Bacterial strains and culture media

Mycobacterium bovis NCTC 10772 is a bovine lymphnode isolate (http://www.straininfo2.ugent.be/culture/99859/catalog;jsessionid=C2B6AA33B1E3024D2889F173B08BDBF8) and M. bovis 04-303 is an isolate from wild boar tuberculous lesions [8]. M. bovis 04-303 has been previously characterized by Aguilar León et al. [8]. Both isolates were grown at 37°C on Middlebrook 7H9 (BD, USA) liquid medium plus 0.5% Tween 80 enriched with 0.4% pyruvic acid, and 1% albumin dextrose complex (ADC). Viable bacteria in the inocula were enumerated with the Live/Dead BacLight™ Bacterial Viability kit (Invitrogen, Molecular Probes, Carlsbad, California).

Infection and sampling

Eight uninfected healthy Fresian calves of approximately 3 months of age were obtained from a TB-free herd (TB-free for the last five years). These calves were negative for the tuberculin skin test and showed absence of an in vitro gamma interferon (IFN-γ) response to both avian tuberculin PPD (PPDA) and bovine tuberculin PPD (PPDB). Animal experimentations were performed inside the biosafety facilities of the National Institute of Agricultural Technology (INTA), Argentina, in compliance with the regulations of the Ethical Committee of INTA and authorized by the National Service of Agricultural and Food Health and Quality (SENASA). Calves were intratracheally inoculated with 10⁷ colony forming units (CFU) of M. bovis strains (four animals with NCTC 10772 and four animals with 04-303) as described previously [10]. Heparinized blood samples were taken at 15, 30, 60, 90, 120 and 150 days after inoculation (dpi), whereas nasal swabs were taken at 15, 30, 60, 90 and 120 dpi. The disease status of the animals was examined at post-mortem by the presence of lesions typical of bTB in lungs, liver, and retropharyngeal, mediastinal, and tracheobronchial lymph nodes. Tissue samples were processed for bacterial culture in Stonebrink medium as described elsewhere [11]. Cultures were processed by Ziehl-Neelsen staining when colonies appear and IS 6110-PCR was performed on DNA from colonies to confirm its identity as M. tuberculosis complex.

DNA extraction from swabs and PCR

Nasal swabs were collected using 50-cm long sterile swabs. Swabs were washed with 20 ml of sterile PBS in 50 ml centrifuge tubes. These were centrifuged at 4500 g for 20 min. The pellets were resuspended in 400 μl of 1 x Tris-EDTA (TE) buffer. DNA was prepared as described by Van Soolingen et al. [12]. The oligonucleotides 5'-cgtgagggcatcgaggtggc-3' and 5'-gcgtaggcgtcggtgacaaa-3' specific for a 245-bp IS 6110 fragment were used. Touch-down amplification was performed with an initial step of 96°C for 3 min, followed by eight cycles of 96°C for 1 min, annealing temperatures starting at 72°C for 1 min (decreasing by 1°C/cycle), and extension at 72°C for 1 min [13]. This step was followed by 30 cycles of 96°C for 1 min, 65°C for 1 min, 72°C for 2 min, and extension at 72°C for 8 min.

Gamma Interferon (IFN-γ) release assay

Heparinized blood samples were dispensed in 200 μl aliquots into individual wells of a 96-well plate. Wells contained whole blood plus 20 μg/ml M. bovis PPD (Prionics), 20 μg/ml M. avium PPD (Prionics), or 4 μg/ml of ESAT6 or CFP10 recombinant antigens. Blood cultures were incubated for 18 h, and plasma was harvested and stored at -80°C. IFN-γ concentrations in stimulated plasma were determined using a commercial ELISA-based kit (Bovigam™; Prionics). Absorbance of standards and test samples were read at 450 nm. The optical density (OD) for the PBS controls, which was usually approximately 0.1 OD units, was used to normalize individual readouts and to calculate optical density indexes (ODIs), where the results obtained by antigen stimulation were divided by the results of the PBS-stimulated cultures. Duplicate samples for individual antigens were analyzed. The interaction between time (days post infection, dpi) and strain (04-303 and NCTC 10772) was significant (p < 0.001) for all antigens. To analyze those interactions, a multiple comparison of treatment means was performed using DGC procedure 22 at a significance level of 0.05. This method was preferred because it prevents the occurrence of overlapping groups of means, and thus facilitates interpretation.

Tuberculin testing

All animals were tested for skin tuberculin test (DTH) both before the M. bovis challenge and three months after the challenge. Animals were intradermally injected with 0.1 ml of PPDB and the thickness of the caudal fold tuberculin skin test were measured using calipers, and measured again 72 h after injection. PPDB (50,000 IU) was obtained from the National Service of Agricultural and Food Health and Quality (SENASA, Buenos Aires, Argentina).

