indicates the threshold defined to divide vaccine responders (VR) and vaccine non-responders (VNR). Symbols represent vaccine cross-protection outcome: (■) unvaccinated controls (UC), (○) VR goats, (●) VNR goats. (B) Specific IFN-γ responses at week 28 of the experiment as determined by ELISA after stimulation of whole blood with PPD-B, ESAT-6/CFP-10, or Rv3615c. Results are represented in relation to vaccine cross-protective outcome and are expressed as mean ΔOD450 ± 95% confidence interval. Significances determined by non-parametric Kruskal-Wallis test: *p < 0.05.

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To further analyze the reductions in post-mortem parameters associated with vaccination, a cross-sectional study was performed on the basis of the criteria described in a previous TB vaccine efficacy trial [

Methods

Experimental animals

The experiment was carried out in 20 Murciano-Granadina female goats between 2 and 3 months of age from a herd free of TB and PTB in the Region of Murcia (southwest Spain). Before the experiment, goats were submitted to standard tests for diagnosis of TB: the CIT and IFN-γ assay (Bovigam™; Prionics AG, Schlieren, Switzerland). Experimental animals were confirmed to be negative for both tests. In addition, all goats had negative results for PTB (Paratub.Serum-S; Institut Pourquier, Montpellier, France).

Vaccination and infection

A group of 10 goats was subcutaneously inoculated with a single dose of 1 ml (2.5 mg/ml) of Silirum® (CZ Veterinaria, Porriño, Pontevedra, Spain), a commercial heat-inactivated and oil-adjuvanted Map vaccine. Another group of 10 goats was maintained as unvaccinated controls. After 14 weeks, all animals were housed and acclimatized in two experimental boxes (level 3 biocontainment) for 1 week prior to experimental infection.

The field strain of M. caprae SB0416 (http://www.mbovis.org) was used as the inoculum and was prepared as previously described [24]. Goats were anesthetized by intravenous administration of 4 to 6 mg/kg of propofol (Propofol Lipuro®) and 0.2 mg/kg of midazolam (Dormicum®), and subsequently inoculated with approximately 1.5 × 103 cfu of M. caprae suspended in 0.5 ml of phosphate-buffered saline (PBS) by the endobronchial route [24].

All experimental procedures were approved by the Animal Welfare Committee of the Universitat Autònoma de Barcelona in agreement with the European Union Laws for protection of experimental animals.

Sampling and clinical observations

Animals were observed twice daily at feeding time. Prior to vaccination and infection and every 2 weeks throughout the experiment, animals were weighed and their rectal temperature was taken and blood samples were collected from the jugular vein into heparinized blood tubes for immunological studies.

Diagnostic tests

Skin tests

Skin tests were carried out in all goats at 26 wpv and 12 wpi. Four goats were also tested at 14 wpv (the remaining goats were not tested to minimize the potential effect of the intradermal tuberculin inoculation on the IFN-γ assay). Tests were performed by inoculating 0.1 ml (2500 IU) of bovine (PPD-B) and avian (PPD-A) tuberculins (Porriño, Pontevedra, Spain) on the left and the right side of the neck, respectively. The skin-fold thickness was recorded just before inoculation and after 72 h. Results were interpreted either by considering only the increase in thickness for PPD-B (as in a single intradermal test, SIT) or for both PPD-B and PPD-A (as in a comparative intradermal test, CIT). The results were read with the strict interpretation used in bovine TB eradication programs. Goats were considered positive to SIT if the increase in skin-fold thickness at the PPD-B site was ≥2 mm. For the CIT interpretation, goats were considered positive if the increase in skin-fold thickness at the PPD-B site was ≥2 mm and higher than the increase at the PPD-A site.

