J. Comp. Path. 2015, Vol. 152, 217e226
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Early Lesions following Aerosol Challenge of Rhesus Macaques (Macaca mulatta) with Mycobacterium tuberculosis (Erdman Strain) E. L. Rayner*, G. R. Pearson*, G. A. Hall*, F. Gleeson†, A. McIntyre†, D. Smyth*, M. J. Dennis* and S. A. Sharpe* * Public Health England, Microbiology Services Division, Porton Down, Salisbury and † Churchill Hospital, Headington, Oxford, UK
Summary Three rhesus macaques (Macaca mulatta) were challenged with Mycobacterium tuberculosis (Mtb), Erdman strain, as part of studies to investigate lesion development at early time points in tuberculosis (TB) and to assess computed tomography (CT) as a method of monitoring disease progression in vivo. Animals were challenged with either a high, mid or low dose of aerosolized Mtb. The low-dose animal was killed humanely at 24 days post challenge (dpc) and the remaining animals at 25 dpc. Abnormalities in clinical parameters were observed in all animals, but clinical signs relating to respiratory disease were not seen. Pulmonary changes consistent with TB infection were detected by CT at 21 dpc and magnetic resonance imaging (MRI) post mortem. Pulmonary nodule counts obtained from both imaging techniques were directly proportional to the challenge dose and correlated with gross and microscopical lesion counts. On gross and microscopical examination, lesions of similar size and morphology were observed in the lungs of all three animals, with the majority containing necrotic foci. Concomitant gross and microscopical, granulomatous lesions were observed in the tracheobronchial lymph nodes of all animals together with evidence of systemic spread. These findings further contribute to our understanding and knowledge of early lesion formation in the lungs of non-human primates. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: early granulomas; lung; macaque; tuberculosis
Introduction Tuberculosis (TB) remains a major threat to public health (World Health Organisation, 2014) and knowledge of the pathogenesis of the disease at early time points is limited. The use of animal models has contributed greatly to the understanding of hostepathogen interactions in this disease and nonhuman primates most closely resemble human beings with respect to immunological and genetic processes (Qiu et al., 2008), as well as to disease progression (Langermans et al., 2001). There are few reports of lesion formation early in infection in non-human primates. Recently, descriptions of gross and microscopical lesions in the lung Correspondence to: E. L. Rayner (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.10.002
and tracheobronchial (hilar) lymph nodes were reported in rhesus macaques (Macaca mulatta) exposed to an aerosol of Mycobacterium tuberculosis (Mtb) H37Rv strain, at varying doses and examined at 2e3 weeks post challenge (wpc) (Rayner et al., 2013). Early involvement of intrapulmonary lymphatics was reported in animals receiving high and mid doses of Mtb and concomitant lesions were present in the tracheobronchial lymph nodes in all animals in the high-dose groups at 2 and 3 wpc, all animals in the mid-dose group at 3 wpc and two out of three animals in the low-dose group at 3 wpc. Lin et al. (2006) reported lesion development in cynomolgus macaques (Macaca fasicularis) challenged with Mtb Erdman strain via the intratracheal route. Lung lesions were not observed until 4 wpc (microscopically) or 5 wpc (grossly). These lesions comprised Ó 2014 Elsevier Ltd. All rights reserved.
E.L. Rayner et al.
