Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis

Abstract

With the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well-characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04-303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04-302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)-γ and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-γ, tumour necrosis factor-α and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD-4); whereas, the control strain AN5 induced progressive expression of this anti-microbial peptide, peaking at day 120. The less virulent strains induced high mBD-4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.

Introduction

Bovine tuberculosis is a significant worldwide disease of cattle. Mycobacterium bovis, the causative agent of this disease, is also a pathogenic organism for humans and several economically important animals [1–3]. In humans, zoonotic tuberculosis (TB) produced by M. bovis is difficult to distinguish clinically from the disease caused by M. tuberculosis. Indeed, before pasteurization of milk, M. bovis was an important cause of human tuberculosis, especially intestinal TB in children. After the generalized adoption of pasteurization of milk and other dairy products, zoonotic TB dropped sharply.

The M. bovis belongs to the M. tuberculosis complex (MTBC) that consisted traditionally of M. tuberculosis, the main agent of human tuberculosis, M. africanum that causes human tuberculosis and is prevalent in the African continent, and M. microti which is pathogenic for the vole. More recently, three new members of the complex have been described: M. canetti[4], a smooth variant that was isolated first from a Somali patient; M. bovis subsp. caprae, related closely to ‘classical’M. bovis, infecting primarily goats in Spain and humans and cattle in Central Europe [5,6], causing one-third of all human M. bovis cases of tuberculosis in Germany; and finally, M. pinnipedii, which causes tuberculosis in marine mammals [7–9].

Despite the high overall genetic relationship, the species of the MTBC show variability in their phenotypes, host range and importance for human TB. The epidemiology of M. bovis TB is very complex and includes transmission within and between domestic and wildlife animals, as well as from animals to humans and vice versa [2,10]. In many regions of the globe, wild mammals act as a reservoir of M. bovis, which prevents the complete eradication of the disease [10].

In cattle, the main route of infection is through the respiratory system. In classical studies, tuberculous lesions were found most frequently in the dorso-caudal region of the lungs, frequently close to the pleural surface [11], the most distant region from the upper respiratory tract in the Bos genus anatomy. In advanced control campaigns, disseminated disease is observed less frequently, and lesions are confined to lymph nodes associated with the respiratory tract. Lesions are observed frequently in retropharyngeal lymph nodes [11,12].

Several groups have reported different levels of virulence among M. tuberculosis isolates. Lopez et al.[13] studied the virulence of major genotypes of M. tuberculosis and found that some prevalent strains from the Beijing family are highly pathogenic in a mouse model of pulmonary tuberculosis, whereas other mycobacterial families showed reduced growth capacity and induced low mortality. Other authors [14] used the rabbit model of tuberculosis and observed that different strains produced different forms of tuberculosis. Medina et al.[15] studied the severity of TB caused by M. tuberculosis and M. bovis and found that the latter species produced more extrapulmonary forms of the disease. In spite of these studies, little is known about the intrinsic virulence properties of M. bovis isolates and the type of immune response that they produce using well-defined models. In this work, we used a well-characterized murine model of progressive pulmonary tuberculosis to study the virulence (survival, bacillary loads, histopathology) and immune response [cytokine expression determined by real-time polymerase chain reaction (PCR)] produced by selected M. bovis genotypes from an epidemiological study performed in Argentina.

Material and methods

Epidemiological and genetic characteristics of M. bovis strains

Table 1 shows the strains that we used in the present study. All the selected strains came from Argentinean cases of tuberculosis caused by M. bovis, except for the reference strain AN5 first isolated in England, which was used as control. Strains were characterized by biochemical tests and gave the typical M. bovis profile by spoligotyping [16,17] and mycobacterium interspersed repetitive unit (MIRU) typing [18].

Table 1.

Code, host source, geographical origin, spoligotype and mycobacterium interspersed repetitive unit (MIRU) patterns of the Mycobacterium bovis isolates used in this study.

