Early lesion formation in cattle experimentally infected with Mycobacterium bovis

J. Comp. Path. 1998 Vol. 119, 27-44

Early Lesion Formation in Cattle Experimentally Infected with Mycobacterium hovis J. P. Cassidy, D. G. Bryson, J. M. Pollock, R. T. Evans, F. Forster and S. D. Neill Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Stoney Road, Stormont, Belfast BT4 3SD, UK

Summary Early lesion formation was examined in 13 calves inoculated intranasally with 2x10^ colony-forming units oiMycobacterium bovis and killed either singly or in pairs at intervals of < 7 days from post-inoculation day (pid) 3 to pid 42. Immunological examinations were carried out before and after infection, and sequential necropsies were performed. M. bovis was recovered as early as pid 3, from the upper respiratory tract mucosae, retropharyngeal lymph nodes and caudal lung lobe. Gross tuberculous lesions were detected in both the upper respiratory tract mucosae and in the lungs of the calves killed from pid 14 onwards. Lesions were also present in the lymph nodes draining these areas. On histological examination, neutrophils appeared to play a key role in the earliest stages of lesion formation, and lesion mineralization was observed for the first time at pid 35. The contemporaneous development of lesions and cellular immunity, as demonstrated by in-vitro lymphocyte proliferation and interferon-y assay responses, provided further evidence of the role of immunopathogenic mechanisms in the development of bovine tuberculosis.

© 1998 W.B. Saunders Company Limited

Introduction Bovine tuberculosis remains a significant disease in a number of countries (de Kantor and Ritacco, 1994; Dabourn, 1995; Neill, 1995). In regions such as N. Ireland, frequent intradermal tuberculin testing results in the detection of most infected cattle in the early stages of the disease. However, there is little published information on the early pathogenesis of bovine tuberculosis (Neill et al, 1994a). Much of the current understanding of the development of bovine tuberculosis is derived from studies of the disease in man and experiments in small laboratory animals (Rich, 1951; Brown, 1983; Dannenberg, 1994; McMurray, 1994; Orme and Collins, 1994). Since the pathogenetic mechanisms in tuberculosis may vary significantly from species to species (Pritchard, 1988; Dannenberg, 1994; McMurray, 1994; O r m e and Collins, 1994), direct extrapolation of the results of such studies to cattle may not always be valid. The objectives of this study were to investigate the sequential development of early lesions in 13 calves inoculated intranasally with Mycobacterium bovis, and to relate lesion development to the results of immunodiagnostic tests. 0021-9975/98/050027 + 18 112.00/0

© 1998 W.B. Saunders Company Limited

28

J. P. Cassidy et al.

P r e v i o u s studies (Neill et al., 1988) i n d i c a t e d t h a t i n o c u l a of 10^ or IC^ colonyf o r m i n g units (cfu) of Af. bovis w e r e necessary to i n d u c e lesions. In the p r e s e n t study, to e n s u r e the consistent r e p r o d u c t i o n of lesions a dose of 2 x 1 0 ' c f u was c h o s e n , e v e n t h o u g h this r e p r e s e n t e d a g r e a t e r c h a l l e n g e t h a n w o u l d b e expected under natural conditions.

Materials and Methods Experimental Calves Fourteen Friesian-crossbred castrated male calves were purchased from a commercial farm with no previous history of bovine tuberculosis. Thirteen of the calves (nos 1-13) were infected and a single control calf (no. 14) was housed separately. At the time of experimental inoculation the animals were 7-8 months old. Pre-inoculation Procedure Blood samples from all calves were taken for immunological examination 14 and 7 days before infection, and immediately before inoculation. Inoculum Calves 1-13 were each inoculated intranasally with 2 x 10'cfu (10'cfu on each of two consecutive days) of a field isolate of M. bovis. T h e inoculum was prepared as previously described (Neill et al., 1988) and delivered (10 ml per nostril) via a syringe through a 12-cm length of fine-bore rubber tubing inserted into the nostril, with the animal's head tilted upwards at an angle of approximately 45° to the ground. T h e head was then held in this position for 10 sec before releasing. The control calf received sterile broth.