218
of primarily caseous granulomas, which was in contrast to the range of granulomatous lesions described for early infection of Mtb H37Rv in rhesus macaques (Rayner et al., 2013). In addition, lesions in the tracheobronchial lymph nodes were not observed until 4 wpc (Lin et al., 2006). Early lesion formation with the Erdman strain of Mtb, via the aerosol route, has not been described in rhesus macaques. Increasingly, imaging techniques are being employed to detect and monitor disease progression in the lungs of non-human primates both post mortem and, more recently, in vivo. Magnetic resonance imaging (MRI) has been used to determine lesion volume post mortem in agar-embedded, formalin-infused lungs in rhesus macaques challenged with Mtb Erdman by aerosol (Sharpe et al., 2009, 2010). Furthermore, in-vivo imaging of TB disease progression over a 3-month period using computed tomography (CT) with H37Rv (Lewinsohn et al., 2006) was reported. Recently, positron emission tomography with CT (PET-CT) has been employed to evaluate novel therapeutics in TB infection of cynomolgus macaques (Lin et al., 2013). Imaging techniques can be important in detecting early disease. By MRI of lungs post mortem, lesions were detected as early as 2 wpc following aerosol infection with a high-dose of Mtb H37Rv in rhesus macaques (Rayner et al., 2013). CT scanning may be more helpful in detecting lesions in vivo as early as 24 days as a previous study has described lesions from one to three months post-challenge [Lewinsohn et al., (2006)]. The aims of the present study were to (1) define the lesions present at 24 and 25 days post challenge (dpc), following aerosol infection with the Erdman strain of Mtb, (2) compare these lesions with early lesions produced by aerosol challenge with Mtb H37Rv strain (Rayner et al., 2013) and (3) compare the sensitivity of CT scanning in vivo with MRI post mortem at this early time point.
Materials and Methods Experimental Animals
One male and two female rhesus macaques of Indian origin, approximately 10 years old, were obtained from an established UK breeding colony. They were na€ıve in terms of prior exposure to mycobacterial antigens (Mtb infection or environmental mycobacteria) as demonstrated by a negative tuberculin test while in their original breeding colony. The Project Licence enabling these studies was approved by the Ethical Review Process of Public Health England (PHE), Porton, Salisbury, UK, and the Home Office, UK. Animals were housed according to the Home Of-
fice (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989), the National Committee for Refinement, Reduction and Replacement (NC3Rs) and the Guidelines on Primate Accommodation, Care and Use, August 2006 (NC3Rs, 2006). For procedures requiring removal from their housing, animals were sedated by intramuscular (IM) injection with ketamine hydrochloride (Ketaset, 100 mg/ml, Zoetis UK Ltd, Southampton, UK; 10 mg/kg). Challenge Procedures
Each animal was challenged by aerosol inhalation with a presented dose estimated of 2,333 (high), 583 (mid) or 226 (low) colony forming units (cfus) of Mtb (Erdman strain), providing estimated retained doses of 111, 28 and 11 cfu, as described previously (Sharpe et al., 2009). The Mtb Erdman strain K 01 challenge stock was kindly provided by Dr A. Yang (Center for Biologics Evaluation and Research, US Food and Drug Administration), generated as described previously (Sharpe et al., 2009). The animals were killed humanely at 24 dpc (low dose) and 25 dpc (mid and high dose) (Table 1). Clinical Procedures
Animals were sedated, weighed and examined prior to challenge and at 14 and 21 dpc. In addition, blood samples were collected prior to challenge, at 14 dpc and at the time of death. Animals were monitored daily for behavioural changes and clinical signs of disease. For CT imaging, animals were anaesthetised with an IM injection of ketamine hydrochloride (Ketaset, 100 mg/ml, Zoetis UK Ltd, UK; 12 mg/ kg), medetomidine hydrochloride (Sedator, 1 mg/ ml, Dechra Veterinary Products, Shrewsbury, UK; 50 mg/kg) and atropine sulphate (Atrocare, 600 mg/ ml, Animalcare Ltd, York, UK, 15 mg/kg). Haematology. Haemoglobin concentrations were measured using a HaemaCue haemoglobinometer (Haemacue Ltd, Dronfield, UK) and erythrocyte
Table 1 Summary of imaging and gross lesions counts Estimated retained CT scan total T1-weighted Total gross lesion dose (colony lesion count MRI lesion count count (from sectioned forming units) lung lobes) 98 (high) 28 (mid) 11 (low)
138 47 20
334 44 24
717 210 45
219
Mycobacteriosis in Rhesus Macaques
