Light and electron-microscopic localization of CD9 and surfactant protein A and D in normal lungs of the horse

The lung is a complex organ, and its physiology and immunology are regulated by various immune molecules and cells. Lung surfactant, a mixture of phospholipids and proteins produced by the bronchiolar and type II alveolar epithelial cells, is one such important player in lung physiology. Compared to knowledge about the biology of the surfactant in rodents and humans, only limited data are available on the surfactant in the horse. Although there are data linking levels of surfactant proteins with respiratory disease in the horse, there are no data on the cellular localization of surfactant protein A (SP-A) and surfactant protein D (SP-D). A member of the tetraspanin family of proteins, CD9 is a cell-signaling and adhesion protein and its expression has been detected in both normal and cancer cells, including those in the lung. Because there are no immunolocalization data on SP-A, SP-D, and CD9 in the normal lungs of the horse, our objective was to conduct a light and electron microscopic immunocytochemical study on normal lungs of the horse. The data showed SP-A and SP-D in bronchiolar epithelial and type II alveolar epithelial cells. These proteins were also localized in type I alveolar epithelial cells, pulmonary intravascular macrophages, and neutrophils, which is likely an outcome of endocytosis of the proteins by these cells. CD9 was present in the airway and vascular smooth muscle cells, endothelium, and blood cells, but not in the airway epithelium. These new data provide a baseline to further examine the expression and functions of SP-A, SP-D, and CD9 proteins in inflammation associated with respiratory diseases in the horse.     

Cattle Infected with Bovine Leukaemia Virus may not only Develop Persistent B‐cell Lymphocytosis but also Persistent B‐cell Lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non‐persistent lymphocytotic, PL^?) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL^? cattle and compared with six age‐matched animals with persistent lymphocytosis (PL⁺) and five non‐infected healthy controls (BLV^?). In the PL^? group, the percentage and number of surface immunoglobulin‐positive (sIg⁺) B cells were significantly reduced. Whereas in BLV^? cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg⁺ and 24% were sIgM⁺ B cells. In the PL^? group, less than 20% of the PBL were sIg⁺ and sIgM⁺ B cells. Only 5% of the PBL co‐expressed sIgM⁺ and CD5⁺ versus 16% in BLV^?. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM⁺ B cells co‐expressing CD5 and CD11b; and (iii) equally both λ‐ and κ‐type light chain B‐cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5^? and CD11b^? sIgM⁺ B cells and CD2⁺ T cells did not differ. In PL⁺ animals, about 75% of the PBL were sIgM⁺CD5⁺ B cells. These cells were of polyclonal origin, as light chains of the λ‐ and κ‐type were expressed in a ratio of 4:1 (57.7% of PBL λ⁺, 14% κ⁺) as in BLV^? animals (33.6% of PBL λ⁺, 8.7% κ⁺). In PL⁺ cattle the absolute number of B‐cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T‐cell numbers, the relative percentage of T‐cells could be lower than in normal controls. The cause for the observed B cell decrease in PL^? cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B‐cell lymphopenia.     

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4⁺, CD8⁺, γδ, B lymphocytes and plasma, CD25⁺, CD68⁺, MHC-II⁺, Ki67⁺ and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4⁺ T cells, but only few epitheloid macrophages were observed in severely sick goats at 2–3 mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13 mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.     

肺表面活性蛋白研究进展

Pulmonary surfactant protein （SP） is a carrier which plays an important role in phospholipids, mainly in the bronchial surface and alveolar gas-liquid interface, with lower surface tension properties, prevents alveolar collapse, is beneficial to gas exchange and to maintain the lungs pulmonary residual volume and to keep pulmonary compliance. Four kinds of proteins have been found, namely SP-A, SP-B, SP-C and SP-D. These four proteins in lung disease defensive and in maintaining lung function play important roles. Therefore, this article reviews SP from its structure, function and the correlation with the disease, to provide a basis for future research.     

Reference ranges of hematology and lymphocyte subsets in healthy Korean native cattle (Hanwoo) and Holstein dairy cattle

There are no accurate reference ranges for hematology parameters and lymphocyte subsets in Korean native beef cattle (Hanwoo). This study was performed to establish reliable reference ranges of hematology and lymphocyte subsets using a large number of Hanwoo cattle (n = 350) and to compare differences between Hanwoo and Holstein dairy cattle (n = 334). Additionally, age‐related changes in lymphocyte subsets were studied. Bovine leukocyte subpopulation analysis was performed using mono or dual color flow cytometry. The leukocyte subpopulations investigated in healthy cattle included: CD2⁺ cells, sIgM⁺ cells, MHC class II⁺ cells, CD3⁺CD4⁺ cells, CD3⁺CD8⁺ cells, and WC1⁺ cells. Although Hanwoo and Holstein cattle are the same species, results showed several differences in hematology and lymphocyte subsets between Hanwoo and Holstein cattle. This study is the first report to establish reference ranges of hematology and lymphocyte subsets in adult Hanwoo cattle.     

Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control infection.     