Strain (source)	Origin (isolation year)	Pattern	Spoligotype number	MIRU pattern
02-2B (human)	Buenos Aires (2002)	0101101000001110111111111011111111111100000	60	232224253122
04-302 (wild boar)	La Pampa (2004)	1100011101111110111111111111111111110100000	10	232224263222
04-303 (wild boar)	La Pampa (2004)	1101101000001110111111111111111111111100000	34	232224263322
534 (cattle)	Santa Fe (1997)	1101101000001110111111111111111111111100000	34	232224163322
V2 (cattle)	Buenos Aires (2003)	1101101000001110111111111111111111111100000	34	232224253322
AN5 (reference strain)	England (1948)	1101011101001110111110111111111111111100000	55	232224243322

Spoligotyping was performed as described previously by Kamerbeek et al.[17]. Cluster analysis of the spoligotype patterns was performed with the BioNumerics software (Windows NT, version 2·5; Applied Maths, Kortijk, Belgium). The categorical coefficient was used to calculate the similarity of spoligotype patterns, and the unweighted pair-group method with arithmetic averages (UPGMA) method was applied to calculate a dendrogram. Clusters of isolates were defined as two or more M. bovis strains with identical spoligotypes. M. tuberculosis H37Rv [American Type Culture Collection (ATCC), Manassas, VA, USA; ATCC 27294] and M. bovis bacillus Calmette–Guérin (BCG) (ATCC 27289) were included as reference strains in each spoligotype determination.

Different polymorphic MIRU loci (MIRUs 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 and 40) were amplified with the oligonucleotides described previously [19]. The amplification mix consisted of 1× buffer (10 mM Tris-HCl, pH 9·0; 50 mM KCl, 0·1% Triton X-100) supplemented with Q buffer (Qiagen, GmbH, Hilden, Germany), 0·25 mM of each 2′-deoxynucleosides 5′-triphosphate, 0·1 µM of each primer and 1·25 U Taq polymerase (Hot Star; Qiagen). Different final concentrations of MgCl₂ were used: 2·5 mM MgCl₂ (MIRUs 23 and 39), 2 mM MgCl₂ (MIRUs 2, 4, 10, 16, 31 and 40) or 1·5 mM MgCl₂ (MIRUs 20, 24, 26 and 27) in a final volume of 50 µL. A culture lysate (5 µl) of each strain was used as template. Amplification was carried out in a PTC-100 thermal cycler (Programmable Thermal Controller; M. J. Research, Inc., Waltham, MA, USA) under the following conditions: initial step 95°C for 15 min; 40 cycles at 94°C for 1 min; 59°C for 1 min; 72°C for 1 min 30 s; and a final extension at 72°C for 10 min. The amplification size product was determined through the comparison with the 50 base pairs (bp) and 100 bp molecular weight markers (Promega, Madison, WI, USA) along with the pattern of the reference strain M. tuberculosis H37Rv. Additionally, Gel Compar version 4·1 (Applied Maths) was used.

Murine model of progressive pulmonary tuberculosis

The M. bovis strains were grown in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) enriched with OADC (oleic acid, albumin, dextrose and catalase; Becton Dickinson, Sparks, MD, USA) under agitation at 37°C. Growth was measured by densitometry up to day 18. Cell suspensions were aliquoted and frozen at −70°C as soon as they reached the stationary phase. Reculture was kept to a minimum to avoid virulence loss.

The experimental BALB/c model of progressive pulmonary tuberculosis has been described in detail previously [13,20–23]. Briefly, bacillary suspensions were adjusted to 2·5 × 10⁵ viable cells in 100 µl phosphate-buffered saline. Pathogen-free male BALB/c mice were used at 6–8 weeks of age. Each animal was anaesthetized with 56 mg/kg of intraperitoneal thiopental (Anestesal; Smith Kline Mex, Calz. México-Xochimilco, México D.F.). The trachea was exposed via a small midline incision, and the bacterial suspension was injected. The incision was sutured with sterile silk, and the mice were maintained in a vertical position until the effect of anaesthesia passed.

Six groups of 84 mice were infected, each group with a different M. bovis strain. The whole experiment was repeated and results were pooled. Twenty mice in each group were left undisturbed and survival was recorded up to day 120. Eight mice (all survivors, if less than eight) were killed by exsanguination at 1, 3, 7, 14, 21, 28, 60 and 120 days after infection. Lungs from four mice were prepared for histopathological studies. After eliminating hilar lymph nodes and thymic tissue, lungs of the remaining four animals were frozen in liquid nitrogen and kept at −70°C for microbiological and immunological studies. All procedures were performed in a laminar flow cabinet in a biosafety level III facility. The protocol was approved by the institutional Ethics Committee for Experimentation in Animals.

Colony-forming unit counts

Right or left lungs from four mice per each killing time-point were used in two different experiments. Lungs were homogenized with a Polytron homogenizer (Kinematica, Lucerne, Switzerland) in sterile tubes containing 3 ml of isotonic saline. Four dilutions of each homogenate were spread onto duplicate plates containing Bacto Middlebrook 7H11 agar (Difco) enriched with oleic acid, BSA, dextrose and catalase (OADC). The number of colonies was counted 21 days post-inoculation.