Post-inoculation Procedure Blood sampling of the calves was carried out at weekly intervals. The animals were killed sequentially and subjected to post-mortem examination at the timepoints detailed in Table 1. The term "post-inoculation day" (pid) refers to days after administration of the second infective dose. Post-mortem Examination After euthanasia by intravenous injection of pentobarbitone sodium, the lungs, trachea and larynx were removed and carefully examined for gross lesions. Each lung lobe was then serially sectioned transversely at approximately 5-mm intervals with a 200m m Macro preparation knife; the lung slices were then examined for lesions. T h e head was sectioned longitudinally along its midline, and the nares, nasal chambers, pharynx and tonsils were examined closely. All lymph nodes were inspected. The retropharyngeal, parotid, submandibular, cervical, bronchial, mediastinal and mesenteric lymph nodes were dissected free and serially sliced at 2-mm intervals, to facilitate thorough examination. T h e following tissues were taken for histopathological and bacteriological examination: nasal mucosa, turbinates and septum, nasopharynx, palatine and pharyngeal tonsils, larynx, trachea, thymus, sections from the cranial, middle and caudal lung lobes, lymph nodes (parotid, submandibular, retropharyngeal, cervical, bronchial, mediastinal and mesenteric), spleen, liver, kidney and small intestine.

Early Lesions in Bovine

29

Tuberculosis

Table 1 D i s t r i b u t i o n o f m a c r o s c o p i c t u b e r c u l o u s l e s i o n s in t h e r e s p i r a t o r y t r a c t Gross tuberculous lesions in Calf no. 1 2 3 4 5 6 7 8 9 10 11 12 13

Killed on pid

URT mucosa

palatine tonsil

URTLN

trachea

stated lung lobe Cranial Middle

3 7 7 11 14 14 17 21 21 24 28 35 42

-

-

-

-

-

-

-

+ + + + + + +

+ + + +

+ + + + + + +

+ + + + + +

-

Caudal

bronchomediastinal LN

-

-

-

-

-

+ ~ + + +

+ +

-

+

+ — +* +

+ + + + + + +

+ = Present; — = absent, pid = Post-inoculation day. U R T mucosa = upper respiratory tract mucosa {includes: nasal mucosa, turbinates, septum and nasopharynx). U R r - L N = upper respiratory tract lymph nodes (retropharyngeal, parotid and submandibular). LN = lymph node. * Single focal lesion present, < 10 m m in diameter.

Histopathological Examination Tissue samples were fixed in 10% neutral buffered formalin and processed by standard paraffin wax technique. Sections were cut and stained with haematoxylin and eosin (HE) and also by the Ziehl-Neelsen (ZN) method. They were then examined for the presence of tuberculous lesions and acid-fast bacteria. Immunological Examination The in-vitro production of interferon-y (IFN-y) and the lymphocyte transformation assay were used as measures of cell-mediated immune responses. For IFN-y responses, aliquots of heparinized blood were stimulated with bovine and avian purified protein derivative (PPD) (Central Veterinary Laboratory, Weybridge) and maintained for 24 h at 37°C, as described previously (Neill et at, 1992). IFN-y concentrations in supernates from stimulated and control cultures were measured by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Commonwealth Serum Laboratories, Australia). Optical density values were determined at 450 nm with an ELISA reader and 630-nm reference filter. Values above OTOO were considered significant. The lymphocyte proliferation assay was carried out as described by Pollock et al. (1994). In summary, peripheral blood mononuclear cells (PBMC) were harvested from heparinized blood samples by density centrifugation over FicoU-Paque (Pharmacia, Uppsala, Sweden), washed by centrifugation and resuspended in phosphate-buffered saline. The PBMC were then placed in 200-|a,l cultures at a concentration of 10^ cells/ ml in RPMI-1640 medium containing fetal calf serum 5%. Bovine and avian PPD antigens were added to triplicate wells at optimal concentrations. Triplicate wells remained unstimulated as controls. The cultures were incubated for 5 days at 37°C in 5% C O j . For the final 16 h, each culture was pulsed with 1 |aCi tritiated thymidine (Amersham Inc., Amersham, UK). Incorporated radioactivity as a measure of cellular