IFN-γ assay

Whole-blood cultures were performed every 2 weeks in 96-well cell culture plates. Heparinized blood (1 ml per well) was incubated for 24 h at 37°C and 5% CO2 with either PPD-B or PPD-A at a final concentration of 10 μg/ml. Phytohemagglutinin (PHA) (Sigma-Aldrich, Steinheim, Germany) was used as positive control at 10 μg/ml; PBS was used as a negative control. In addition, at 0, 2, 6, 10, and 14 wpi, the peptide cocktail ESAT-6/CFP-10 (E/C) and Rv3615c (Animal Health and Veterinary Laboratories Agency, Weybridge, UK) were used at a final concentration of 5 μg/ml. Plasma supernatants were collected after centrifugation and transferred to a 96-well plate. The IFN-γ enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s instructions (Bovigam™). Optical density was measured at 450 nm (OD450) using a microplate reader (PowerWave XS; BioTek, Winooski, VT). Results were expressed as ΔOD450 (OD450 of antigen-stimulated sample minus OD450 of non-stimulated sample). A result was positive when ΔOD450 > 0.05, whereas in the case of the standard test (using PPD-A and PPD-B as stimuli), a result was positive when ΔOD450 of PPD-B > 0.05 and OD450 of PPD-B > OD450 of PPD-A.

Map IgG ELISA

Plasma samples were analyzed in duplicate for antibodies to Map with Paratub.Serum-S™ ELISA before vaccination (week 0), before infection (week 14), and before the end of the experiment (week 28). The assay was performed according to the manufacturer’s instructions. The ELISA reaction was also measured at OD450. The results were expressed as S/P (%), calculated as (mean of sample OD450 - mean of negative control OD450)/(mean of positive control OD450 - mean of negative control OD450) × 100. According to the criterion described by the manufacturer, a sample with an S/P of <45% was considered to be negative, of <55% and ≥45% to be doubtful, and of ≥55% to be positive.

Post-mortem examination

All goats were euthanized at 14 wpi (28 wpv) with an overdose of sodium pentobarbital administered intravenously. They were immediately necropsied to assess the presence and volume of tuberculous lesions in the lungs and pulmonary LN.

Lungs

Ten percent-buffered formalin was poured into the trachea, which then was tied, and the lungs were subsequently immersed in a container with formalin. After fixation, the lungs were sliced at 4- to 5-mm intervals, and each slice was photographed. Gross lesions were analyzed using image analyzer software (ImageJ 1.43u; National Institutes of Health, USA). The volume of gross lesions in each slice was calculated by multiplying the affected area and slice thickness. The total volume of gross lesions was calculated by adding the partial volumes of gross lesions obtained for each slice.

Lymph nodes

The number of gross lesions and their diameter were recorded for each LN at the time of necropsy. Data were recorded by the same pathologist to ensure measurement consistency. LN pathology scoring was calculated by the approximated total volume of granulomas per sample, calculated using the sphere formula (4/3 × π × r3). After pathological evaluation, the whole LN was processed for bacterial culture to calculate the bacterial load.

Bacterial count

Cranial and caudal mediastinal, tracheobronchial, and retropharyngeal LN were individually weighed and homogenized with 10 ml of sterile distilled water using a tissue homogenizer (Masticator; IUL Instruments, Barcelona, Catalonia, Spain). Homogenates were decontaminated with a final concentration of 0.35% w/v hexadecylpyridinium chloride [42] for 15 min with orbital shaking. Decontaminated homogenates were centrifuged at 2471 × g for 30 min, and pellets were resuspended in 10 ml of PBS containing 0.05% Tween 80. Aliquots of 0.1 ml of a 10-fold serial dilution of each homogenate were plated on Middlebrook 7H11 agar (BD Diagnostics, Sparks, MD). Plates were incubated at 37°C for 28 days, and colonies were then counted and the bacterial load (cfu/g) for each sample was calculated.

Statistical analysis

Student’s unpaired two-sample t-test was used for comparisons between the groups in terms of differences in thickness increases in the CIT, S/P values of Map IgG ELISA, number of affected lung lobes, logarithm-transformed data of volumes of gross lesions (log10 mm3), and bacterial loads in the LN (log10 cfu/g). Correlations between pathology and bacterial burden were assessed using linear regression analysis, whereas the non-parametric Spearman rank test was used to analyze correlations between IFN-γ responses (ΔOD450) and post-mortem data. After cross-sectional analysis, IFN-γ responses and post-mortem parameters in the resulting groups were compared by applying the non-parametric Kruskal-Wallis test and Dunn’s post hoc multiple comparison test. The normality of the data and the homogeneity of variances between treatment groups were assessed using the Shapiro-Wilk and Levene tests, respectively. Statistical analysis of the data was performed using SPSS Statistical Package version 17.0 (IBM Inc., Chicago, IL).