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Pathology of bovine tuberculosis
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Pathology of bovine tuberculosis M. Domingo a,b,*, E. Vidal a, A. Marco b a b
Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Catalonia, Spain Departament de Anatomia I Sanitat Animals, Universitat Autònoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Catalonia, Spain
A R T I C L E
I N F O
Article history: Received 7 October 2013 Accepted 28 March 2014 Keyword: Bovine tuberculosis pathology
A B S T R A C T
Bovine tuberculosis (bTB) is a chronic granulomatous caseous-necrotising inflammatory process that mainly affects the lungs and their draining lymph nodes (Ln.). The pathological changes associated with bTB infection reflect the interplay between the host defence mechanisms and the mycobacterial virulence factors and the balance between the immunologic protective responses and the damaging inflammatory processes. Inhalation is the most common infection route and causes lesions of the nasopharynx and lower respiratory tract, including its associated lymph nodes. The initial infection (primary complex) may be followed by chronic (post-primary) tuberculosis or may be generalised. Goat tuberculosis often produces liquefactive necrosis and caverns, similarly to human TB. The assessment of the severity of TB lesions is crucial for vaccine trials. Semi-quantitative gross lesion scoring systems have been developed for cattle, but imaging technology has allowed the development of more standardised, objective, and quantitative methods, such as multi-detector computed tomography (MDCT), which provides quantitative measures of lesion volume. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bovine tuberculosis (bTB) is an infectious disease of cattle that is caused by Mycobacterium bovis and Mycobacterium caprae1 and also affects domestic small ruminants, humans, and a wide range of other domestic and wild mammals (O’Reilly and Daborn, 1995). The recognition of the macro- and microscopic lesions of tuberculosis in cattle has aided the understanding of the disease process and has helped eradicate the infection from herds and countries. The basis of our tuberculosis pathology knowledge was settled during the successful eradication campaigns conducted during the first half of the 20th century in many European countries, North America, and Australia (Francis, 1958; Schulz et al., 1991). Eradication was prompted by the zoonotic risk of M. bovis and the great economic impact of this disease on farms and trade (Pollock and Neill, 2002; Wedlock et al., 2002). According to a recent EFSA assessment, milk pasteurisation has presently rendered the risk of transmission of M. bovis through milk and milk products negligible, and the risk of the (bovine) meat-borne transmission of tuberculosis to humans is currently also considered negligible, at least in industrialised countries (EFSA, 2013). However, the recognition of bTB during meat in-
* Corresponding author. Tel.: +34 935814567; fax: +34 935814490. E-mail address: [email protected] (M. Domingo). 1 Both M. bovis and M. caprae (Aranaz, 2003) cause tuberculosis in ruminants and other mammals, including humans. In the following, any reference to M. bovis also includes M. caprae unless otherwise stated.
spection is still highly relevant for the surveillance and control of this infection in animals and herds, both in countries with high to low prevalences of infection and in countries that are officially infection-free, where meat inspection is a cornerstone for granting and sustaining the official TB-free (OTB) status. Therefore, knowledge of the pathology of tuberculosis is a keystone for the recognition and identification of bTB-infected animals and herds. Bovine tuberculosis manifests as a chronic granulomatous caseous-necrotising inflammatory process that primarily affects the lungs and their draining lymph nodes (Ln.) but also affects many other organs depending on the portal of entry of the infection. The infection remains subclinical for months or years until the organ involvement is sufficiently severe to cause functional impairment. The lesions may remain localised or may generalise to other tissues and organs. The different stages of infection and morphopathological appearances of lesions have given rise to a complex nomenclature in classic reference textbooks (Francis, 1958; Schulz et al., 1991). However, only a few of the names and stages of bTB that were initially recognised are currently used in veterinary pathology reference textbooks, and the nomenclature is now greatly simplified (Dahme and Weiss, 2007; Jubb et al., 2007; Zachary and McGavin, 2012). This simplification may be partially due to the changes in lesion presentation that have been elicited through the continuous testing and eradication strategies, which have led to a simplification of the overall pathological picture. Currently, most infected animals are found in the initial stages of infection. Thus, advanced forms of bTB are currently rarely found in countries with on-going eradication campaigns (Liébana et al., 2008; Menzies and Neill, 2000;
http://dx.doi.org/10.1016/j.rvsc.2014.03.017 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
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Neill et al., 2001). Advanced forms of bTB usually appear only in infected herds that have not been previously subjected to the tuberculin skin test or in species that are not subjected to eradication campaigns, such as goats, sheep, or farmed game. Regardless of the terms that are applied to tuberculous lesions and the stages of the disease, the pathological changes associated with infection by pathogenic mycobacteria are inseparable from the interplay between the host defence mechanisms and the mycobacterial virulence factors, which can be considered a delicate equilibrium between the immunological protective responses and the inflammatory damaging processes (Pollock and Neill, 2002; Welsh et al., 2005). 2. Pathology of tuberculosis in cattle The pathological features of tuberculosis in cattle are well known from natural and experimental studies, and, with few particular exceptions, most of this knowledge can be applied to other domestic species, such as goats, sheep, pigs, and horses. However, in companion animals, the usually caseous exudative character of TB is not observed, and lesions are of a more proliferative nature. The following description is focused on tuberculosis in cattle, and some specific features of tuberculosis in other species are described in a different section. 2.1. Portal of entry of Mycobacterium bovis The route of transmission determines the location and spectrum of the lesions observed in bTB. Inhalation is the most common route of infection and causes lesions of the nasopharynx and lower respiratory tract, including the lungs and associated Ln. (Neill et al., 1994, 2001). In contrast, the ingestion of M. bovis from contaminated pastures, feed, or water usually causes lesions of the mesenteric lymph nodes (Menzies and Neill, 2000) and few or absent visible lesions on the intestinal wall. Other routes of infection, such as transplacental, genital, or intramammary, are now infrequent due to the epidemiological situations of most countries with active eradication programmes. Transplacental transmission to the foetus probably results from tuberculous endometritis and leads to a primary complex in the liver and/or in portal lymph nodes. Calves born with congenital infections usually develop generalised tuberculosis in the first weeks or months of life (Vural and Tunca, 2001). However, tuberculous endometritis is now a rare event due to the detection of infected animals and the removal of these animals from herds. Not surprisingly, in a recent cohort study that quantified the risk of bTB in the progeny of cows with confirmed infection, the relative risk was estimated to be 1.2 (95% confidence interval 0.8–1.79), and the study concluded that the progeny of infected dams are not at a significantly increased risk of M. bovis infection compared with the progeny of uninfected cows (Menzies et al., 2012). Coital transmission may occur if bTB is established in the genital organs, but this form of transmission is now also uncommon. Furthermore, mammary tuberculosis is usually transmitted iatrogenically from cow to cow through intramammary treatments and is now very rare. Alimentary bTB in suckling calves was often a consequence of the presence of M. bovis in the dam’s milk. Recent investigations of field and experimental cases of bTB have drawn attention to the tonsils as a site of infection by M. bovis. A significant number of naturally bTB-infected cattle (up to 20%) have tuberculous lesions in the palatine tonsils, and M. bovis can be isolated from animals that are not exhibiting lesions on the tonsils (Cassidy et al., 1999; Liébana et al., 2008; Menzies and Neill, 2000). Furthermore, the experimental inoculation of cattle with M. bovis by intratonsilar instillation also leads to granulomatous tuberculous lesions in the retropharyngeal lymph nodes six and eight weeks after inoculation; this finding highlights the relevance of this infection pathway (Palmer et al., 1999). Intranasal inoculation with