30

J. P. Cassidy et al.

proliferation was determined as counts per minute (cpm) by scintillation counting. Results were expressed as stimulation indices (Sis), which were the ratio (mean cpm) of stimulated wells to control wells. A response was considered positive when the diflFerence between the bovine and avian SI was > 2 ' 0 . Sera were tested for the presence of mycobacterial antibodies by ELISA. Microtitre plates were coated with bovine PPD, and the binding of IgG antibodies from the bovine sera was detected with an enzyme-conjugated polyclonal antiserum and colorimetric detection system (Hanna etal., 1989). Positive and negative serum controls and reagent blanks were included in each test plate. Each sample was tested in triplicate and the results were expressed as a mean value. T h e ELISA value was calculated with the following formula: ^ , .,„ . , (Mean reading of sample —mean reading of blank) .„^ ELISA value = ^ ,. ^ ^ r*7 7^ T >^ 100 (Mean readmg of positive — mean reading of blank) Values greater than 54% were considered significant. T h e proportions of the major T-cell subpopulations in peripheral blood were determined by flow cytometry. C D 4 T cells, CD8 T cells, WC1 T cells (the dominant subpopulation of circulating 78 T cells) and cells expressing immunoglobulin on their membrane (B cells) were labelled by monoclonal antibodies C C 8 , CC63, CC15 and IL-A59, as described previously (Pollock et at, 1996). T h e percentages of the individual populations were determined by flow cytometric analysis (FAGS Vantage, Becton Dickinson). Routine haematology, incorporating total and differential white cell counting, was also performed on parallel blood samples to allow subsequent calculation of the absolute numbers of T-cell subpopulations in the blood. T h e statistical significance of any changes in the various lymphocyte subpopulations with time was tested by applying the Wilcoxon Signed-Ranks test. Bacteriological

Examination

Tissue samples were prepared for culture as described previously (Mcllroy et al., 1986). T h e resulting deposit was inoculated into a BAGTEC 460 culture system (Becton Dickinson) and on to four slants of Lowenstein-Jensen medium, three with pyruvate and one with glycerol. The BACTEG 460 system is a semi-automatic radiometric detection system that incorporates a broth culture medium radioactively labelled with carbon 14. T h e inoculated medium is incubated at 37°G and read periodically for up to 84 days. T h e inoculated Lowenstein-Jensen slants were incubated at 37°G and examined after 28 days.

Results Gross Post-mortem

Findings

N o lesions w e r e d e t e c t e d in calves 1 or 2, killed o n pids 3 a n d 7. A few small p l u m - c o l o u r e d a r e a s of c o n s o l i d a t i o n w e r e o b s e r v e d in the c r a n i o v e n t r a l r e g i o n of t h e l u n g in calves 3, 4 a n d 5 (pids 7, 11 a n d 14). T h e s e w e r e s u b s e q u e n t l y s h o w n to b e areas of c h r o n i c l y m p h o c y t i c ("cuffing") p n e u m o n i a (Pirie a n d A l l a n , 1975). Lesions confirmed as t u b e r c u l o u s o n s u b s e q u e n t h i s t o p a t h o l o g i c a l e x a m i n a t i o n w e r e o b s e r v e d in the r e s p i r a t o r y tracts of calves 6 to 13 (pids 1 4 - 4 2 ) ; t h e i r distribution is s u m m a r i z e d in T a b l e 2. T o facilitate t h e p r e s e n t a t i o n of results, the t e r m " u p p e r r e s p i r a t o r y t r a c t ( U R T ) " has b e e n used to g r o u p t o g e t h e r the n a s a l m u c o s a , t u r b i n a t e s , s e p t u m a n d n a s o p h a r y n x . U R T lesions consisted of m u l t i p l e raised pale-yellow p l a q u e s , 1-3 m m in

Early L e s i o n s in Bovine Tuberculosis

31

Table 2 Distribution of microscopically observed lesions or acid-fast bacteria, or both, in the respiratory tract Microscopical lesions or bacteria^or both, in Calf