sedimentation rate, (ESR) was determined using the Sediplast system (Guest Medical, Edenbridge, UK). Computed Tomography. A 16-slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA) was used. Each animal was transported to the scanner in a containment pod. All axial scans were performed at 120 KVp, with Auto mA (ranging between 10 and 120) and were acquired using a small scan field of view. Rotation speed was 0.8 sec. Images were displayed as an 11 cm field of view. Niopam 300 (Bracco, Milan, Italy), a non-ionic, iodinated contrast medium, was administered intravenously (IV) at 2 ml/kg body weight and scan sequences were repeated at 90 sec after half of the total volume had been injected. The data were interpreted with the observer blinded to the treatment group. Post-mortem Procedures Necropsy Examination. Animals were sedated and a dorsoventral chest radiograph was taken. The level of anaesthesia was deepened prior to exsanguination by cardiac puncture, followed by termination via IV pentobarbitone sodium overdose. A post-mortem examination was performed immediately. The whole lung, with heart and tracheobronchial (hilar) lymph nodes attached, was removed intact and fixed by intratracheal infusion with 10% neutral buffered formalin (NBF) using a syringe and 13CH Nelaton catheter (JAK Marketing, York, UK). The catheter tip was inserted into each bronchus in turn via the trachea; the lungs were infused until they were expanded to a size considered to be normal inspiratory dimensions and the trachea was ligated to retain the fluid. The infused lung was immersed in 10% NBF. Samples of kidneys, liver, spleen, lymph nodes (axillary, submandibular and mesenteric), tonsil and gastrointestinal tract (stomach, jejunum and transverse colon) were fixed in 10% NBF.
Pathological Studies Gross Examination following Fixation and Magnetic Resonance Imaging. The fixed lungs were removed from the agar, sliced serially and lesions were counted as described previously (Rayner et al., 2013). Briefly, lobes were sliced at 5e10 mm intervals and creamewhite foci, considered to be tuberculous lesions, were identified and counted. The total lesion count was calculated by counting discrete foci measuring 1e5 mm in diameter and coalesced areas, often measuring >5 mm in diameter. Histopathological Examination. Representative samples from each lung lobe and other organs were processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Selected tissues were stained additionally by the ZiehleNeelsen (ZN) method. Lesions in the lung parenchyma were identified, categorized and counted as described previously (Rayner et al., 2013). Briefly, lesions defined as ‘unorganized’ (types 1e3) were those lacking a peripheral cuff of lymphocytes, while ‘organized’ lesions were those with a cuff of lymphocytes (types 4 and 5). Type 1 lesions were typically small, diffuse foci of macrophages and lymphocytes with scattered neutrophils and eosinophils, which lacked clearly defined boundaries. Type 2 unorganized lesions were composed of similar inflammatory cells and were generally larger, circumscribed foci forming a more defined, frequently circular granuloma with variably demarcated borders. Focal necrosis was present in some of these latter granulomas, characterized by nuclear pyknosis and karyorrhexis with loss of cellular architecture, and these were designated type 3 lesions. In one HE-stained section from each lung lobe, granulomas types 1 to 3, as described above, were counted and recorded (Table 2).
Results Magnetic Resonance Imaging. The expanded, fixed lungs were set in 2% agarose (SigmaeAldrich, UK) in water at 40 C, sealed in polythene bags, placed in an eightchannel phased array head coil and MRI was performed with a 3.0 T 750 MRI Scanner (General Electric Healthcare). The parameters of the T1 weighted Fast Spin Echo (FSE) images were: TR: 300, TE: 12, 24 cm field of view, 1.5 mm slice thickness, 0 mm slice gap, 512 224 matrix, 1 NEX. Each acquisition took approximately 3 min. Lung lesions were identified in the images from their signal intensity and nodular morphology relative to normal lung parenchyma. The data were interpreted with the observer blinded to the treatment group.
Clinical Findings
Changes in clinical parameters are shown in Fig. 1. All animals had decreased appetites from 1 to 2 dpc, which returned to normal by 6 dpc. Nevertheless, by the end of the study period, a 10% reduction in body weight loss was noted in the high- and lowdose animals and a 2% reduction in the mid-dose animal. Rectal body temperature increased progressively in all animals and moderate pyrexia was present prior to humane destruction. A progressive decrease in haemoglobin levels was noted in all animals. Erythrocyte sedimentation rate values increased mildly in all three animals by the end of
E.L. Rayner et al.