Histopathology and morphometry

For histological analysis, lungs were perfused with 100% ethanol via the trachea, immersed for 24 h and embedded in paraffin. Five-micrometer transversal sections, taken through the hilus, were stained with haematoxylin and eosin. The percentage of lung surface affected by pneumonia and the granuloma size in square microns were determined using an automated image analyser (Q Win Leica, Milton Keynes, UK) [13].

Real-time PCR analysis of cytokines in lung homogenates

Left or right lung lobes from three different mice per group in two different experiments were used to isolate mRNA using the RNeasy Mini Kit (Qiagen), according to the manufacturer's recommendations. Quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription (RT) of the mRNA was performed using 5 µg RNA, oligo-dT and the Omniscript kit (Qiagen). Real-time PCR was performed using the 7500 real-time PCR system (Applied Biosystems, USA) and Quantitect SYBR Green Mastermix kit (Qiagen). Standard curves of quantified and diluted PCR product, as well as negative controls, were included in each PCR run. Specific primers were designed using the programme Primer Express (Applied Biosystems) for the following targets: glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′-CATTGTGGAAGGGCTCATGA-3′, 5′-GGAAGGCCATGCCAGTGAGC-3′, tumour necrosis factor (TNF)-α: 5′-TGTGGCTTCGACCTCTACCTC-3′, 5′-GCCGAGAAAGGCTGCTTG-3′, interferon (IFN)-γ: 5′-GGTGACATGAAAATCCTGCAG-3′, 5′-CCTCAAACTTGGCAATACTCATGA-3′, interleukin (IL)-4: 5′-CGTCCTCACAGCAACGGAGA-3′, 5′-GCAGCTTATCGATGAATCCAGG-3′, inducible nitric oxide synthase (iNOS): 5′-AGCGAGGAGCAGGTGGAAG-3′, 5′-CATTTCGCTGTCTCCCCAA-3′ and murine beta defensin-4 (mBD-4): 5′-TCTGTTTGCATTTCTCCTGGTG-3′, 5′-TTTGCTAAAAGCTGCAGGTGG-3′. Cycling conditions used were: initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 20 s, 60°C for 20 s and 72°C for 34 s. Quantities of the specific mRNA in the sample were measured according to the corresponding gene-specific standard. The mRNA copy number of each cytokine was related to 1 million copies of mRNA encoding the GAPDH gene.

Statistics

A one-way anova and Student's t-test were used to compare morphometry and colony-forming unit (CFU) counts. Survival curves were compared with the Kaplan–Meier test. Differences were considered significant when P < 0·05.

Results

Strains and genotyping

Five hundred and forty-two M. bovis isolates were collected from different sources in Argentina and genotyped. From these, five strains were selected for the mouse model experiments according to their prevalence in cattle and occurrence in other species. Table 1 shows that three isolates had the Sp34 spoligotype prevalent in Argentina, which accounts for 45% of all isolates ([16], Martin Zumarraga, PhD thesis). Two strains with this spoligotype were from cattle (V2 and 534) coming from dairy farms in the humid Pampas region, and the third strain (04-303) was isolated from a wild boar. In spite of the identical spoligotypes of these strains, the MIRU pattern was different, indicating recent genetic divergence of these isolates. Strain 04-302 was isolated from another wild boar, but it had a different spoligotype (Sp10) that has a frequency of 0·5% of all isolates and was included for comparison with strain 04-303. Both wild boars were captured in free-ranging fields during testing for classical swine fever. Strain 02-2B had another spoligotype, Sp 60 (frequency 0·2%), and was isolated from a woman with chronic glenohumeral monoarthritis after a 6-week history of pain and swelling of the right shoulder [17]. One year previously, the patient had suffered fever with a left pleural effusion that resolved spontaneously. This patient lived with many cats that she fed daily with ‘raw beef lung’[17]. The reference strain AN5 was used as a control and its spoligotype was never found in Argentina. The relatedness of spoligotypes was analysed using the Dice similarity coefficient, and a dendrogram (data not shown) was constructed using UPGMA in BioNumerics (Applied Maths). The global relatedness of four spoligotypes was determined as 85·55%. The more related spoligotype Spo 34 and Spo 60 are 94·9% similar. Concerning MIRU typing, that corresponding to strain 04-303 was also observed in cattle isolates, while those of strains 04-302 and 02-2B were found exclusively in boars and humans respectively.