1 2 3 4 5 6 7 8 9 10 11 12 13

Killed on pid

3 7 7 11 14 14 17 21 21 24 28 35 42

URT rriucosa

palatine tomil

+ + + — + + + + + + +

URTLN

Cranial

Middle

Caudal

bronchomediastinal LN

stated lung lobe

trachea

-

-

-

-

-

~

-

_ — — — + + + +

_ + + + + + + + + + +

_ — — + + + + + + +

— — + — + + +

— +* + +t + +

+ — + +t -

+ + + + + + + +

+§

+

* Single acid-last organism observed. N o associated inflammation. I Single local lesion present, < 1 m m in diameter. ;|; Lesions restricted to a few lobules. § Single focal lesion present, < 1 0 m m in diameter. For abbreviations, see T a b l e 1.

diameter, on the mucosae of calves 7 to 13 (Fig. 1). Lung lesions in calves 9, II and 13 consisted of areas of plum-coloured consolidation of variable extent, with a cranioventral distribution (Figs 2 and 3). Within all these areas, multiple fawn-coloured nodules, 1-4 mm in diameter, with caseous cores, were present. Calf 6 had areas of plum-coloured consolidation in both cranial and caudal lobes without associated nodules. In calf 13, a single small consolidated area containing a caseous core was present near the dorsal surface of the right caudal lobe. No abnormalities were detected in the lymph nodes of calves 1 to 5. T h e left bronchial and the mediastinal lymph nodes of calf 6 were enlarged, and on section were seen to contain multiple fawn-coloured foci, 1-2 m m in diameter, with central caseous necrosis. In calves 7 to 13, similar foci were seen in various enlarged lymph nodes associated with the respiratory tract. No lesions were observed in other organs of the experimentally infected calves or in any organs of the control calf. Histopathological Findings Lungs. No acid-fast organisms or tuberculous lesions were observed in calf 1, killed on pid 3. Unequivocal lesions with associated acid-fast organisms were first seen in calf 3 (pid 7). Macrophages and macrophage giant cells, many of which contained neutrophilic debris and acid-fast organisms, were noted within alveoli (Fig. 4), and additional small aggregates of often degenerate neutrophils, with associated acid-fast organisms, were also observed.

32

Fig. 1.

J. P. C a s s i d y et al.

Multiple raised tuberculous plaques (arrow), 1-3 m m in diameter, extending over nasopharynx (calf 13).

By pid 14 (calf 6), multifocal aggregates of neutrophils had become larger and more numerous than in calf 3, with a distinct mantle of macrophages, many of which were "epithelioid" in appearance (Figs 5 and 6). Material suggestive of phagocytized neutrophil debris was present within the cytoplasm of some of the surrounding macrophages. Large numbers of acid-fast organisms were observed within the central neutrophil aggregates and in the cytoplasm of the surrounding macrophages. In calf 9 (pid 21), extensive central necrosis of neutrophil and macrophage aggregates had occurred, resulting in an amorphous mass of nuclear debris and eosinophilic coagulated cytoplasm. A mantle of intact and degenerate neutrophils, macrophages, macrophage giant cells and lymphocytes surrounded necrotic areas (Fig. 7). A limited degree of circumscribing fibrosis was noted in some areas. Similar lesions but with larger necrotic cores were present in calves 11 and 13 (pids 28 and 42). In calf 13, some mineralization of necrotic areas and a moderate degree of fibrous encapsulation of lesions had occurred. No tuberculous lesions or acid-fast organisms were observed in calves 2, 4

Early L e s i o n s in Bovine Tuberculosis

Fig. 2.

Extensive tuberculous consolidation of right middle lung lobe (calf 11).

Fig. 3.

Cross-section of consolidated right middle lung lobe (calf 11).