220
Table 2 Summary of microscopical changes in lung and tracheobronchial lymph nodes Retained aerosol dose (colony forming units)
Lung Parenchymal granulomas
Lymphatic granulomatous lesions
HE lesion types ZN 98 (high) 28 (mid) 11 (low)
1, 2, 3 1, 2, 3 1, 3
Tracheobronchial lymph nodes granulomatous lesions
+ + +
BALT/perivascular lymphoid tissue granulomatous lesions
HE
ZN
HE
ZN
HE
ZN
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+, present; BALT, bronchus-associated lymphoid tissue; lesion type 1, unorganized diffuse; lesion type 2, unorganized circumscribed; lesion type 3, unorganized circumscribed with focal necrosis; HE, haematoxylin and eosin; ZN, ZiehleNeelsen stain.
the study period. Clinical signs related to respiratory disease were not observed in any animal. Computed Tomography
12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
High Mid Low
High Mid Low
42
o
Rectal temperature ( C)
Body weight (kg)
Total lesion counts are shown in Table 1. At 21 dpc, nodules (Figs. 2AeC) were detected in all
lung lobes in all animals without evidence of calcification or cavitation. Total nodular counts were proportional to the challenge dose. In addition, the subcarinal (inferior tracheobronchial) lymph nodes appeared enlarged in the mid-dose animal. In the high- and low-dose animals, lymph nodes draining the lung appeared normal. Evidence of
40 38 36 34
-1
0
1
2
3
4
-1
Time post challenge (weeks)
Haemoglobin (g/l)
130 120 110 100 90 80 70 0
2
3
3.5
Time post challenge (weeks)
Erythocyte sedimentation rate (mm/h)
High Mid Low
140
1
2
3
4
Time post challenge (weeks)
160 150
0
30 High Mid Low
25 20 15 10 5 0 -1
0
1
2
3
4
Time post challenge (weeks)
Fig. 1. Clinical parameters indicating changes in body weight, rectal temperature, haemoglobin values and erythrocyte sedimentation rate.
Mycobacteriosis in Rhesus Macaques
221
Fig. 2. Computed tomography (A, B and C), magnetic resonance imaging (D, E and F) and gross images of formalin-fixed lungs (G, H and I) from high- (A, D, G), mid- (B, E and H) and low-dose (C, F and I) animals.
changes consistent with Mtb infection was not detected in other organs. Magnetic Resonance Imaging
Total T1-weighted MRI lesion counts are shown in Table 1. In the lungs of the low-dose animal at 24 dpc and the mid- and high-dose animals at 25 dpc, lesions were observed; these were predominantly discrete, high signal intensity nodules, approximately 1e3 mm in diameter, identified on the high resolution, T1-weighted images (Figs. 2DeF). Total nodule counts were proportional to the challenge dose. Pathology Gross Findings. Lesions were present in the lungs (Figs. 2GeI) and tracheobronchial lymph nodes (Fig. 3) of all animals and in the liver of the middose animal; the number of gross lesions counted in the lungs is shown in Table 1. Firm, cream-coloured
nodules, ranging from 2 to 5 mm in diameter, some of which had coalesced, were observed frequently in all lung lobes of the high-dose animal. Lesions of similar appearance and dimensions, but fewer in number, were noted in the mid- and low-dose animals, correlating with the lower challenge dose. Tracheobronchial lymph nodes from all three animals were moderately enlarged, with fibrous adhesions to surrounding soft tissue structures in all animals. The cortical and medullary parenchyma contained irregular, cream-coloured foci (Fig. 3), which were larger and more frequent in the highand mid-dose animals than in the low-dose animal. Scattered, small, dark greyeblack foci were also present; these were interpreted as anthracosis. In the liver of the mid-dose animal, a single, cream-coloured lesion,