Differences in survival, lung pathology and bacillary loads in BALB/c mice infected with different M. bovis strains

BALB/c mice infected with the M. bovis reference strain AN5 were used as control. This strain showed intermediate virulence, producing 50% survival after 4 months of infection with moderate bacillary counts in the lungs (Fig. 1). The AN5 strain induced a strong inflammatory response with numerous well-formed granulomas after 2 weeks of infection and progressive pneumonia that affected 60% of the lung surface after 4 months of infection in co-existence with large granulomas (Figs 1 and 2). In contrast, mice infected with strain 04-303 showed higher mortality. Mice started to die at 3 weeks of infection and by 6 weeks all the mice had died, with twofold more lung CFU than seen in animals infected with the control strain. Interestingly, this strain induced a mild inflammatory response during the first and second weeks post-infection, followed by sudden extensive lung consolidation with massive areas of necrosis (Figs 1 and 2). Thus, this strain was the most virulent and its most striking feature was the sudden development of pneumonia with extensive necrosis (Fig. 2).

Fig. 1.

Survival, lung bacillary loads and morphometry (% of lung surface area affected by pneumonia and granuloma size) in BALB/c mice infected by an intratracheal injection (2·5 × 10⁵ bacilli) of Mycobacterium bovis with different genotypes and isolated from different hosts. Reference strain AN5 was used as control. Survival curves were constructed with 20 infected mice. Bacillary loads and morphometry curves were performed with four animals per time-point in two different experiments. Asterisks represent statistical significance (P < 0·005) compared with infection with the AN5 strain.

Fig. 2.

Representative mouse lung histopathology after 1 month of infection with selected Mycobacterium bovis strains with different genotypes and isolated from diverse hosts. (a) Reference strain AN5 induced large granulomas (arrows), moderate pneumonic patches and abundant perivascular inflammation. (b) In contrast, animals infected with the high virulent strain 04-303 showed massive pneumonia with extensive necrosis at the same infection day. (c) Mice infected with strain 04-302 also showed extensive pneumonia but mild necrosis. (d) In comparison, the lungs of mice after 4 months of infection with attenuated strain 02-2B showed small patches of pneumonia (all micrographs 100×, haematoxylin and eosin staining).

Strain 04-302 also induced higher mortality, killing all the animals after 2 months of infection. Lung histopathology showed a strong inflammatory response from day 3 post-infection, with progressive pneumonia after day 21 and large granulomas. Pneumonic areas at days 28 and 60 post-infection showed mild necrosis, producing 10% more lung consolidation at day 60 post-infection than was seen in mice infected with the control strain AN5 (Figs 1 and 2).

In contrast, strains 534, V2 and 02-2B were less virulent, permitting 100% and 80% survival after 4 months of infection, with lower bacillary loads. These strains induced small patches of pneumonia that affected less than 20% of the lung surface and smaller granulomas than in control animals infected with M. bovis strain AN5 (Figs 1 and 2).

Differences in immune responses evoked by different M. bovis strains

To understand more clearly the molecular and cellular basis of the observed bacterial virulence, the expression of cytokines and other relevant molecules was measured by real-time RT–PCR. Reference strain AN5 induced rapid, high and stable expression of IFN-γ. Lower expression but with similar kinetics was observed for IL-4 (Fig. 3). This strain also induced high expression of TNF-α, peaking on days 3 and 28 post-infection, whereas iNOS expression increased progressively, peaking on day 14 post-infection, followed by a slight decrease until the experiment concluded after 4 months of infection (Fig. 3). In contrast, the more virulent strain 04-303 induced significantly lower and stable expression of IFN-γ, TNF-α, iNOS and IL-4 from days 1 to 28 post-infection (Fig. 3). Similarly, low IFN-γ and iNOS expression was induced by the other highly virulent strain 04-302, but in contrast to strain 04-303, strain 04-302 induced higher TNF-α expression and the highest IL-4 levels, fourfold higher than IFN-γ (Fig. 3). We also studied the expression of mBD-4 as a significant factor of innate immunity. In this animal model, when infected with M. tuberculosis, H37Rv mBD-4 seems to have a significant role in the control of bacillary growth [20]. Interestingly, infection with either 04-303 or 04-302 induced very low mBD-4 expression, whereas the control strain AN5 induced progressively increasing expression of this anti-microbial peptide, peaking at day 120 (Fig. 3).