a high dose of M. bovis also produces lesions in the upper respiratory tract mucosa, retropharyngeal lymph nodes, and the lungs (Cassidy et al., 1999). The oropharyngeal mucosa and retropharyngeal Ln. should probably be viewed as a common pathway for both upper respiratory and alimentary infections. Indeed, in a classic study examining the organ locations of tuberculous lesions in bovine animals with a single lesion (Corner et al., 1990), the most frequently affected organ was found to be the medial retropharyngeal Ln. (29.4%), followed by the mediastinal Ln. (28.2%), the tracheobronchial Ln. (18.0%), the lungs (8.0%), the mesenteric Ln. (2.9%), the parotid Ln. (2.4%), and the caudal cervical Ln. (2.4%). In summary, although the aerogenous route is the most common pathway of infection in domestic ruminants and in human beings and most cases of bTB involve the lungs and respiratory Ln. (O’Reilly and Daborn, 1995; Smith and Moss, 1994), the oropharyngeal route is apparently also a frequent pathway of entry that is probably more relevant than previously believed. 2.2. Primary infection The entry of mycobacteria through mucous membranes or into alveolar spaces leads to the recognition of bacterial cell wall components and the activation of inflammatory signalling pathways in phagocytes. The mycobacteria are then phagocytised by macrophages, and neutrophils are attracted to and accumulate at the site of initial infection. These cells interact with other cells involved in the innate and acquired immunological responses (Arentz and Hawn, 2007). In immunocompetent human beings, approximately 90% of infections are controlled through this initial immune response, and specific CD4+ T-cells and activated macrophages eliminate the mycobacteria or control its multiplication for years or decades (latent tuberculosis). Consequently, only a small proportion of infected individuals develop active tuberculosis (Smith and Moss, 1994, see O’Garra et al., 2013 for a recent review). It is unknown whether latent infections or even the elimination of mycobacteria after a primary infection occur in cattle. It has been hypothesised that some cattle with positive skin tests may be latently infected, and this hypothesis is based on the failure to detect tuberculous lesions or to culture M. bovis from several organs (Pollock and Neill, 2002). However, there is no practical way to detect latent infections in cattle, and the examination and culture of many different lymph nodes throughout the body would be necessary before declaring a carcass uninfected. Regardless of the real status of animals exhibiting cell-mediated immunity (CMI) to M. bovis antigens, these animals are generally slaughtered as a measure to control and eradicate infection in herds. Furthermore, this assessment is mixed and confounded with the well-known problems of the specificity of the tuberculin skin test in bovines (de la Rua-Domenech et al., 2006). After the initial infection, the viable mycobacteria are readily transported by phagocytic cells through lymphatic capillary vessels to the draining lymph node, where they establish a new infection focus. This dual infection is known as the primary complex, and it is usually subclassified as complete or incomplete depending on whether both lesions are present or the lesion at the site of entry is missing. The primary complex is most frequently found in the lower respiratory tract of domestic ruminants, although the initial lesion in the lung may frequently be absent or so small that it is missed at necropsy if it is not carefully searched for (Fig. 1). However, the slicing of the lungs into thin sections and careful examination may reveal small lesions in almost 70% of infected animals (McIlroy et al., 1986). The typical gross lesion of tuberculosis is known as a tubercle, which is a circumscribed yellowish granulomatous inflammatory nodule approximately 2–20 mm in diameter that is more or less encapsulated by connective tissue and often contains central caseous necrosis and mineralisation. Histologically, small tuberculous granulomas are formed by neutrophils, epithelioid
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Fig. 1. A small tuberculous granuloma in the lung of a goat. The lesion is subpleural and easy to identify, but internal lesions may go unnoticed if the lung is not carefully inspected after slicing.
macrophages that sometimes have foamy cytoplasm, and a few Langhans-type multinucleated giant cells (Fig. 2). This lesion grows over time, and caseous necrosis develops in the centre of the tubercle and appears as amorphous eosinophilic material with necrotic cell debris and central mineralisation (Neill et al., 2001). Epithelioid macrophages, Langhans-type multinucleate giant cells, and lymphocytes surround this central zone of necrosis. Progressively, a more or less complete connective tissue capsule forms through the cytokine-induced proliferation of fibroblasts (Marshall et al., 1996) and the apposition of the pre-existing fibrous tissue of the interlobular septa. A low or very low number of acid-fast bacteria may be found in the caseous eosinophilic material or within the epithelioid cells or the multinucleated giant cells in most bTB lesions in cattle and other domestic ruminants. However, the apparent absence of acid-fast bacteria in histological slides with typical
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Fig. 3. Chronic lung tuberculosis in a cow. The large area of the diaphragmatic lung lobe affected by tuberculous lesions is sharply delimitated from the nonaffected lung tissue by the main lobulillar septa. The infection extends through the intracanalicular route and affects contiguous alveolar groups sharing the same airway ramifications.
tuberculous lesions is not sufficient to rule out the morphological diagnosis of tuberculosis. 2.3. Chronic organ tuberculosis In some animals, the existence of a vigorous CMI may prevent the growth of lesions and the extension of lesions to other organs, but the systemic immune response, which acts in the blood and lymphatic compartments, is usually unable to stop the spread of the infection via pre-existing anatomical channels in the organs, such as alveolar spaces and pulmonary airways. Therefore, tissue damage progresses, and the initially small granulomatous lesion at the portal of entry becomes larger with time. The presence of large caseous necrotic lesions that are mineralised, fibrotic, and often confluent in one organ (either a parenchymatous organ or a lymph node) results in the classification of chronic (or post-primary) tuberculosis that includes the tissue or organ affected in the name. In cattle, these chronic lesions frequently occur in the lung or in the mediastinal Ln. and may become large. In the lung, the chronic lesion is characterised by extension through the bronchiolar and bronchial tree with multifocal confluent caseous necrosis and, eventually, the formation of cavernous lesions inside the initially affected lobe. The affected zone shows a sharp delimitation that respects the architectural stromal lobular barriers (Fig. 3). Ulcerative lesions in the bronchi and the trachea may occur. The mineralisation of lesions is not prominent at this stage. Similarly, chronically affected lymph nodes may become considerably enlarged, may be fully occupied by granulomatous caseous material surrounded by irregular fibrous tissue trabeculae, and may expand with the unique limit of the thick peripheral fibrous capsule of the lymph node (Fig. 4). 2.4. Generalisation
Fig. 2. A histological section of a mediastinic lymph node of a cow showing a tuberculous granuloma in its initial stage. Neutrophils are abundant in the centre of the granuloma and are surrounded by macrophages, Langhans-type multinucleated cells, and lymphocytes. Many macrophages exhibit a foamy cytoplasm. H&E staining was used.