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
EXPERIMENTALLY INDUCED DISEASE
Early Lesions following Aerosol Challenge of Rhesus Macaques (Macaca mulatta) with Mycobacterium tuberculosis (Erdman Strain) E. L. Rayner*, G. R. Pearson*, G. A. Hall*, F. Gleeson†, A. McIntyre†, D. Smyth*, M. J. Dennis* and S. A. Sharpe* * Public Health England, Microbiology Services Division, Porton Down, Salisbury and † Churchill Hospital, Headington, Oxford, UK
Summary Three rhesus macaques (Macaca mulatta) were challenged with Mycobacterium tuberculosis (Mtb), Erdman strain, as part of studies to investigate lesion development at early time points in tuberculosis (TB) and to assess computed tomography (CT) as a method of monitoring disease progression in vivo. Animals were challenged with either a high, mid or low dose of aerosolized Mtb. The low-dose animal was killed humanely at 24 days post challenge (dpc) and the remaining animals at 25 dpc. Abnormalities in clinical parameters were observed in all animals, but clinical signs relating to respiratory disease were not seen. Pulmonary changes consistent with TB infection were detected by CT at 21 dpc and magnetic resonance imaging (MRI) post mortem. Pulmonary nodule counts obtained from both imaging techniques were directly proportional to the challenge dose and correlated with gross and microscopical lesion counts. On gross and microscopical examination, lesions of similar size and morphology were observed in the lungs of all three animals, with the majority containing necrotic foci. Concomitant gross and microscopical, granulomatous lesions were observed in the tracheobronchial lymph nodes of all animals together with evidence of systemic spread. These findings further contribute to our understanding and knowledge of early lesion formation in the lungs of non-human primates. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: early granulomas; lung; macaque; tuberculosis
Introduction Tuberculosis (TB) remains a major threat to public health (World Health Organisation, 2014) and knowledge of the pathogenesis of the disease at early time points is limited. The use of animal models has contributed greatly to the understanding of hostepathogen interactions in this disease and nonhuman primates most closely resemble human beings with respect to immunological and genetic processes (Qiu et al., 2008), as well as to disease progression (Langermans et al., 2001). There are few reports of lesion formation early in infection in non-human primates. Recently, descriptions of gross and microscopical lesions in the lung Correspondence to: E. L. Rayner (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.10.002
and tracheobronchial (hilar) lymph nodes were reported in rhesus macaques (Macaca mulatta) exposed to an aerosol of Mycobacterium tuberculosis (Mtb) H37Rv strain, at varying doses and examined at 2e3 weeks post challenge (wpc) (Rayner et al., 2013). Early involvement of intrapulmonary lymphatics was reported in animals receiving high and mid doses of Mtb and concomitant lesions were present in the tracheobronchial lymph nodes in all animals in the high-dose groups at 2 and 3 wpc, all animals in the mid-dose group at 3 wpc and two out of three animals in the low-dose group at 3 wpc. Lin et al. (2006) reported lesion development in cynomolgus macaques (Macaca fasicularis) challenged with Mtb Erdman strain via the intratracheal route. Lung lesions were not observed until 4 wpc (microscopically) or 5 wpc (grossly). These lesions comprised Ó 2014 Elsevier Ltd. All rights reserved.
E.L. Rayner et al.