33

or 7. In calf 5, a single acid-fast organism was observed in an alveolar macrophage in the right middle pulmonary lobe, with no inflammatory response. Chronic lymphocytic ("cuffing") pneumonia of varying severity was observed in nine of the calves. T h e lesions were classified in calves 3, 10, 11 and 13 as mild, in calves 7 and 8 as mild to moderate, and in calves 4, 5 and 6 as moderate. With the exception of calf 5 (see above), no acid-fast organisms were observed in association with these intercurrent lesions, which could be

34

J. P. Cassidy et al.

3^ -^^ m'&

^smt^-^^

^'^^

:::. I

Fig. 4.

Macrophage giant cell containing neutrophilic debris (black arrow) and acid-fast organisms (white arrow) in alveolar lumen. Z N . X 167.

Fig. 5.

Neutrophil aggregate (N) with mantle of macrophages (m), many of which are epithelioid in appearance. H E . X 104.

distinguished with confidence from the developing granulomatous tuberculous lesions. No significant abnormalities were observed in the lungs of the control calf URT. In calf 1 (pid 3), the histological appearance of the U R T mucosae was similar to that of the control calf. In calf 2 (pid 7), clusters of acid-fast organisms were present in sub-epithelial areas, many associated with lymphoid

Early L e s i o n s in Bovine Tuberculosis

35

Fig. 6.

Detail of" central neutrophil aggregate (N). H E . x 167.

Fig. 7.

Central necrosis within developing lesion (N) surrotinded by macrophages (m) a n d lymphocytes (1). H E . X 125.

aggregates. In calves 4 and 5 (pids 11 and 14), large numbers of acid-fast organisms were observed within aggregates of neutrophils and macrophages in sub-epithelial lymphoid tissue and within the surface epithelial cells in the U R T mucosa. In calves 8 and 9 (pid 21), central areas of necrosis were observed within the neutrophil and macrophage aggregates. In calves 10 to 13 (pids 24, 28, 35 and 42, respectively), these necrotic centres had enlarged, and mineralization was noted in calves 12 and 13 (pids 35 and 42).

36

J. P. Cassidy et al.

Table 3 Distribution of microscopically observed lesions or acid-fast bacteria, or both, in lymph nodes Microscopical lesions or hactena, or both, in stated lymph node

Calf no.

Killed on pid

Retropharyngeal

Parotid

Submandihular

Cervical

Bronchial

Mediastinal

Mesenteric

3 7 7 11 14 14 17 21 21 24 28 35 42

-

-

-

-

-

-

-

— + + + + + + + + + +

_ + +

— -

—

4-

4-

4-

+

44-

+ + -

1 •2 3

4 5 6 7 8 9 10 11 12 13

+ = Present;

+ 4444-

44-

-

44-

-

+

444-

44-

4-

-

44444-

44444-

+

+

— 4-

— ~ —

— —absent.

T h e distribution of histologically observed lesions or acid-fast organisms, or both, in the respiratory tract is detailed in Table 2. Lymph nodes. The sequence of development of lesions in the lymph nodes was similar to that in the lungs and U R T . Table 3 shows the distribution of acid-fast organisms or lesions, or both. Immunological Examination Tables 4, 5 and 6 detail the results of the IFN-y, lymphocyte proliferation and humoral assays, respectively. Before infection, all 14 calves had negative IFN-y and antimycobacterial antibody responses. Calf 13 had a positive lymphocyte proliferation response on the day of inoculation. The control calf had a positive lymphocyte proliferation response on pid 28. A Wilcoxon Signed-Ranks test indicated statistically significant decreases in the numbers of circulating yS T lymphocytes between pids 0 and 7 (P<0-01) (Table 7). No significant changes were found in the CD4, CDS, CD4:CD8 and B-cell responses. Bacteriological Examination M. bovis was recovered from the respiratory tract of all 13 infected calves post mortem (Table 8), but all tissues from the control calf were negative.

Discussion This sequential study revealed (1) the prominent neutrophil component of the host response to M. bovis in early-stage lesions, (2) the contemporaneous

Early Lesions in Bovine

37

Tuberculosis

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38

J. P. Cassidy et al.

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39

Early Lesions in Bovine Tuberculosis

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J . P. C a s s i d y et

al.