If the initial immune response is ineffective, a primary infection may generalise during this initial stage; this process is known as early generalisation. Similarly, generalisation may occur in the post-primary phase or after reinfection, in which case it is called late generalisation. Generalisation results from haematogenous or lymphatic dissemination of the mycobacteria following the erosion of small blood or lymphatic vessels by growing tubercles. The most common form of generalisation is miliary tuberculosis, which is characterised by a large number of small grey to white-yellowish caseous foci resembling millet seeds (Fig. 5) without clear-cut
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Fig. 4. Chronic lymph node tuberculosis in a cow. A mediastinic lymph node fully occupied by lesions showing confluent granulomas with caseous necrosis and mineralisation. Note the fibrous tissue proliferation that appears whitish striations interspersed among the granulomas.
Fig. 6. Severe caseous-necrotising acinous tuberculosis in the lung of a cow (“rupture” form). Whole acini are caseous necrotised with minimal or absent mineralisation. Nearly the entire lung is affected.
delimitations. These forms are probably the consequence of a massive release of mycobacteria into the circulation accompanied by the haematogenous invasion of the lungs and many other organs. Microscopically, acid-fast bacteria are easy to find in these lesions. Generalised lesions may also vary in size, which suggests more protracted bacteraemia. Some forms of generalisation appear to be particularly fulminant and extensive, and cause diffuse caseation of lesions with minimal mineralisation. These forms occur frequently in the lung and are usually called “rupture forms”, and it is assumed that the host’s CMI has waned (Fig. 6). During the generalisation phase, some animals may be “anergic” and show no reaction in CMI tests (either tuberculin tests or blood IFN-γ tests). Generalisation to the serosal surfaces, especially the pleura, pericardium, or peritoneum, may also occur, and is characterised by multiple small tubercles of approximately 0.5 to 1 cm in diameter that resemble pearls. Effusions in the serosal surfaces are very rare in tuberculosis in cattle. This form of generalisation to the serosal surfaces may occur by lymphohaematogenous dissemination or by local extension into the cavitary spaces from eroding granulomas in the
affected organs (Fig. 7). Serosal generalisation occurs in cattle but is uncommon in other domestic ruminants. 3. Tuberculosis in other domestic species M. bovis has a wide range of hosts and infects a large number of domestic and wild mammals (O’Reilly and Daborn, 1995). Although the general pathological features of bTB in cattle are shared with most other affected domestic species, there are some differences in the types and distributions of lesions (Thoen, 1994). TB in goats and sheep is closely related to cattle TB in terms of immunological responses and pathological characteristics (Bezos et al., 2011; Marianelli et al., 2010); however, due to the lack of TB eradication campaigns for these species in most countries, chronic and advanced TB forms are not uncommon in affected small ruminant herds. Although TB in small ruminants is considered an infrequent disease, it has been recognised for many years in some Mediterranean countries (Gutiérrez et al., 1995), particularly in goats. Recent reports of TB in both goats and sheep in several EU
Fig. 5. Miliary tuberculosis in the lung of a goat. A. Lateral view of the right lung after removal of the right chest wall. Multiple, small, confluent granulomas can be found throughout the lung. B. Transverse section of the same lung showing miliary granulomas. C. Histological section of the same lung at the same magnification as in B. Note the multifocal distribution of the miliary granulomas, which probably originated from a massive haematogenous dissemination of mycobacteria. H&E staining was used.
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Fig. 7. Miliary generalised tuberculosis on the peritoneal serosa (tuberculous “pearls”).
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countries (Álvarez et al., 2008; Daniel et al., 2009; Quintas et al., 2010; Sharpe et al., 2010) have renewed attention to these domestic species as possible TB reservoirs for cattle (Napp et al., 2013). TB in small ruminants is primarily a chronic infection that causes exudative granulomatous caseous inflammatory lesions in the lungs and associated Ln. (Fig. 8); however, lesions may also occur in the upper respiratory tract Ln. Disseminated forms in other organs, such as the spleen, liver, or mesenteric lymph nodes, are infrequently found (Daniel et al., 2009). Generalisation to serosal surfaces seldom occurs. Goats exhibit a strong tendency to develop liquefactive necrosis and caverns inside tuberculous granulomas (Fig. 9D) that is remarkably similar to that observed in human TB. Histologically, the lesions are similar to those observed in cattle (Marianelli et al., 2010) with the aforementioned difference. Swine and horses may be infected by M. bovis, M. tuberculosis, or M. avium, and the lesions caused by these mycobacteria may not be morphologically differentiable (Thoen, 1994). The digestive route of infection is common in these two species, and lesions are frequently found in oropharyngeal Ln, the gastrointestinal tract, and mesenteric and portal Ln. Lesions in pigs and horses are often disseminated, and tubercles or miliary granulomatous inflammation can be found in the liver, lungs, and spleen (Fig. 9). Lesions in pigs
Fig. 8. Tuberculosis in small domestic ruminants. A. Tracheobronchial and mediastinal lymph nodes from sheep. Confluent granulomas with caseous necrosis and mineralisation. B and C. Multifocal granulomatous lesions in (B) the liver of a sheep and (C) the lung of a goat. The enlargement of the thoracic lymph nodes and multiple granulomatous lesions involving all pulmonary lobes are evident. D. Extensive cavitary lesions in the lung of a goat.
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Fig. 9. A. Miliary tuberculosis in the lung of a pig. Caseous necrosis is usually minimal or absent. B. Multifocal hepatic tuberculosis in the liver of a pig. The typical manifestation of digestive alimentary tuberculosis in pigs is shown. C. Miliary tuberculosis in the lung of a horse. Small granulomatous lesions that resulted from haematogenous dissemination are shown. D. Multiple tuberculous lesions in the spleen of a horse. The lesions have a productive character and resemble mesenchymal tumours.
and horses show a more proliferative character than those in cattle, are less prone to caseous necrosis and mineralisation, and may be mistaken for neoplastic processes. Dogs and cats are susceptible to M. bovis, M. tuberculosis, and M. avium, which may cause lesions and disease in immunosuppressed cats. In these species, lesions correspond to solid granulomatous inflammatory infiltrations that lack caseation and are grossly similar to neoplastic disease (Zachary and McGavin, 2012) (Fig. 10). Serohaemorrhagic effusions may occur in the thoracic and pericardial cavities in dogs (Dahme and Weiss, 2007). 4. Evaluation of tuberculous lesions in experimental vaccine efficacy studies Animal models are a fundamental tool for progress in tuberculosis vaccine research, and vaccine strategies are first developed using laboratory animals, such as mice, rabbits, guinea pigs, and nonhuman primates (Young, 2009), and then tested in clinical trials (Rowland and McShane, 2011). In the last decade, research efforts towards the development of vaccines against bovine TB that seek to control infection either in cattle or in the wildlife reservoirs of bTB, such as badgers and wild boars (Buddle et al., 2011; Hewinson et al., 2003; Vordermeier et al., 2006), have been renewed. Research on bTB vaccines has benefited from human vaccine research, and the promising vaccination strategies that have been developed for protection against M. tuberculosis may be
Fig. 10. Tuberculosis in the lung of a dog. The left apical lobe is affected by a chronic lesion. Miliary tuberculous dissemination to the lung tissue with multiple small nodules of approximately 2 mm in diameter is apparent. Tuberculosis lesions in dogs are productive, and mineralisation is rare or absent.