218
of primarily caseous granulomas, which was in contrast to the range of granulomatous lesions described for early infection of Mtb H37Rv in rhesus macaques (Rayner et al., 2013). In addition, lesions in the tracheobronchial lymph nodes were not observed until 4 wpc (Lin et al., 2006). Early lesion formation with the Erdman strain of Mtb, via the aerosol route, has not been described in rhesus macaques. Increasingly, imaging techniques are being employed to detect and monitor disease progression in the lungs of non-human primates both post mortem and, more recently, in vivo. Magnetic resonance imaging (MRI) has been used to determine lesion volume post mortem in agar-embedded, formalin-infused lungs in rhesus macaques challenged with Mtb Erdman by aerosol (Sharpe et al., 2009, 2010). Furthermore, in-vivo imaging of TB disease progression over a 3-month period using computed tomography (CT) with H37Rv (Lewinsohn et al., 2006) was reported. Recently, positron emission tomography with CT (PET-CT) has been employed to evaluate novel therapeutics in TB infection of cynomolgus macaques (Lin et al., 2013). Imaging techniques can be important in detecting early disease. By MRI of lungs post mortem, lesions were detected as early as 2 wpc following aerosol infection with a high-dose of Mtb H37Rv in rhesus macaques (Rayner et al., 2013). CT scanning may be more helpful in detecting lesions in vivo as early as 24 days as a previous study has described lesions from one to three months post-challenge [Lewinsohn et al., (2006)]. The aims of the present study were to (1) define the lesions present at 24 and 25 days post challenge (dpc), following aerosol infection with the Erdman strain of Mtb, (2) compare these lesions with early lesions produced by aerosol challenge with Mtb H37Rv strain (Rayner et al., 2013) and (3) compare the sensitivity of CT scanning in vivo with MRI post mortem at this early time point.
Materials and Methods Experimental Animals
One male and two female rhesus macaques of Indian origin, approximately 10 years old, were obtained from an established UK breeding colony. They were na€ıve in terms of prior exposure to mycobacterial antigens (Mtb infection or environmental mycobacteria) as demonstrated by a negative tuberculin test while in their original breeding colony. The Project Licence enabling these studies was approved by the Ethical Review Process of Public Health England (PHE), Porton, Salisbury, UK, and the Home Office, UK. Animals were housed according to the Home Of-
fice (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989), the National Committee for Refinement, Reduction and Replacement (NC3Rs) and the Guidelines on Primate Accommodation, Care and Use, August 2006 (NC3Rs, 2006). For procedures requiring removal from their housing, animals were sedated by intramuscular (IM) injection with ketamine hydrochloride (Ketaset, 100 mg/ml, Zoetis UK Ltd, Southampton, UK; 10 mg/kg). Challenge Procedures
Each animal was challenged by aerosol inhalation with a presented dose estimated of 2,333 (high), 583 (mid) or 226 (low) colony forming units (cfus) of Mtb (Erdman strain), providing estimated retained doses of 111, 28 and 11 cfu, as described previously (Sharpe et al., 2009). The Mtb Erdman strain K 01 challenge stock was kindly provided by Dr A. Yang (Center for Biologics Evaluation and Research, US Food and Drug Administration), generated as described previously (Sharpe et al., 2009). The animals were killed humanely at 24 dpc (low dose) and 25 dpc (mid and high dose) (Table 1). Clinical Procedures
Animals were sedated, weighed and examined prior to challenge and at 14 and 21 dpc. In addition, blood samples were collected prior to challenge, at 14 dpc and at the time of death. Animals were monitored daily for behavioural changes and clinical signs of disease. For CT imaging, animals were anaesthetised with an IM injection of ketamine hydrochloride (Ketaset, 100 mg/ml, Zoetis UK Ltd, UK; 12 mg/ kg), medetomidine hydrochloride (Sedator, 1 mg/ ml, Dechra Veterinary Products, Shrewsbury, UK; 50 mg/kg) and atropine sulphate (Atrocare, 600 mg/ ml, Animalcare Ltd, York, UK, 15 mg/kg). Haematology. Haemoglobin concentrations were measured using a HaemaCue haemoglobinometer (Haemacue Ltd, Dronfield, UK) and erythrocyte
Table 1 Summary of imaging and gross lesions counts Estimated retained CT scan total T1-weighted Total gross lesion dose (colony lesion count MRI lesion count count (from sectioned forming units) lung lobes) 98 (high) 28 (mid) 11 (low)
138 47 20
334 44 24
717 210 45
219
Mycobacteriosis in Rhesus Macaques