Table 7 y6 T-cell r e s p o n s e s w i t h t i m e p o s t - i n f e c t i o n ( c e l l s x lO'/ml) y6 T-cell reapomes (cells Iff /ml) in calf no. 5

pid 0 7 14 21 28 35 42

0-44

0-56 0-52

0-39 0-22 0-20

0-61 0-33

10

11

12

13

0-30 0-30 0-32 N

1-21 0-36 0-45 0-73 1-33

0-58 0-32 0-45 0-66 0-89 0-67

0-87 0-54 0-35 0-62 0-73 0-69 1-23

8 0-33 0-10 0-16

0-88 0-85 1-00

0-30 0-35 1-43

0-23 0-20 018 0-52

N = result not a^ 'ailable. ...= N o entry (calf already killed). pid =-Post-inocu ation day.

Table 8 D i s t r i b u t i o n of Af. bovis M. bovis isolated from Calf

Killed on pid

URT mucosa

palatine tonsil

URTLN

trachea

stated lung lobe Cranial Caudal

1 2 3 4 5 6 7 8 9 10 11 12 13

3 7 7 11 14 14 17 21 21 24 28 35 42

+ + + + + + + + + + + + +

+ + — + + + + + + + + + +

+ + + + + + + + + + + + +

— + + + + + + + + + +

— + + + + + + + +

+ + + + + + + + + + +

bronchomediastinal LN

— + + + + + + + + + + +* +

mesenteric Ui

~ + + + + + + +

+ =M. bovis isolated; — = n o t isolated. For abbreviations, see Table 1.

development of lesions and positive in-vitro cellular immune responses (lymphocyte proliferation and IFN-y assay responses), and (3) the appearance of mineralization within central areas of necrosis by pid 35. The infecting dose, although high, failed to induce pronounced lesions in the lungs of seven of the calves (nos 2, 4, 5, 7, 8, 10 and 12). The neutrophil response to M. bovis infection, particularly prominent in calves 5 and 6, has previously received limited attention and emphasis. However, in a previous experimental study by Eastwood in 1911, in which the subcutaneous route of inoculation was used, the neutrophil was considered to play an important early role in the ingestion of organisms (Francis, 1958). More recently, in-vitro and in-vivo studies have also demonstrated the role of neutrophils in mycobacterial infections (Brown et al., 1987; Silva et al., 1989;

Early Lesions in Bovine Tuberculosis

41

Appelbcrg et at, 1995). Mechanisms to explain neutrophil participation in the early response to infection include the chemotactic activity of interleukin-8 released from tumour necrosis factor-a-primed tissue cells (Kunkel et al., 1996) and the influence of IFN-y and granulocyte-macrophage colony-stimulating factor, produced by activated T cells (Weisbart et al., 1987; Ainsworth et al., 1996). The aggregation of neutrophils around mycobacteria, and their subsequent degeneration to form necrotic cores, highlight an important role for these cells in lesion formation. The presence of degenerate neutrophils around the necrotic cores of lesions in tuberculin reactor cattle suggests that a similar process may occur in naturally infected animals. Further proposed neutrophil responses to mycobacterial infection include macrophage chemotaxis (Brown, 1983) and the donation of molecules such as lactoferrin and myeloperoxidase to macrophage giant cells (Silva et al., 1989). Neutrophils may thus have a more significant role in the control of mycobacterial infections than has previously been appreciated. It is possible that in cattle, as has been suggested in man, they control some subclinical mycobacterial infections without additional significant macrophage intervention (Rook, 1988). The temporal association of cellular immunity (in-vitro lymphocyte proliferation and IFN-y assay responses) with lesion development supports previous assertions that tuberculous lesion formation has an immunological basis (Collins and Campbell, 1982; Dannenberg, 1991; Dungworth, 1993; Barnes et al., 1994). It was noteworthy that positive lymphocyte proliferation responses in five of the six calves surviving at pid 21 coincided with the appearance of pronounced central areas of necrosis within the developing lesions of the two calves (nos 8 and 9) killed at this timepoint. IFN-y has been identified as a T cell-derived cytokine which can trigger anti-mycobacterial mechanisms in murine macrophages (Flesch and Kaufmann, 1987). The findings of this study suggest that this mediator plays a similar role in cattle. The control animal (calf 14) had a positive lymphocyte proliferation response at pid 28, suggestive of prior exposure to M. bovis or other mycobacteria. However, this was highly unlikely because the calf, which came from a herd with no previous history of tuberculosis, was housed well away from the M. ^oyzj^-infected animals and was fed and sampled separately from them. Furthermore, M. bovis was not cultured from this animal j&o.y/ mortem. Antigenic cross-reactivity as a result of contact with environmental mycobacteria has previously been demonstrated as a cause of false-positive responses in other assays of cellular immunity to bovine tuberculosis (the IFN-y assay and tuberculin skin test) (Monaghan et al, 1994; Wood and Rothel, 1994). The mineralization of lesions by pid 35 (calf 12) was notable insofar as this is usually considered a more chronic change. However, Lepper et al. (1977) observed mineralized lesions in cattle 36 days after intravenous inoculation with M. bovis. In man, tuberculous mineralization has been described in lesions as early as 58 days after infection (Rich, 1951). Increased knowledge of the timescale within which mineralization can occur might prove helpful in estimating the age of lesions during epidemiological investigations. The extensive lesions in the U R T mucosa of calves 7 to 13 may have been related to the high infecting dose or the inoculation technique, or both.