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Fig. 11. MDCT. Experimental tuberculosis infection in a goat four months after endobronchial infection with 1.53 M. caprae CFU. A. Right lung showing a large tuberculous lesion in the diaphragmatic lobe. B. Three-dimensional reconstruction of the entire lung. The total volume of the lung is indicated. C. Volume-rendering image of the entire lung showing different tissue densities with different colours: air is shown in black, fluids are shown in grey, and tuberculous lesions are shown in red. D. Volume-rendering image of the entire lung showing only the tuberculous lesions in white. The total volume of the lesions is indicated. H: head, F: foot, R: right, L: left.
candidates for efficient testing in cattle. Moreover, bTB vaccine research in farm animal models (cattle, goats, and pigs) may provide additional relevant data on the safety and efficacy of candidate human vaccines (de Val Pérez et al., 2011; Gil et al., 2010; Pollock et al., 2006; Van Rhijn et al., 2008; Waters et al., 2012) because of the similarities in the pathological and immunological responses to pathogenic mycobacteria (Buddle et al., 2005; Perez de Val et al., 2012) between humans and large mammal hosts. The severity of the observed lesions and their distributions are highly dependent on the mycobacterial species that is inoculated, the dose applied, and the infection pathway (Buddle et al., 2005; Palmer et al., 2002).
Ideally, the models used should mimic as closely as possible the natural conditions in terms of route, dose, type, and distribution of lesions found in natural disease. From this perspective, models that employ low challenge doses that do not quickly overwhelm the host immune system and result in localised, circumscribed lesions in the lower respiratory tract are preferred (Vordermeier et al., 2006). In the majority of recent vaccine efficacy trials in cattle, the vaccines have been administered to young calves up to six months of age and are followed by mycobacterial challenge three to four months postvaccination and necropsy three to five months after M. bovis challenge. Natural aerogenous transmission through contact with infected
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Fig. 12. Histological sections from the lung of goats with an experimental tuberculosis infection. Small lesions illustrating the four different granuloma stages (according to Wangoo et al., 2005) that are used to classify granulomas in vaccine efficacy studies are shown. All of the sections were stained through the H&E method and are shown at the same magnification. A. Stage I: the granuloma is in its initial stage and exhibits poor organisation, few epithelioid macrophages and lymphocytes, and the absence of central caseous necrosis and mineralisation. B. Stage II: greater numbers of epithelioid macrophages are present, necrosis is minimal or absent, and encapsulation is incomplete or absent. C. Stage III: caseous necrosis in the centre of the granuloma with incipient mineralisation. Encapsulation is absent. D. Stage IV: granuloma with evident necrosis and mineralisation. Confluent lesions are occasionally present at this stage, and encapsulation is complete.
cattle has been shown to have poor efficacy for experimental studies (Costello et al., 1998; Khatri et al., 2012), and more standardised routes of infection, such as aerosolisation, intranasal, tonsilar, and endotracheal routes, are preferred both in ruminants and in badgers (Buddle et al., 1994; Corner et al., 2007; Dean et al., 2005). The aerosolisation of mycobacteria is frequently used in large and small laboratory animal models, and this method of delivering mycobacteria to the lungs is efficient at producing lesions but has the disadvantage of often causing lesions with multifocal distributions (Gonzalez-Juarrero et al., 2013; Rayner et al., 2013; Rodgers et al., 2007; Waters et al., 2009, see Young, 2009 for a recent review). None of the bTB vaccines that have been assessed to date is able to fully prevent infection and lesion formation, although they may induce variable reductions in lesions and/or reductions in the bacterial burdens of target organs, and these reductions are interpreted as being indicative of protection (Waters et al., 2012). Therefore, the assessment of lesion severity in vaccine trials should be performed using robust, objective, and if possible, quantitative
scoring systems. A semi-quantitative scoring system for gross lesions in the lungs and lymph nodes that is based on lesion distribution and extension has been developed for cattle and is frequently used in ruminants and other species to assess the efficacy of vaccines (Vordermeier et al., 2002; Waters et al., 2009). This method requires careful slicing of the lungs and lymph nodes into 0.5- to 1-mm-thin sections and careful observation, and provides a semiquantitative grading of the size and distribution of the lesions (Waters et al., 2009; Wedlock et al., 2005). In recent years, imaging technology and analytical software have allowed the development of more standardised, objective, and quantitative methods, such as radiographic morphometry (Maue et al., 2004), magnetic resonance imaging (MRI) (Sharpe et al., 2009), and multi-detector computed tomography (MDCT) (de Val Pérez et al., 2011). In our experience, MDCT is a robust quantitative system for assessing the intensity of pathological changes in the goat model of tuberculosis. The lungs have to be carefully removed from the thoracic cavity while ensuring that the pleural surface is not incised. The mediastinic and
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tracheobronchial Ln. are also carefully removed, semi-quantitatively scored for pathological changes under sterile conditions, and used, as a whole, for the determination of the mycobacterial load by titration. After removal of the pericardium, the heart, and the large vessels, the lungs are fixed with 10% buffered formalin by pouring the fixative into the trachea (perfusion is accomplished by gravity through the use of a vertical column that extends approximately 40– 50 cm above the lungs) while holding the lungs in a vertical position until the trachea is filled with fixative. The trachea is then tied, and the whole lungs are immersed in a container with formalin for complete fixation. After fixation, the lungs may be scanned, and the MDCT data are analysed and processed. The main outcomes of this process model is a measure of the volume of the lesions relative to the total pulmonary volume (Fig. 11) and a measure of the total mycobacterial burden in the respiratory tract lymph nodes. Goats with good health statuses that are free of other infections that can induce lung lesions (such as lung nematode parasites and caseous lymphadenitis, which can be mistaken for tuberculous lesions in MDTC analysis) should be used. The microscopic features of tuberculous granulomas in lymph nodes have been described in detail and are used to stage these granulomas according to morphological criteria, such as the presence or absence of necrosis, mineralisation, and fibrous capsules (Wangoo et al., 2005). In this classification, four types of granulomas, namely type I to type IV, are recognised. Type I granulomas represent the initial stage and contain clusters of epithelioid macrophages with multinucleated Langhans-type cells and a thin rim of lymphocytes. Necrosis is absent at this stage. Neutrophil infiltration in the centres of the granulomas is not a feature of the lesions in lymph nodes, but neutrophils are often observed in early lung granulomas, at least in cattle. In type II granulomas, epithelioid macrophages, multinucleated Langhans-type cells, and lymphocytes are more numerous, and caseous necrosis starts to develop in the centres of the tubercles. Caseous necrosis is well developed in type III granulomas, but mineralisation is minimal. Type IV lymph node granulomas are “mature” and have extensive, often confluent, caseous necrosis with mineralisation and well-developed fibrous encapsulations (Fig. 12). This classification system can likely also be applied to lung granulomas. The motivation behind this classification system is to provide a framework within which the development and chronicity of lesions can be interpreted, which helps the identification of stages and the recognition of deviations from the normal granuloma progression, such as those induced by vaccination, immunosuppression, and mycobacterial virulence variation.
Acknowledgements The studies of the goat model of TB were supported by two EU-7th-FP projects: NADIR and TB-STEP. We thank Bernat Perez from CReSA for providing the MDCT images and the Institute of Veterinary Pathology, Giessen, Germany, for providing some of the macroscopic pictures.