sedimentation rate, (ESR) was determined using the Sediplast system (Guest Medical, Edenbridge, UK). Computed Tomography. A 16-slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA) was used. Each animal was transported to the scanner in a containment pod. All axial scans were performed at 120 KVp, with Auto mA (ranging between 10 and 120) and were acquired using a small scan field of view. Rotation speed was 0.8 sec. Images were displayed as an 11 cm field of view. Niopam 300 (Bracco, Milan, Italy), a non-ionic, iodinated contrast medium, was administered intravenously (IV) at 2 ml/kg body weight and scan sequences were repeated at 90 sec after half of the total volume had been injected. The data were interpreted with the observer blinded to the treatment group. Post-mortem Procedures Necropsy Examination. Animals were sedated and a dorsoventral chest radiograph was taken. The level of anaesthesia was deepened prior to exsanguination by cardiac puncture, followed by termination via IV pentobarbitone sodium overdose. A post-mortem examination was performed immediately. The whole lung, with heart and tracheobronchial (hilar) lymph nodes attached, was removed intact and fixed by intratracheal infusion with 10% neutral buffered formalin (NBF) using a syringe and 13CH Nelaton catheter (JAK Marketing, York, UK). The catheter tip was inserted into each bronchus in turn via the trachea; the lungs were infused until they were expanded to a size considered to be normal inspiratory dimensions and the trachea was ligated to retain the fluid. The infused lung was immersed in 10% NBF. Samples of kidneys, liver, spleen, lymph nodes (axillary, submandibular and mesenteric), tonsil and gastrointestinal tract (stomach, jejunum and transverse colon) were fixed in 10% NBF.
Pathological Studies Gross Examination following Fixation and Magnetic Resonance Imaging. The fixed lungs were removed from the agar, sliced serially and lesions were counted as described previously (Rayner et al., 2013). Briefly, lobes were sliced at 5e10 mm intervals and creamewhite foci, considered to be tuberculous lesions, were identified and counted. The total lesion count was calculated by counting discrete foci measuring 1e5 mm in diameter and coalesced areas, often measuring >5 mm in diameter. Histopathological Examination. Representative samples from each lung lobe and other organs were processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Selected tissues were stained additionally by the ZiehleNeelsen (ZN) method. Lesions in the lung parenchyma were identified, categorized and counted as described previously (Rayner et al., 2013). Briefly, lesions defined as ‘unorganized’ (types 1e3) were those lacking a peripheral cuff of lymphocytes, while ‘organized’ lesions were those with a cuff of lymphocytes (types 4 and 5). Type 1 lesions were typically small, diffuse foci of macrophages and lymphocytes with scattered neutrophils and eosinophils, which lacked clearly defined boundaries. Type 2 unorganized lesions were composed of similar inflammatory cells and were generally larger, circumscribed foci forming a more defined, frequently circular granuloma with variably demarcated borders. Focal necrosis was present in some of these latter granulomas, characterized by nuclear pyknosis and karyorrhexis with loss of cellular architecture, and these were designated type 3 lesions. In one HE-stained section from each lung lobe, granulomas types 1 to 3, as described above, were counted and recorded (Table 2).
Results Magnetic Resonance Imaging. The expanded, fixed lungs were set in 2% agarose (SigmaeAldrich, UK) in water at 40 C, sealed in polythene bags, placed in an eightchannel phased array head coil and MRI was performed with a 3.0 T 750 MRI Scanner (General Electric Healthcare). The parameters of the T1 weighted Fast Spin Echo (FSE) images were: TR: 300, TE: 12, 24 cm field of view, 1.5 mm slice thickness, 0 mm slice gap, 512 224 matrix, 1 NEX. Each acquisition took approximately 3 min. Lung lesions were identified in the images from their signal intensity and nodular morphology relative to normal lung parenchyma. The data were interpreted with the observer blinded to the treatment group.
Clinical Findings
Changes in clinical parameters are shown in Fig. 1. All animals had decreased appetites from 1 to 2 dpc, which returned to normal by 6 dpc. Nevertheless, by the end of the study period, a 10% reduction in body weight loss was noted in the high- and lowdose animals and a 2% reduction in the mid-dose animal. Rectal body temperature increased progressively in all animals and moderate pyrexia was present prior to humane destruction. A progressive decrease in haemoglobin levels was noted in all animals. Erythrocyte sedimentation rate values increased mildly in all three animals by the end of
E.L. Rayner et al.