Fig. 3.

Expression of cytokine genes determined by real-time reverse transcription–polymerase chain reaction in the infected lungs. BALB/c mice were infected with the indicated Mycobacterium bovis strain and killed at different time-points. The lungs from four different animals at each time-point were used to determine the gene expression of the indicated factor. In comparison with the control strain AN5 (black and white bars), the clinical isolate 04-303 (black bars) induced lower expression of cytokines, inducible nitric oxide synthase (iNOS) and murine beta defensin-4 (mBD-4). Strain 04-302 (grey bars) also induced similar low interferon (IFN)-γ, iNOS and mBD-4 expression but with high interleukin (IL)-4 levels. In contrast, less virulent strains 02-2B (white bars), V2 (diagonal bars) and 534 (hatched bars) induced progressive increase of IFN-γ and iNOS, and high expression of IL-4 and mBD-4 during the first month followed by a sharp decrease during late infection, and constant high expression of tumour necrosis factor-α. Data are the means and standard deviations. Asterisks represent statistical significance (P < 0·05) when compared with mice infected with the AN5 strain.

The low virulence strain 02-2B induced progressive IFN-γ expression, peaking at day 120 post-infection (Fig. 3). This strain also induced rapid and high IL-4 expression during the first month post-infection, followed by a progressive decrease, reaching its lowest expression at day 120 post-infection. This strain induced relatively low TNF-α expression but it increased progressively, peaking at day 120 post-infection. Higher expression, but with similar kinetics, was observed for iNOS, which also peaked at day 120. The other low virulence strain V2 induced a progressive increase of IFN-γ and iNOS expression, which was significant during late disease, at days 60 and 120 post-infection (Fig. 3). This strain induced higher TNF-α expression than the control strain AN5 during the first month post-infection, followed by similar amounts to those exhibited by control animals during late disease. Similarly to strain 02-2B, isolate V2 also induced high IL-4 expression during the first month of infection, followed by a sharp decrease during late disease at days 60 and 120 post-infection. Strain 534, which also permitted prolonged survival of the mice, induced progressive expression of IFN-γ peaking at day 120 and high expression of IL-4 during the first month followed by a marked decrease, whereas expression of TNF-α and iNOS was higher than that induced by the control strain AN5 during late disease (Fig. 3). All these less virulent strains induced high expression of mBD-4 during early infection, followed by a progressive decrease during late disease (Fig. 3).

Discussion

Much of the information currently available on the pathogenesis of M. bovis comes from experimentally or naturally infected rabbits and cattle respectively [24,25]. However, several experimental models using different animals including mice have been established [25], and the mouse model has been totally validated. We took advantage of a well-characterized model of progressive pulmonary tuberculosis in BALB/c mice, induced by a high intratracheal challenge dose, to study the virulence and immune responses induced by selected M. bovis strains from a wide epidemiological/molecular study in Argentina. M. bovis strain AN5 is used worldwide for bovine purified protein derivative production because of its high bacterial mass yield in glycerinated media. This phenotype was selected by repeated subcultures in laboratory growth medium following a procedure similar to that used to generate BCG [26]. Interestingly, our results show that even after many subculture passages, strain AN5 is not highly attenuated. It exhibited an intermediate level of virulence producing 50% mortality after 2 months of infection.

When BALB/c mice are infected through the intratracheal route with a high dose of M. tuberculosis strain H37Rv, there is a predominant T helper type 1 (Th1) response peaking after 3 weeks that controls bacterial growth temporarily [21]. Then, bacterial proliferation resumes accompanied by an increment in Th2 cytokines and a decrease in IFN-γ, TNF-α and iNOS concomitantly with progressive pneumonia and small granulomas [22,23]. This experimental murine model resembles closely human pulmonary tuberculosis in developing countries, where there are high challenge doses and a tendency for elevated Th2 responses in progressive disease [27]. The present study shows that in spite of its high genome sequence similarity to M. tuberculosis H37Rv, M. bovis AN5 induced different immunopathology. The bovine strain induced more and bigger granulomas during late disease, with higher IFN-γ than IL-4 expression throughout the infection, as well as stable TNF-α expression. This cytokine profile could explain the production of large granulomas during late disease, but this response was not able to control growth of bacilli or tissue damage totally and eventually infected animals died. Differences in gene expression and regulation during infection could be related to these differences between the bovine and human bacilli. In comparison with M. tuberculosis, the bovine strain has a smaller genome, lacking several regions called RDs (regions of difference) [28]. Indeed, the greatest degree of sequence variation between the human and bovine bacillus occurs in genes encoding cell wall compounds or secreted proteins [26,29–31].