42

J. P. Cassidy et al.

However, evidence for the occurrence of U R T mucosal lesions in naturally infected cattle, although on a considerably reduced scale, is available. In two separate studies, lesions were found to be restricted to the U R T lymph nodes of 2 3 % (Neill et al., 1994b) and 32% (Corner, 1994) of naturally infected cattle, suggesting the possibility of primary U R T mucosal lesions. Such lesions in naturally infected cattle would be potentially significant foci of mycobacterial excretion. However, their small number and microscopic size might make their detection difficult. A pre-existing chronic lymphocytic "cuffing" pneumonia, ranging in severity from mild to moderate, was observed in nine of the calves (3, 4, 5, 6, 7, 8, 10,11 and 13), but these lesions were histologically distinct from the developing tuberculous lesions. Unequivocal tuberculous lesions with acid-fast organisms were also present in the lungs of calves 3, 6, 8, 11 and 13. Although M. bovis was isolated from the lungs of calves 4, 5, 7 and 10 and a single acid-fast organism was observed in an alveolar macrophage in the right middle lung lobe of calf 5, the pneumonic lesions observed were not considered to be the result of M. bovis infection. The possibility that the "cuffing" pneumonia contributed to the statistically significant fall in circulating y8 T lymphocytes between pids 0 and 7 (P<0-01) deserves consideration. However, the close temporal association of this fall with experimental infection and developing tuberculous pathology, and the finding of a similar decrease by Pollock et al. (1996) after experimental infection with M. bovis, suggests that the fall was due to M. bovis. If we had attempted to vaccinate these calves against "cuffing" pneumonia before inoculation with M. bovis, this would have complicated the interpretation of the immunological results. There is evidence to suggest a particular predilection of yS T lymphocytes for mycobacteria (Kaufmann, 1990) and in ruminants these cells are thought to play a role in the protection of epithelial surfaces (Hein and Mackay, 1991). Other studies have suggested that they play a role in stimulating granuloma formation (Modlin et al., 1989). Therefore, the decrease in the circulating numbers of these cells may reflect their migration to sites of early lesion formation, possibly within epithelial surfaces. Whether the cell-mediated or humoral immune response is activated after M. bovis infection is dictated by the mycobacterial antigen load and the duration of the period since infection (Ritacco et al., 1991; Neill et al., 1994b). In early infection, when the antigen load is low, cell-mediated immune responses dominate. Later, as this load increases, the humoral response becomes ascendant. Antigen loads may have been high in the present study, but the fact that the early post-infection period was under investigation may explain why only two of the calves (10 and 13) exhibited ELISA responses. Acknowledgments The authors gratefully acknowledge the excellent technical assistance of the staff of the Pathology and Bacteriology Departments of the Veterinary Sciences Division. Thanks are also due to Ms Deirdre McConaghy for the statistical analysis.

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