Vet Immunol Immunopathol. 2015 Dec 15;168(3-4):249-57. doi: 10.1016/j.vetimm.2015.10.005.
Development of an in vitro model of the early-stage bovine tuberculous granuloma using Mycobacterium bovis-BCG.
Laura Garza-Cuarteroa, Elaine McCarthya, Joseph Bradya, Joseph Cassidya, Clare Hamiltonb, Mary Sekiyaa, Jim NcNairc, Grace Mulcahya
aSchool of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
bMoredun Research Institute, Penicuik, Midlothian, Edinburgh EH260PZ, Scotland, United Kingdom
cAgri-Food and Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland, United Kingdom
Corresponding Author: Laura Garza Cuartero, e-mail: firstname.lastname@example.org
Mycobacterium bovis causes 3.1% of human tuberculosis cases, as described by the World Health Organisation. In cattle, this organism causes bovine tuberculosis (BTB) which can have a prevalence of up to 39.5% in some developing countries. In developed countries, although the prevalence of BTB has been reduced through eradication programmes, complete eradication has in some cases proved elusive, with prevalences in cattle of 0.5% in the Republic of Ireland and of 4.3% in the UK. As the tuberculous granuloma is the fundamental lesion that reflects the pathogenesis, immune control and progression of BTB, we aimed to develop an in vitro model of the early-stage bovine tuberculous granuloma, in order to model the early stages of BTB, while also reducing the use of experimentally infected animals. In vitro models of human and ovine mycobacterial granulomas have previously been developed; however, so far, there is no model for the BTB granuloma. As the disease in cattle differs in a number of ways from that in other species, we consider this to be a significant gap in the tools available to study the pathogenesis of BTB. By combining bovine monocyte-derived macrophages infected with M. bovis-BCG and autologous lymphocytes we have developed an early-stage tuberculous bovine granuloma model. In the model, 3D cell aggregations formed a spherical-shape that grew for up to 11 days post-infection. This bovine tuberculous granuloma model can aid in the study of such lesion development, and in comparative studies of pathogenesis, such as, for example, the question of mycobacterial latency in bovine tuberculosis. Copyright © 2015 Elsevier B.V.
KEYWORDS: Bovine tuberculosis; In vitro; M. bovis-BCG; Model; Monocyte-derived macrophage; Tuberculous granuloma
Mycobacterium bovis, is the causative agent of Bovine tuberculosis (BTB) and it can also cause progressive disease in most warm-blooded vertebrates including man (1). As an estimate, the World Health Organization (WHO) reported that 3.1% of human tuberculosis is caused by M. bovis (2,3). The tuberculous granuloma is the fundamental lesion in tuberculosis and is the reflection of disease pathogenesis, and host immune response. This tuberculous granuloma is formed by aggregations of multinucleated, epithelioid cells and foamy macrophages at sites of infection, and together with neutrophils create the caseous necrotic centre (4). In addition, cytokines recruit lymphocytes to the tubercle. As the granuloma matures, a circumscribing fibrous capsule forms (5). In cattle, the disease is subclinical, potentially for many years, until substantial lesion development may induce clinical signs (6,7). In humans, such subclinical stage is known to be induced by a phenomenon of latency. However, in cases of immunosuppression or concurrent infection the tuberculous granuloma can be reactivated; which then develops a necrotic cavitated core, releasing the bacilli into the airways and disseminating the infection (8). To date, as the progression of the disease in bovine TB is significantly different from that on human TB, we considered crucial to develop a model of the early – stage granuloma of bovine TB.
Materials and Methods
For a detailed protocol see the manuscript Garza-Cuartero et al. 2015 (9). We used Holstein Friesian male cattle as sources of blood for culture of lymphocytes and monocyte-derived macrophages. We carried out the density gradient centrifugation for isolation of Peripheral Blood Mononuclear Cells (PBMC), and for macrophage differentiation we cultured monocytes over a period of 7 days (9). Macrophages were infected with Mycobacterium bovis-BCG at a ratio of 0.5:1 (bacteria:macrophage) for 12 hours and we added autologous lymphocytes for granuloma formation. Non-infected macrophages were included as negative controls throughout the experiments. We used ultra-low adherent plates to allow macrophage migration to form the cell aggregations; in previous experiments where tissue treated wells were used instead, cells attached to the wells, did not migrate, and therefore, did not form granulomas. Next, we monitored, counted and measured granuloma formation by microscopy. At selected time points we carried out histological analysis of granulomas to examine the tuberculous granuloma internal structure as well as to confirm intracellular BCG infection of cells (9). For this, we fixed first granulomas on the wells and then added haematoxylin in order to localize the granulomas later on. In parallel, we prepared 0.2% semi-liquid agar and poured it into fresh 24- well plates without any cells. Next, we collected with a transfer pipette the previously fixed and haematoxylin stained-granulomas and placed them gently into the centre of the semi-liquid agar. Once the agar plugs containing the granulomas were solidified we processed them as normal tissue for histology. Finally, we stained them with Eosin and Ziehl–Neelsen (ZN) to confirm the granuloma structure and localize the mycobacteria in the granuloma(9).
Results and discussion
In our experiments we found that M. bovis-BCG infected bovine macrophages and autologous lymphocytes aggregated to form defined 3D spheres, which increased in size and number over time. In comparison, uninfected cells formed flatter aggregations which did not increase in size or number. As the granuloma-like structures developed, they grew more compact in appearance, in comparison to the looser cell aggregations found in some control wells (9). The BCG-infected granulomas showed optimal integrity at Day 7-9 post-infection in some cases and in others, granulomas were well maintained until 21 days post-infection. Foamy macrophages characterised by the inclusion of intra-cytoplasmatic vacuoles were observed in the preparations from BCG-infected cells. However, cells in uninfected control wells did not develop such vacuoles (9). Those foamy macrophages have been shown to be present in mycobacterial infections (10,11).
The appearance of our tuberculous granuloma model is similar to that of previous early-stage tuberculous granulomas formed in vivo (12). Histological sectioning of agar-embedded granulomas, stained with H&E and counterstained with ZN, showed the presence of bacilli surrounded by macrophages and lymphocytes (Figure 1). See other examples in the original paper (9). Even though other in vitro models using human and ovine PBMC have shown induction of granuloma formation (13–17), here we show for the first time, an in vitro model of the bovine TB granuloma. The importance of this model laids in the fact that pathogenesis of the BTB in cattle differs from TB in humans as well as the disease in other hosts; for instance the well-known phenomenon of latency in human TB does not seem to occur frequently in BTB cattle. Hence, the availability of this specific in vitro model of the bovine tuberculous granuloma provides with a new tool for comparative studies of both diseases, such as the study of activation/dormancy genes in mycobacteria. For detailed information see Garza-Cuartero et al 2015 (9).
Figure 1. Histological examination of tuberculous granulomas. H & E and subsequent ZN staining of parafin-embedded agar containing granulomas (a, b) showed mycobacteria (arrow in b) and macrophages and lymphoctyes surrounding them. Squared granuloma in (a) at 20x magnification is shown in (b) at 40x.
This work was supported by Science Foundation Ireland (SFI) grant number 09/IN.1/B2625
We acknowledge the help of the manager and staff of Lyons Estate UCD farm for assistance in obtaining donor bovine blood. We would like to thank Stephen Gordon’s group in UCD, School of Veterinary Medicine, for their valuable advice on aspects of mycobacterial biology.
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