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Pathology of bovine tuberculosis M. Domingo a,b,*, E. Vidal a, A. Marco b a b
Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Catalonia, Spain Departament de Anatomia I Sanitat Animals, Universitat Autònoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Catalonia, Spain
A R T I C L E
I N F O
Article history: Received 7 October 2013 Accepted 28 March 2014 Keyword: Bovine tuberculosis pathology
A B S T R A C T
Bovine tuberculosis (bTB) is a chronic granulomatous caseous-necrotising inflammatory process that mainly affects the lungs and their draining lymph nodes (Ln.). The pathological changes associated with bTB infection reflect the interplay between the host defence mechanisms and the mycobacterial virulence factors and the balance between the immunologic protective responses and the damaging inflammatory processes. Inhalation is the most common infection route and causes lesions of the nasopharynx and lower respiratory tract, including its associated lymph nodes. The initial infection (primary complex) may be followed by chronic (post-primary) tuberculosis or may be generalised. Goat tuberculosis often produces liquefactive necrosis and caverns, similarly to human TB. The assessment of the severity of TB lesions is crucial for vaccine trials. Semi-quantitative gross lesion scoring systems have been developed for cattle, but imaging technology has allowed the development of more standardised, objective, and quantitative methods, such as multi-detector computed tomography (MDCT), which provides quantitative measures of lesion volume. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bovine tuberculosis (bTB) is an infectious disease of cattle that is caused by Mycobacterium bovis and Mycobacterium caprae1 and also affects domestic small ruminants, humans, and a wide range of other domestic and wild mammals (O’Reilly and Daborn, 1995). The recognition of the macro- and microscopic lesions of tuberculosis in cattle has aided the understanding of the disease process and has helped eradicate the infection from herds and countries. The basis of our tuberculosis pathology knowledge was settled during the successful eradication campaigns conducted during the first half of the 20th century in many European countries, North America, and Australia (Francis, 1958; Schulz et al., 1991). Eradication was prompted by the zoonotic risk of M. bovis and the great economic impact of this disease on farms and trade (Pollock and Neill, 2002; Wedlock et al., 2002). According to a recent EFSA assessment, milk pasteurisation has presently rendered the risk of transmission of M. bovis through milk and milk products negligible, and the risk of the (bovine) meat-borne transmission of tuberculosis to humans is currently also considered negligible, at least in industrialised countries (EFSA, 2013). However, the recognition of bTB during meat in-
* Corresponding author. Tel.: +34 935814567; fax: +34 935814490. E-mail address: [email protected] (M. Domingo). 1 Both M. bovis and M. caprae (Aranaz, 2003) cause tuberculosis in ruminants and other mammals, including humans. In the following, any reference to M. bovis also includes M. caprae unless otherwise stated.
spection is still highly relevant for the surveillance and control of this infection in animals and herds, both in countries with high to low prevalences of infection and in countries that are officially infection-free, where meat inspection is a cornerstone for granting and sustaining the official TB-free (OTB) status. Therefore, knowledge of the pathology of tuberculosis is a keystone for the recognition and identification of bTB-infected animals and herds. Bovine tuberculosis manifests as a chronic granulomatous caseous-necrotising inflammatory process that primarily affects the lungs and their draining lymph nodes (Ln.) but also affects many other organs depending on the portal of entry of the infection. The infection remains subclinical for months or years until the organ involvement is sufficiently severe to cause functional impairment. The lesions may remain localised or may generalise to other tissues and organs. The different stages of infection and morphopathological appearances of lesions have given rise to a complex nomenclature in classic reference textbooks (Francis, 1958; Schulz et al., 1991). However, only a few of the names and stages of bTB that were initially recognised are currently used in veterinary pathology reference textbooks, and the nomenclature is now greatly simplified (Dahme and Weiss, 2007; Jubb et al., 2007; Zachary and McGavin, 2012). This simplification may be partially due to the changes in lesion presentation that have been elicited through the continuous testing and eradication strategies, which have led to a simplification of the overall pathological picture. Currently, most infected animals are found in the initial stages of infection. Thus, advanced forms of bTB are currently rarely found in countries with on-going eradication campaigns (Liébana et al., 2008; Menzies and Neill, 2000;
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Neill et al., 2001). Advanced forms of bTB usually appear only in infected herds that have not been previously subjected to the tuberculin skin test or in species that are not subjected to eradication campaigns, such as goats, sheep, or farmed game. Regardless of the terms that are applied to tuberculous lesions and the stages of the disease, the pathological changes associated with infection by pathogenic mycobacteria are inseparable from the interplay between the host defence mechanisms and the mycobacterial virulence factors, which can be considered a delicate equilibrium between the immunological protective responses and the inflammatory damaging processes (Pollock and Neill, 2002; Welsh et al., 2005). 2. Pathology of tuberculosis in cattle The pathological features of tuberculosis in cattle are well known from natural and experimental studies, and, with few particular exceptions, most of this knowledge can be applied to other domestic species, such as goats, sheep, pigs, and horses. However, in companion animals, the usually caseous exudative character of TB is not observed, and lesions are of a more proliferative nature. The following description is focused on tuberculosis in cattle, and some specific features of tuberculosis in other species are described in a different section. 2.1. Portal of entry of Mycobacterium bovis The route of transmission determines the location and spectrum of the lesions observed in bTB. Inhalation is the most common route of infection and causes lesions of the nasopharynx and lower respiratory tract, including the lungs and associated Ln. (Neill et al., 1994, 2001). In contrast, the ingestion of M. bovis from contaminated pastures, feed, or water usually causes lesions of the mesenteric lymph nodes (Menzies and Neill, 2000) and few or absent visible lesions on the intestinal wall. Other routes of infection, such as transplacental, genital, or intramammary, are now infrequent due to the epidemiological situations of most countries with active eradication programmes. Transplacental transmission to the foetus probably results from tuberculous endometritis and leads to a primary complex in the liver and/or in portal lymph nodes. Calves born with congenital infections usually develop generalised tuberculosis in the first weeks or months of life (Vural and Tunca, 2001). However, tuberculous endometritis is now a rare event due to the detection of infected animals and the removal of these animals from herds. Not surprisingly, in a recent cohort study that quantified the risk of bTB in the progeny of cows with confirmed infection, the relative risk was estimated to be 1.2 (95% confidence interval 0.8–1.79), and the study concluded that the progeny of infected dams are not at a significantly increased risk of M. bovis infection compared with the progeny of uninfected cows (Menzies et al., 2012). Coital transmission may occur if bTB is established in the genital organs, but this form of transmission is now also uncommon. Furthermore, mammary tuberculosis is usually transmitted iatrogenically from cow to cow through intramammary treatments and is now very rare. Alimentary bTB in suckling calves was often a consequence of the presence of M. bovis in the dam’s milk. Recent investigations of field and experimental cases of bTB have drawn attention to the tonsils as a site of infection by M. bovis. A significant number of naturally bTB-infected cattle (up to 20%) have tuberculous lesions in the palatine tonsils, and M. bovis can be isolated from animals that are not exhibiting lesions on the tonsils (Cassidy et al., 1999; Liébana et al., 2008; Menzies and Neill, 2000). Furthermore, the experimental inoculation of cattle with M. bovis by intratonsilar instillation also leads to granulomatous tuberculous lesions in the retropharyngeal lymph nodes six and eight weeks after inoculation; this finding highlights the relevance of this infection pathway (Palmer et al., 1999). Intranasal inoculation with