Guinea pig virulence testing

Groups of five guinea pigs (females of 300-350 g) were intratracheally inoculated with 10³ CFU of either M. bovis NCTC 10772 or M. bovis 04-303 strains. Survival was followed for 110 days. After death or sacrifice the lesions were observed and recorded. DTH testing was performed in surviving animals at 40 dpi by intradermal injection at the back with 0.3 μg and 1 μg of PPDB and PBS as a control. The induration diameter was read at 72 h.

Histopathology: cattle and guinea pigs

Lungs, and retropharyngeal, mediastinal, and tracheobronchial lymph nodes in all cases and any other organ with lesions compatible with TB were collected and in cattle and guinea pigs and were fixed in 10% formaldehyde, sectioned and embedded in paraffin. Then, 5 μm-thick sections were stained with hematoxylin and eosin for histopathological examination. References were classified to a degree of I to IV according to Wangoo's criteria [14].

Statistical analysis

To analyze the ODI results, a mixed model was fit for each antigen. In all cases, a correlation structure was estimated to take into account the correlation between the observations made in the same animal. All models included a variance function to take into account variance dependence on fitted values. Models were fit using the nlme R library through a user-friendly interface implemented in InfoStat [15, 16].

Experimental infection of bovines and surveillance of M. bovisinfection by PCR in nasal swabs and blood samples

IFN-γ response

The bovine immune cellular response against both strains was evaluated by IFN-γ production in response to PPDB and recombinant antigens (Figure 1). Although all infected animals developed strong antigen-specific IFN-γ responses to PPDB, animals inoculated with the M. bovis 04-303 strain developed an early stronger response at 15 dpi. IFN-γ released by whole blood samples in response to ESAT6 and CFP10 followed a similar pattern, with a high response at 15 dpi in animals infected with 04-303 compared to those infected with the NCTC 10772 isolate. In the group infected with M. bovis 04-303, the responses to PPDB at 60 dpi showed higher ODIs in the IFN-γ test than in the group infected with the NCTC 10772 strain.

Delayed-type hypersensitivity response

The injection of M. bovis PPD resulted in negative tuberculin skin test reactions in all animals before the M. bovis infection. All animals were positive in the single intradermal tuberculin test at 90 dpi and M. bovis 04-303-inoculated cattle showed a stronger reaction than M. bovis NCTC 10772-inoculated ones (p < 0.05) by Student' t-test.

Pathological, histopathological tests and microbiological studies after challenge

Animals were killed after 5 months of infection. Typical tuberculosis lesions were found in three M. bovis 04-303-infected cattle. In this experimental group, lesions were granulomatous and pneumonic. In the three animals, lesions were localized in the left and right apical lobe lung, and in mediastinal lymph node and liver. The remaining animal showed no lesions. In the group inoculated with NCTC 10772 only one animal showed granulomatous lesions in the retropharyngeal lymph nodes (data not shown). The histopathological analysis revealed that while animals infected with M. bovis-04-303 showed type II and III granulomas in lung and lymphnodes in two animals and even type IV in another animal, no granulomas were observed in M. bovis NCTC 10772-infected cattle. In M. bovis 04-303 but not in M. bovis NCTC 10772-infected cattle also presented pneumonic areas and bronchiole infiltration (data not shown).

Virulence testing of both M. bovisstrains in guinea pigs

In order to confirm the results observed in cattle, groups of guinea pigs were infected with either M. bovis 04-303 or M. bovis NCTC 10772 and the survival rate was used as a main parameter of virulence. In the group infected with M. bovis 04-303, four animals died at 17 dpi and one animal died at 55 dpi. In the case of guinea pigs inoculated with NCTC 10772, two animals died before 30 dpi, whereas the remaining animals survived until the end of the experiment. A survival curve is shown in Figure 2. DTH was performed with PPDB at 40 dpi in surviving animals. Induration reaction after intradermal inoculation of 0.3 μg of PPDB was detected. In animals infected with M. bovis 04-303, the reaction was 3 mm, while in those infected with M. bovis NCTC 10772 it was 0.7 ± 0.19 mm. When 1.0 μg of the antigen was intradermally inoculated, the induration of animals infected with M. bovis 04-303 was 6.5 mm and in those infected with M. bovis NCTC 10772 was 1.8 ± 0.02. At necropsy, animals infected with M. bovis 04-303 that died at 17 dpi presented small purulent granulomas in lungs, while the one that died at 55 dpi showed large granulomas (data not shown).

The histopathological analysis of three out of four animals infected with NCTC 10772 showed extensive pneumonic areas with incipient type I or II granulomas. The other animal showed a less extended pneumonic area with cell infiltration in bronchioles. At macroscopic examination, two animals showed granulomatous lesions and the two other no apparent lesions (data not shown). Four out of five of the animals experimentally infected with M. bovis 04-303 had extensive pneumonic areas covering almost all the lung surface with incipient granulomas. Most of these animals died at 17 dpi. Another animal presented small areas with cell infiltration and pneumonia. At macroscopic examination, three of the animals that had died at 17 dpi did not present macroscopic TB lesions (data not shown). The animal that had died at 55 dpi presented numerous lesions.