Previous studies in mice, comparing infection with M. tuberculosis (H37Rv) and M. bovis (strain Ravanel), have shown that the latter induced higher mortality and tissue damage, as well as a greater capacity for extrapulmonary dissemination [15,32], suggesting that M. bovis is actually more virulent than M. tuberculosis in this animal model. Our results, using several clinical isolates of M. bovis, showed for the first time wide variability in virulence. Strain 04-303 was the most virulent, killing all the animals after 4 weeks of infection with high lung bacillary loads and extensive pneumonia with massive necrosis. Similar results for survival and bacillary loads were obtained with the prevalent hypervirulent M. tuberculosis Beijing strain 9501000 [13]. However, this Beijing strain and many others that we have studied did not produce such massive necrosis as that observed with M. bovis 04-303. The cytokine expression profile induced by this M. bovis strain was also distinctive, evoking the lowest levels of both IFN-γ and IL-4 expression but with similar mRNA copies. Thus, this strain induced a rapid and stable mixed Th1/Th2 balance with higher TNF-α expression throughout the infection, which in this model has been associated with necrosis [33,34]. A similar Th0 profile has been observed in experimental models of bovine tuberculosis associated with reduced cellular protective immunity and extensive disease [35–37]. Another interesting observation was the low expression of mBD-4 in the lungs of mice infected with the highly virulent strains 04-303 and 04-302. Mycobacterial infection induces production of relevant molecules of the innate immune system, such as β-defensins [38,39]. These molecules are cationic natural anti-microbial peptides that can kill the microbes and some of them have chemotactic activities on immune cells [39]. In this animal model, we have shown that after infection with strain M. tuberculosis H37Rv there is rapid and high expression of mBD-4 during the early phase, while there is efficient control of bacillary replication. Then, during the progressive phase, there is a pronounced decrease in expression of mBD-4, while proliferation of the bacilli increases [20]. Thus, the low expression of mBD-4 in mice infected with highly virulent M. bovis strains could contribute to a high rate of bacillary replication and extensive tissue damage.

Several wildlife species are infected naturally with M. bovis, and the wild boar is a major reservoir in some regions in Europe, such as the south central area of Spain, where the prevalence of macroscopic tuberculous lesions observed in these animals reached 100% [40]. Although there is host genetic variability [41], wild boars seem to be infected easily by M. bovis, but apparently they are resistant to the development of clinical disease [42]. In spite of the fact that we only studied two isolates, it is interesting to speculate that such natural resistance of the host could be a selective force for organisms with higher virulence, similar to long-term massive BCG vaccination, which has been suggested as a contributory factor in the successful spread of the M. tuberculosis Beijing genotype [43].

The M. bovis strain 02-2B was isolated from another uncommon host and showed a non-prevalent spoligotype. This strain was isolated from an elderly lady, who was apparently infected after handling raw lung tissue from infected cows [19]. Strain 02-2B was clearly attenuated and induced the highest IFN-γ expression, peaking after 4 months of infection. Interestingly, strain 02-2B also induced high IL-4 expression in comparison with strain M. bovis AN5 during the first month of infection, followed by a sharp decrease during late disease, indicating that during chronic infection a predominant Th1 response was established that correlated with the efficient control of bacilli growth. A similar cytokine pattern but with the highest iNOS expression was induced by the other low virulence strain 534. Natural and experimental infection of cattle with M. bovis also induces efficient IL-4 production that usually decreases after 16 weeks post-infection [44]. Moreover, experimental infection of cows with a very low dose that did not result in lesions also induced a rapid and high IL-4 production which then decreased rapidly, as observed in mice infected with these low virulence strains. This IL-4 response has been considered an efficient anti-inflammatory response.

In conclusion, this study seeks to integrate the kinetics of pathological changes with the host immune responses, induced by selected M. bovis strains with different genotypes and isolated from diverse hosts. Our results show for the first time that M. bovis isolates exhibit different levels of virulence and immune response patterns. Strains isolated from wild animals showed higher virulence and induced non-protective immune responses, whereas strains isolated from humans or cattle exhibited lower virulence and induced a rapid Th1/Th2 cytokine profile that was switched to a predominant Th1 profile during late infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also shows variable virulence. The molecular basis for this differential virulence is currently being evaluated by expression microarray technology.

Disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.