a high dose of M. bovis also produces lesions in the upper respiratory tract mucosa, retropharyngeal lymph nodes, and the lungs (Cassidy et al., 1999). The oropharyngeal mucosa and retropharyngeal Ln. should probably be viewed as a common pathway for both upper respiratory and alimentary infections. Indeed, in a classic study examining the organ locations of tuberculous lesions in bovine animals with a single lesion (Corner et al., 1990), the most frequently affected organ was found to be the medial retropharyngeal Ln. (29.4%), followed by the mediastinal Ln. (28.2%), the tracheobronchial Ln. (18.0%), the lungs (8.0%), the mesenteric Ln. (2.9%), the parotid Ln. (2.4%), and the caudal cervical Ln. (2.4%). In summary, although the aerogenous route is the most common pathway of infection in domestic ruminants and in human beings and most cases of bTB involve the lungs and respiratory Ln. (O’Reilly and Daborn, 1995; Smith and Moss, 1994), the oropharyngeal route is apparently also a frequent pathway of entry that is probably more relevant than previously believed. 2.2. Primary infection The entry of mycobacteria through mucous membranes or into alveolar spaces leads to the recognition of bacterial cell wall components and the activation of inflammatory signalling pathways in phagocytes. The mycobacteria are then phagocytised by macrophages, and neutrophils are attracted to and accumulate at the site of initial infection. These cells interact with other cells involved in the innate and acquired immunological responses (Arentz and Hawn, 2007). In immunocompetent human beings, approximately 90% of infections are controlled through this initial immune response, and specific CD4+ T-cells and activated macrophages eliminate the mycobacteria or control its multiplication for years or decades (latent tuberculosis). Consequently, only a small proportion of infected individuals develop active tuberculosis (Smith and Moss, 1994, see O’Garra et al., 2013 for a recent review). It is unknown whether latent infections or even the elimination of mycobacteria after a primary infection occur in cattle. It has been hypothesised that some cattle with positive skin tests may be latently infected, and this hypothesis is based on the failure to detect tuberculous lesions or to culture M. bovis from several organs (Pollock and Neill, 2002). However, there is no practical way to detect latent infections in cattle, and the examination and culture of many different lymph nodes throughout the body would be necessary before declaring a carcass uninfected. Regardless of the real status of animals exhibiting cell-mediated immunity (CMI) to M. bovis antigens, these animals are generally slaughtered as a measure to control and eradicate infection in herds. Furthermore, this assessment is mixed and confounded with the well-known problems of the specificity of the tuberculin skin test in bovines (de la Rua-Domenech et al., 2006). After the initial infection, the viable mycobacteria are readily transported by phagocytic cells through lymphatic capillary vessels to the draining lymph node, where they establish a new infection focus. This dual infection is known as the primary complex, and it is usually subclassified as complete or incomplete depending on whether both lesions are present or the lesion at the site of entry is missing. The primary complex is most frequently found in the lower respiratory tract of domestic ruminants, although the initial lesion in the lung may frequently be absent or so small that it is missed at necropsy if it is not carefully searched for (Fig. 1). However, the slicing of the lungs into thin sections and careful examination may reveal small lesions in almost 70% of infected animals (McIlroy et al., 1986). The typical gross lesion of tuberculosis is known as a tubercle, which is a circumscribed yellowish granulomatous inflammatory nodule approximately 2–20 mm in diameter that is more or less encapsulated by connective tissue and often contains central caseous necrosis and mineralisation. Histologically, small tuberculous granulomas are formed by neutrophils, epithelioid
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Fig. 1. A small tuberculous granuloma in the lung of a goat. The lesion is subpleural and easy to identify, but internal lesions may go unnoticed if the lung is not carefully inspected after slicing.
macrophages that sometimes have foamy cytoplasm, and a few Langhans-type multinucleated giant cells (Fig. 2). This lesion grows over time, and caseous necrosis develops in the centre of the tubercle and appears as amorphous eosinophilic material with necrotic cell debris and central mineralisation (Neill et al., 2001). Epithelioid macrophages, Langhans-type multinucleate giant cells, and lymphocytes surround this central zone of necrosis. Progressively, a more or less complete connective tissue capsule forms through the cytokine-induced proliferation of fibroblasts (Marshall et al., 1996) and the apposition of the pre-existing fibrous tissue of the interlobular septa. A low or very low number of acid-fast bacteria may be found in the caseous eosinophilic material or within the epithelioid cells or the multinucleated giant cells in most bTB lesions in cattle and other domestic ruminants. However, the apparent absence of acid-fast bacteria in histological slides with typical
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Fig. 3. Chronic lung tuberculosis in a cow. The large area of the diaphragmatic lung lobe affected by tuberculous lesions is sharply delimitated from the nonaffected lung tissue by the main lobulillar septa. The infection extends through the intracanalicular route and affects contiguous alveolar groups sharing the same airway ramifications.
tuberculous lesions is not sufficient to rule out the morphological diagnosis of tuberculosis. 2.3. Chronic organ tuberculosis In some animals, the existence of a vigorous CMI may prevent the growth of lesions and the extension of lesions to other organs, but the systemic immune response, which acts in the blood and lymphatic compartments, is usually unable to stop the spread of the infection via pre-existing anatomical channels in the organs, such as alveolar spaces and pulmonary airways. Therefore, tissue damage progresses, and the initially small granulomatous lesion at the portal of entry becomes larger with time. The presence of large caseous necrotic lesions that are mineralised, fibrotic, and often confluent in one organ (either a parenchymatous organ or a lymph node) results in the classification of chronic (or post-primary) tuberculosis that includes the tissue or organ affected in the name. In cattle, these chronic lesions frequently occur in the lung or in the mediastinal Ln. and may become large. In the lung, the chronic lesion is characterised by extension through the bronchiolar and bronchial tree with multifocal confluent caseous necrosis and, eventually, the formation of cavernous lesions inside the initially affected lobe. The affected zone shows a sharp delimitation that respects the architectural stromal lobular barriers (Fig. 3). Ulcerative lesions in the bronchi and the trachea may occur. The mineralisation of lesions is not prominent at this stage. Similarly, chronically affected lymph nodes may become considerably enlarged, may be fully occupied by granulomatous caseous material surrounded by irregular fibrous tissue trabeculae, and may expand with the unique limit of the thick peripheral fibrous capsule of the lymph node (Fig. 4). 2.4. Generalisation
Fig. 2. A histological section of a mediastinic lymph node of a cow showing a tuberculous granuloma in its initial stage. Neutrophils are abundant in the centre of the granuloma and are surrounded by macrophages, Langhans-type multinucleated cells, and lymphocytes. Many macrophages exhibit a foamy cytoplasm. H&E staining was used.
If the initial immune response is ineffective, a primary infection may generalise during this initial stage; this process is known as early generalisation. Similarly, generalisation may occur in the post-primary phase or after reinfection, in which case it is called late generalisation. Generalisation results from haematogenous or lymphatic dissemination of the mycobacteria following the erosion of small blood or lymphatic vessels by growing tubercles. The most common form of generalisation is miliary tuberculosis, which is characterised by a large number of small grey to white-yellowish caseous foci resembling millet seeds (Fig. 5) without clear-cut
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Fig. 4. Chronic lymph node tuberculosis in a cow. A mediastinic lymph node fully occupied by lesions showing confluent granulomas with caseous necrosis and mineralisation. Note the fibrous tissue proliferation that appears whitish striations interspersed among the granulomas.