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4⁺ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4⁺ T cell responses in cattle. Monocytes and CD4⁺ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4⁺ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4⁺ T cell responses in vitro for an important pathogen of livestock.     

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis

The local expression of WC1⁺ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1⁺ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1⁺ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1⁺ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.     

Ultrastructural changes in caprine lungs infected with Mycoplasma capricolum subspecies capripneumonia

Ultrastructural examination of the lungs from goats with natural infections with Mycoplasma capricolum subspecies capripneumonia, the causative agent of contagious caprine pleuropneumonia, revealed extensive hyperplasia of type II pneumocytes. Typically, these type II cells contained abundant numbers of osmiophilic lamellar bodies that had lost most of their characteristic lamellar ultrastructure. These findings were absent in healthy control goats.     

Spontaneous pulmonary alveolar proteinosis in captive "moustached tamarins" (Saguinus mystax)

Pulmonary alveolar proteinosis is a rare human disease characterized by accumulation of surfactant in alveoli without generating an inflammatory response. Lung lesions resembling pulmonary alveolar proteinosis were observed in 7 adult tamarins (5 males and 2 females). Gross lesions were characterized by areas of discoloration, slight bulging over the lung parenchyma, and occasional consolidation. Histologic examination of tamarin lung samples revealed intra-alveolar accumulation of amorphous, amphophilic, periodic acid-Schiff-positive, finely granular to dense material. In some cases, type II pneumocyte hypertrophy and hyperplasia were observed with pleural and septal thickening and fibrosis. Large numbers of intra-alveolar foamy macrophages were noted surrounding and/or in the vicinity of the lesions. Immunohistochemical analysis of the lung lesions using polyclonal (surfactant proteins A, B, and C) and monoclonal (surfactant protein D) antibodies revealed the granular material to be composed largely of surfactant protein B, followed by surfactant protein A. Surfactant proteins C and D were present in lesser quantities, with the latter observed surrounding the lipoproteinaceous deposits. Transmission electron microscopy of the affected lungs showed numerous, irregularly shaped osmiophilic lamellar bodies in type II pneumocytes. The cytoplasm in alveolar macrophages was expanded, containing ingested surfactant with swollen mitochondria and rough endoplasmic reticulum. Thoracic radiographs, available in 1 animal, depicted the lesions as small multifocal opacities randomly distributed in cranial and diaphragmatic lung lobes. This is, to the authors' knowledge, the first report of spontaneous pulmonary alveolar proteinosis in nonhuman primates.     

Expression of inducible nitric oxide synthase in spontaneous bovine bronchopneumonia.

The expression of inducible nitric oxide synthase (iNOS), major histocompatibility class II molecules (MHC-II), CD68, and the calcium-binding proteins S100A8 and S100A9 (also called MRP8 and MRP14, respectively) was assessed in lung tissues from cattle that succumbed to pneumonia. Expression patterns of these markers were related to the types of lung lesion. iNOS expression was only observed in lungs infected with Arcanobacterium pyogenes or Pasteurella haemolytica but not in lungs from cattle with subacute chronic interstitial pneumonia and acute interstitial pneumonia due to Escherichia coli infection. High levels of iNOS were expressed by cells (probably leukocytes) surrounding necrotic foci. Occasionally, iNOS was expressed by intraalveolar macrophages in viable parenchyma, by leukocytes within the airways, and by some chondrocytes in the supporting cartilage of bronchi. Cells expressing MHC-II were distributed relatively evenly throughout areas of inflammation and did not display any clear association with necrotic foci. Cell types expressing MHC-II included type II alveolar epithelial cells, spindle-shaped cells of the interstitium, cells in bronchus-associated lymphoid tissue, and leukocytes in lymph and blood vessels but largely excluded iNOS-positive cells. Likewise, CD68-positive cells were rarely positive for iNOS and were not confined to the areas surrounding necrotic tissue. As with MHC-II and CD68, there was little if any coexpression of iNOS and either of the S100 proteins tested. Thus, in cattle with necrotizing bronchopneumonia, iNOS-expressing cells were largely restricted to the cellular zone surrounding necrotic areas.     

Susceptibility to tuberculosis: composition of tuberculous granulomas in Thorbecke and outbred New Zealand White rabbits

We sought to characterize the lung cellular immune responses to inhaled Mycobacterium tuberculosis (Mtb) of the susceptible inbred Thorbecke rabbit (the genomically sequenced strain, now unavailable) and compare it to outbred, Mtb-resistant, New Zealand White rabbits. Using Mtb CDC1551, we confirmed that the inbred rabbits allowed establishment of infection with this low virulence strain, compared to poor establishment in outbred rabbits. With a more virulent strain, Mtb Erdman, that establishes infection well in both rabbit strains, we analyzed granulomas from rabbit lungs 5 weeks after aerosol infection. The lung granulomas of inbred rabbits had significantly higher frequencies of cells expressing MHC Class II and CD11b, and lower frequencies of CD8+ T cells than the outbred controls. Macrophage-sized cells expressing MHC Class II in inbred rabbit granulomas showed significantly decreased intensity of expression, suggesting impaired maturation. Although the inbred dermal tuberculin reactions were decreased, the in vitro IFN-gamma mRNA responses of hilar node lymphocytes to tuberculin were higher than those of outbred rabbits. Further delineation of the outbred rabbit's resistant immune response to Mtb infection is warranted.     