220
Table 2 Summary of microscopical changes in lung and tracheobronchial lymph nodes Retained aerosol dose (colony forming units)
Lung Parenchymal granulomas
Lymphatic granulomatous lesions
HE lesion types ZN 98 (high) 28 (mid) 11 (low)
1, 2, 3 1, 2, 3 1, 3
Tracheobronchial lymph nodes granulomatous lesions
+ + +
BALT/perivascular lymphoid tissue granulomatous lesions
HE
ZN
HE
ZN
HE
ZN
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+, present; BALT, bronchus-associated lymphoid tissue; lesion type 1, unorganized diffuse; lesion type 2, unorganized circumscribed; lesion type 3, unorganized circumscribed with focal necrosis; HE, haematoxylin and eosin; ZN, ZiehleNeelsen stain.
the study period. Clinical signs related to respiratory disease were not observed in any animal. Computed Tomography
12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
High Mid Low
High Mid Low
42
o
Rectal temperature ( C)
Body weight (kg)
Total lesion counts are shown in Table 1. At 21 dpc, nodules (Figs. 2AeC) were detected in all
lung lobes in all animals without evidence of calcification or cavitation. Total nodular counts were proportional to the challenge dose. In addition, the subcarinal (inferior tracheobronchial) lymph nodes appeared enlarged in the mid-dose animal. In the high- and low-dose animals, lymph nodes draining the lung appeared normal. Evidence of
40 38 36 34
-1
0
1
2
3
4
-1
Time post challenge (weeks)
Haemoglobin (g/l)
130 120 110 100 90 80 70 0
2
3
3.5
Time post challenge (weeks)
Erythocyte sedimentation rate (mm/h)
High Mid Low
140
1
2
3
4
Time post challenge (weeks)
160 150
0
30 High Mid Low
25 20 15 10 5 0 -1
0
1
2
3
4
Time post challenge (weeks)
Fig. 1. Clinical parameters indicating changes in body weight, rectal temperature, haemoglobin values and erythrocyte sedimentation rate.
Mycobacteriosis in Rhesus Macaques
221
Fig. 2. Computed tomography (A, B and C), magnetic resonance imaging (D, E and F) and gross images of formalin-fixed lungs (G, H and I) from high- (A, D, G), mid- (B, E and H) and low-dose (C, F and I) animals.
changes consistent with Mtb infection was not detected in other organs. Magnetic Resonance Imaging
Total T1-weighted MRI lesion counts are shown in Table 1. In the lungs of the low-dose animal at 24 dpc and the mid- and high-dose animals at 25 dpc, lesions were observed; these were predominantly discrete, high signal intensity nodules, approximately 1e3 mm in diameter, identified on the high resolution, T1-weighted images (Figs. 2DeF). Total nodule counts were proportional to the challenge dose. Pathology Gross Findings. Lesions were present in the lungs (Figs. 2GeI) and tracheobronchial lymph nodes (Fig. 3) of all animals and in the liver of the middose animal; the number of gross lesions counted in the lungs is shown in Table 1. Firm, cream-coloured
nodules, ranging from 2 to 5 mm in diameter, some of which had coalesced, were observed frequently in all lung lobes of the high-dose animal. Lesions of similar appearance and dimensions, but fewer in number, were noted in the mid- and low-dose animals, correlating with the lower challenge dose. Tracheobronchial lymph nodes from all three animals were moderately enlarged, with fibrous adhesions to surrounding soft tissue structures in all animals. The cortical and medullary parenchyma contained irregular, cream-coloured foci (Fig. 3), which were larger and more frequent in the highand mid-dose animals than in the low-dose animal. Scattered, small, dark greyeblack foci were also present; these were interpreted as anthracosis. In the liver of the mid-dose animal, a single, cream-coloured lesion,