Fig. 6. Severe caseous-necrotising acinous tuberculosis in the lung of a cow (“rupture” form). Whole acini are caseous necrotised with minimal or absent mineralisation. Nearly the entire lung is affected.
delimitations. These forms are probably the consequence of a massive release of mycobacteria into the circulation accompanied by the haematogenous invasion of the lungs and many other organs. Microscopically, acid-fast bacteria are easy to find in these lesions. Generalised lesions may also vary in size, which suggests more protracted bacteraemia. Some forms of generalisation appear to be particularly fulminant and extensive, and cause diffuse caseation of lesions with minimal mineralisation. These forms occur frequently in the lung and are usually called “rupture forms”, and it is assumed that the host’s CMI has waned (Fig. 6). During the generalisation phase, some animals may be “anergic” and show no reaction in CMI tests (either tuberculin tests or blood IFN-γ tests). Generalisation to the serosal surfaces, especially the pleura, pericardium, or peritoneum, may also occur, and is characterised by multiple small tubercles of approximately 0.5 to 1 cm in diameter that resemble pearls. Effusions in the serosal surfaces are very rare in tuberculosis in cattle. This form of generalisation to the serosal surfaces may occur by lymphohaematogenous dissemination or by local extension into the cavitary spaces from eroding granulomas in the
affected organs (Fig. 7). Serosal generalisation occurs in cattle but is uncommon in other domestic ruminants. 3. Tuberculosis in other domestic species M. bovis has a wide range of hosts and infects a large number of domestic and wild mammals (O’Reilly and Daborn, 1995). Although the general pathological features of bTB in cattle are shared with most other affected domestic species, there are some differences in the types and distributions of lesions (Thoen, 1994). TB in goats and sheep is closely related to cattle TB in terms of immunological responses and pathological characteristics (Bezos et al., 2011; Marianelli et al., 2010); however, due to the lack of TB eradication campaigns for these species in most countries, chronic and advanced TB forms are not uncommon in affected small ruminant herds. Although TB in small ruminants is considered an infrequent disease, it has been recognised for many years in some Mediterranean countries (Gutiérrez et al., 1995), particularly in goats. Recent reports of TB in both goats and sheep in several EU
Fig. 5. Miliary tuberculosis in the lung of a goat. A. Lateral view of the right lung after removal of the right chest wall. Multiple, small, confluent granulomas can be found throughout the lung. B. Transverse section of the same lung showing miliary granulomas. C. Histological section of the same lung at the same magnification as in B. Note the multifocal distribution of the miliary granulomas, which probably originated from a massive haematogenous dissemination of mycobacteria. H&E staining was used.
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Fig. 7. Miliary generalised tuberculosis on the peritoneal serosa (tuberculous “pearls”).
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countries (Álvarez et al., 2008; Daniel et al., 2009; Quintas et al., 2010; Sharpe et al., 2010) have renewed attention to these domestic species as possible TB reservoirs for cattle (Napp et al., 2013). TB in small ruminants is primarily a chronic infection that causes exudative granulomatous caseous inflammatory lesions in the lungs and associated Ln. (Fig. 8); however, lesions may also occur in the upper respiratory tract Ln. Disseminated forms in other organs, such as the spleen, liver, or mesenteric lymph nodes, are infrequently found (Daniel et al., 2009). Generalisation to serosal surfaces seldom occurs. Goats exhibit a strong tendency to develop liquefactive necrosis and caverns inside tuberculous granulomas (Fig. 9D) that is remarkably similar to that observed in human TB. Histologically, the lesions are similar to those observed in cattle (Marianelli et al., 2010) with the aforementioned difference. Swine and horses may be infected by M. bovis, M. tuberculosis, or M. avium, and the lesions caused by these mycobacteria may not be morphologically differentiable (Thoen, 1994). The digestive route of infection is common in these two species, and lesions are frequently found in oropharyngeal Ln, the gastrointestinal tract, and mesenteric and portal Ln. Lesions in pigs and horses are often disseminated, and tubercles or miliary granulomatous inflammation can be found in the liver, lungs, and spleen (Fig. 9). Lesions in pigs
Fig. 8. Tuberculosis in small domestic ruminants. A. Tracheobronchial and mediastinal lymph nodes from sheep. Confluent granulomas with caseous necrosis and mineralisation. B and C. Multifocal granulomatous lesions in (B) the liver of a sheep and (C) the lung of a goat. The enlargement of the thoracic lymph nodes and multiple granulomatous lesions involving all pulmonary lobes are evident. D. Extensive cavitary lesions in the lung of a goat.
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Fig. 9. A. Miliary tuberculosis in the lung of a pig. Caseous necrosis is usually minimal or absent. B. Multifocal hepatic tuberculosis in the liver of a pig. The typical manifestation of digestive alimentary tuberculosis in pigs is shown. C. Miliary tuberculosis in the lung of a horse. Small granulomatous lesions that resulted from haematogenous dissemination are shown. D. Multiple tuberculous lesions in the spleen of a horse. The lesions have a productive character and resemble mesenchymal tumours.
and horses show a more proliferative character than those in cattle, are less prone to caseous necrosis and mineralisation, and may be mistaken for neoplastic processes. Dogs and cats are susceptible to M. bovis, M. tuberculosis, and M. avium, which may cause lesions and disease in immunosuppressed cats. In these species, lesions correspond to solid granulomatous inflammatory infiltrations that lack caseation and are grossly similar to neoplastic disease (Zachary and McGavin, 2012) (Fig. 10). Serohaemorrhagic effusions may occur in the thoracic and pericardial cavities in dogs (Dahme and Weiss, 2007). 4. Evaluation of tuberculous lesions in experimental vaccine efficacy studies Animal models are a fundamental tool for progress in tuberculosis vaccine research, and vaccine strategies are first developed using laboratory animals, such as mice, rabbits, guinea pigs, and nonhuman primates (Young, 2009), and then tested in clinical trials (Rowland and McShane, 2011). In the last decade, research efforts towards the development of vaccines against bovine TB that seek to control infection either in cattle or in the wildlife reservoirs of bTB, such as badgers and wild boars (Buddle et al., 2011; Hewinson et al., 2003; Vordermeier et al., 2006), have been renewed. Research on bTB vaccines has benefited from human vaccine research, and the promising vaccination strategies that have been developed for protection against M. tuberculosis may be
Fig. 10. Tuberculosis in the lung of a dog. The left apical lobe is affected by a chronic lesion. Miliary tuberculous dissemination to the lung tissue with multiple small nodules of approximately 2 mm in diameter is apparent. Tuberculosis lesions in dogs are productive, and mineralisation is rare or absent.