In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25−CD4+ cells

This paper investigates the in vitro effect of dexamethasone on bovine CD25^highCD4⁺, CD25^lowCD4⁺ and CD25⁻CD4⁺ T cells. Only a small percentage of bovine CD25^highCD4⁺ (2–4%) and CD25^lowCD4⁺ (1–2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25⁻CD4⁺ cells, but it increased the relative and absolute numbers of CD25^highCD4⁺ and CD25^lowCD4⁺ lymphocytes, while at the same time reducing the percentage of Foxp3⁺ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25⁺CD4⁺, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4⁺ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25⁻CD4⁺ cells and antiapoptotic effect on CD25^highCD4⁺ and CD25^lowCD4⁺ cells. The results obtained from this study indicate that the involvement of CD4⁺ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25⁻CD4⁺ cells. Secretion of TGF-β and IL-10 by CD4⁺ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10⁺CD4⁺ cells.     

Factors affecting disease manifestation of toxocarosis in humans: Genetics and environment

Toxocara canis is regarded as the main cause of human toxocarosis but the relative contribution of T. cati is probably underestimated; serological and other diagnostic methods used in most studies of this zoonotic disease do not distinguish between the two parasites. The definitive hosts for T. canis are caniidae. Pups generally have higher infection rates than adult animals and are a major source of eggs in the environment. Humans usually acquire T. canis infection by accidental ingestion of embryonated eggs or encapsulated larvae from the environment or contaminated food, such infections may lead to visceral larva migrans (VLM), ocular larva migrans (OLM) or covert toxocarosis (CT). Although a mixed Th1- and Th2-mediated immunological response, particularly with high levels of IgE and eosinophilia is observed, the underlying mechanisms of molecular and immunopathogenesis for the development of the symptomatic syndromes of VLM, OLM, or of asymptomatic CT are largely unclear. Studies have indicated that immunological defences against various infectious diseases may be highly influenced by complex interactions of environmental and host genetic factors e.g. MHC class I and II, also known as human leucocyte antigen (HLA). Toxocara spp. infections are associated with a polarized CD4⁺ Th2 response with high IgE levels and eosinophilia, mediated mainly by HLA class II molecules. Associations have been made between HLA class II and pathological severity and host genetic effects on exposure to infection. Recent research suggests Foxp3⁺ CD4⁺CD25⁺-expressing T regulatory (Treg) cells play a role in regulation of the immunopathology of granulomas in experimental toxocaral granulomatous hepatitis and in enhanced expression of TGF-β1, which is an important factor for the local survival and function of Treg observed during T. canis invasion in the mouse small intestine, liver, muscle, and brain. Since the potential susceptibility loci HLA class II molecules, are considered involved in the regulation of a Th2-dominant immunity which is highly controlled by Foxp3⁺ CD4⁺CD25⁺ Treg cells by stimulation through TGF-β1, which thus provides a beneficial environment to T. canis larvae but severe injuries to local organs. However, TGF-β1 variant Leu10Pro known to be involved in disease severity warrants further elucidation as this too may have a role in the severity of human toxocarosis. Exploration of TGF-β1 polymorphism, Foxp3⁺ CD4⁺CD25⁺ Treg cells, and MHC polymorphisms may allow insight into the contribution made by environmental and genetic factors in influencing disease syndrome type and severity in humans with toxocarosis.     

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6 h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6 h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0⁺CD69⁺CD4⁺ memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

Pathology of naturally occurring bovine tuberculosis in England and Wales

The aim of this study was to obtain a contemporary data set of pathology in tuberculin reactor and in-contact cattle in England and Wales. Four hundred animals (200 reactors and 200 in-contacts) from 242 farms located in 14 counties in Western England and Wales were examined. The mean number of lymph nodes (LNs) with tuberculosis (TB)-like lesions per TB-confirmed animal was 1.7 in reactors and 1.5 in in-contact animals. Tuberculous lesions in both reactor and in-contact animals were most commonly observed in the LNs of the thorax, followed by the head and abdomen, particularly the mediastinal, retropharyngeal and tracheobronchial LNs. Twenty-five reactors had macroscopic lesions in the palatine tonsils. Among TB-confirmed cattle, 27% of reactors and 9% of in-contact animals had gross TB-like lesions in the lungs, particularly in the caudal lobes. Gross lesions that were not TB-confirmed were parasitic granulomas (45%), bacterial or mycotic club-forming pyogranulomas (27%) and bacterial abscesses (23%). Diagnostic sensitivity was maximised when bacteriology and histopathology were used concurrently. Stage IV granulomas, alone or in combination with other stages, constituted 63% of lesions, while 16% of lesions were stage I/II granulomas. Caseous necrosis and calcification were common features of the granulomas encountered in natural Mycobacterium bovis infections, even with pathology limited to a small number of sites. Granulomas often covered large areas of histological sections and typically contained only small numbers of acid fast bacilli.