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Fig. 11. MDCT. Experimental tuberculosis infection in a goat four months after endobronchial infection with 1.53 M. caprae CFU. A. Right lung showing a large tuberculous lesion in the diaphragmatic lobe. B. Three-dimensional reconstruction of the entire lung. The total volume of the lung is indicated. C. Volume-rendering image of the entire lung showing different tissue densities with different colours: air is shown in black, fluids are shown in grey, and tuberculous lesions are shown in red. D. Volume-rendering image of the entire lung showing only the tuberculous lesions in white. The total volume of the lesions is indicated. H: head, F: foot, R: right, L: left.
candidates for efficient testing in cattle. Moreover, bTB vaccine research in farm animal models (cattle, goats, and pigs) may provide additional relevant data on the safety and efficacy of candidate human vaccines (de Val Pérez et al., 2011; Gil et al., 2010; Pollock et al., 2006; Van Rhijn et al., 2008; Waters et al., 2012) because of the similarities in the pathological and immunological responses to pathogenic mycobacteria (Buddle et al., 2005; Perez de Val et al., 2012) between humans and large mammal hosts. The severity of the observed lesions and their distributions are highly dependent on the mycobacterial species that is inoculated, the dose applied, and the infection pathway (Buddle et al., 2005; Palmer et al., 2002).
Ideally, the models used should mimic as closely as possible the natural conditions in terms of route, dose, type, and distribution of lesions found in natural disease. From this perspective, models that employ low challenge doses that do not quickly overwhelm the host immune system and result in localised, circumscribed lesions in the lower respiratory tract are preferred (Vordermeier et al., 2006). In the majority of recent vaccine efficacy trials in cattle, the vaccines have been administered to young calves up to six months of age and are followed by mycobacterial challenge three to four months postvaccination and necropsy three to five months after M. bovis challenge. Natural aerogenous transmission through contact with infected
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Fig. 12. Histological sections from the lung of goats with an experimental tuberculosis infection. Small lesions illustrating the four different granuloma stages (according to Wangoo et al., 2005) that are used to classify granulomas in vaccine efficacy studies are shown. All of the sections were stained through the H&E method and are shown at the same magnification. A. Stage I: the granuloma is in its initial stage and exhibits poor organisation, few epithelioid macrophages and lymphocytes, and the absence of central caseous necrosis and mineralisation. B. Stage II: greater numbers of epithelioid macrophages are present, necrosis is minimal or absent, and encapsulation is incomplete or absent. C. Stage III: caseous necrosis in the centre of the granuloma with incipient mineralisation. Encapsulation is absent. D. Stage IV: granuloma with evident necrosis and mineralisation. Confluent lesions are occasionally present at this stage, and encapsulation is complete.
cattle has been shown to have poor efficacy for experimental studies (Costello et al., 1998; Khatri et al., 2012), and more standardised routes of infection, such as aerosolisation, intranasal, tonsilar, and endotracheal routes, are preferred both in ruminants and in badgers (Buddle et al., 1994; Corner et al., 2007; Dean et al., 2005). The aerosolisation of mycobacteria is frequently used in large and small laboratory animal models, and this method of delivering mycobacteria to the lungs is efficient at producing lesions but has the disadvantage of often causing lesions with multifocal distributions (Gonzalez-Juarrero et al., 2013; Rayner et al., 2013; Rodgers et al., 2007; Waters et al., 2009, see Young, 2009 for a recent review). None of the bTB vaccines that have been assessed to date is able to fully prevent infection and lesion formation, although they may induce variable reductions in lesions and/or reductions in the bacterial burdens of target organs, and these reductions are interpreted as being indicative of protection (Waters et al., 2012). Therefore, the assessment of lesion severity in vaccine trials should be performed using robust, objective, and if possible, quantitative
scoring systems. A semi-quantitative scoring system for gross lesions in the lungs and lymph nodes that is based on lesion distribution and extension has been developed for cattle and is frequently used in ruminants and other species to assess the efficacy of vaccines (Vordermeier et al., 2002; Waters et al., 2009). This method requires careful slicing of the lungs and lymph nodes into 0.5- to 1-mm-thin sections and careful observation, and provides a semiquantitative grading of the size and distribution of the lesions (Waters et al., 2009; Wedlock et al., 2005). In recent years, imaging technology and analytical software have allowed the development of more standardised, objective, and quantitative methods, such as radiographic morphometry (Maue et al., 2004), magnetic resonance imaging (MRI) (Sharpe et al., 2009), and multi-detector computed tomography (MDCT) (de Val Pérez et al., 2011). In our experience, MDCT is a robust quantitative system for assessing the intensity of pathological changes in the goat model of tuberculosis. The lungs have to be carefully removed from the thoracic cavity while ensuring that the pleural surface is not incised. The mediastinic and
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tracheobronchial Ln. are also carefully removed, semi-quantitatively scored for pathological changes under sterile conditions, and used, as a whole, for the determination of the mycobacterial load by titration. After removal of the pericardium, the heart, and the large vessels, the lungs are fixed with 10% buffered formalin by pouring the fixative into the trachea (perfusion is accomplished by gravity through the use of a vertical column that extends approximately 40– 50 cm above the lungs) while holding the lungs in a vertical position until the trachea is filled with fixative. The trachea is then tied, and the whole lungs are immersed in a container with formalin for complete fixation. After fixation, the lungs may be scanned, and the MDCT data are analysed and processed. The main outcomes of this process model is a measure of the volume of the lesions relative to the total pulmonary volume (Fig. 11) and a measure of the total mycobacterial burden in the respiratory tract lymph nodes. Goats with good health statuses that are free of other infections that can induce lung lesions (such as lung nematode parasites and caseous lymphadenitis, which can be mistaken for tuberculous lesions in MDTC analysis) should be used. The microscopic features of tuberculous granulomas in lymph nodes have been described in detail and are used to stage these granulomas according to morphological criteria, such as the presence or absence of necrosis, mineralisation, and fibrous capsules (Wangoo et al., 2005). In this classification, four types of granulomas, namely type I to type IV, are recognised. Type I granulomas represent the initial stage and contain clusters of epithelioid macrophages with multinucleated Langhans-type cells and a thin rim of lymphocytes. Necrosis is absent at this stage. Neutrophil infiltration in the centres of the granulomas is not a feature of the lesions in lymph nodes, but neutrophils are often observed in early lung granulomas, at least in cattle. In type II granulomas, epithelioid macrophages, multinucleated Langhans-type cells, and lymphocytes are more numerous, and caseous necrosis starts to develop in the centres of the tubercles. Caseous necrosis is well developed in type III granulomas, but mineralisation is minimal. Type IV lymph node granulomas are “mature” and have extensive, often confluent, caseous necrosis with mineralisation and well-developed fibrous encapsulations (Fig. 12). This classification system can likely also be applied to lung granulomas. The motivation behind this classification system is to provide a framework within which the development and chronicity of lesions can be interpreted, which helps the identification of stages and the recognition of deviations from the normal granuloma progression, such as those induced by vaccination, immunosuppression, and mycobacterial virulence variation.
Acknowledgements The studies of the goat model of TB were supported by two EU-7th-FP projects: NADIR and TB-STEP. We thank Bernat Perez from CReSA for providing the MDCT images and the Institute of Veterinary Pathology, Giessen, Germany, for providing some of the macroscopic pictures.
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Please cite this article in press as: M. Domingo, E. Vidal, A. Marco, Pathology of bovine tuberculosis, Research in Veterinary Science (2014), doi: 10.1016/ j.rvsc.2014.03.017