Classification of lesions and comparison of immunohistochemical and acid fast staining in diagnosis of naturally occurring paratuberculosis in goats

Lesions associated with paratuberculosis and degrees of positivity by Ziehl-Neelsen (ZN) and Avidin–Biotin Complex peroxidase (ABC) techniques in 26 adult goats grossly or clinically suspected to paratuberculosis were studied. Of these, 17 (65.4%) had microscopic lesions associated with paratuberculosis which were classified into three categories. Diffuse multibacillary lesions (30.8%) characterized by a diffuse granulomatous enteritis with epithelioid macrophages infiltration as main inflammatory cells into lamina propria that arranged mosaic-like appearance, and filled with numerous acid fast bacilli in ZN and highly positive in ABC. Diffuse lymphocytic lesions (11.5%) composed of lymphocytes as main inflammatory infiltrate, with some epithelioid macrophages or giant cells containing few if any mycobacteria. Diffuse mixed lesions (23%) characterized by a diffuse granulomatous enteritis with infiltration of mixture of large numbers of lymphocytes and epithelioid macrophages with varying degrees of positivity in ZN and ABC. In all types of lesions, other lesions such as granulomatous lymphangitis and arteritis, lymphangiectasis, thrombi composed of degenerated epithelioid macrophages were observed. Caseous necrosis, calcification and fibrosis in mesenteric lymph nodes commonly, especially in diffuse lymphocytic form, were observed. In conclusion, the present study showed three categories of lesions in which diffuse multibacillary form was the commonest type. Both ABC and ZN methods can detect (ABC with more positivity) almost all the goats with diffuse multibacillary and mixed lesions; more cases with lymphocytic forms were positive by ABC than by ZN. ABC method seems to be an efficient diagnostic adjunct to conventional ZN staining for diagnosis of paratuberculosis in goats. The importance of sampling the ileum (especially ileocecal valve), jejunum and mesenteric lymph nodes to find microscopic lesions of paratuberculosis in goats is emphasized.     

Bacteriological and Pathological Study of Animals Given Freund Adjuvant

Freund complete adjuvant, with allegedly "killed and dried" Mycobacterium butyricum added (intramuscularly in rabbits and intraperitoneally in mice), produced a local granulomatous lesion and frequently a similar lesion in the viscera. Acid-fast bacilli were present in these lesions, and in some animals M. butyricum was obtained in culture. When this culture was inoculated in mice, a similar lesion developed. Pathological lesions with similar acid-fast organisms also were observed by us in rabbits given Freund complete adjuvant by two other investigators.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> disease in wild red-legged partridges (<Emphasis Type="Italic">Alectoris rufa</Emphasis>)

Three wild red-legged partridges (Alectoris rufa) from intensively managed hunting areas in Spain were received for necropsy. They showed granulomatous lesions in different parts of the body, mainly in liver and spleen. Microscope examination of the granulomas showed central caseous necrosis and large amounts of acid-fast bacilli, surrounded by epitheloid cells, giant cells, and lymphocytes. Attempts to isolate and culture the bacillus in Colestsos medium were unsuccessful, but the polymerase chain reaction technique revealed the presence of microorganisms belonging to the Mycobacterium avium complex in one of the partridges. This is the first report of avian tuberculosis in free-living red-legged partridges.     

Immunohistochemical demonstration of mycobacterial antigens in intracranial tuberculoma.

Mycobacterial antigens have been demonstrated immunohistochemically in the paraffin sections of 10 intracranial tuberculous granulomas and the results were compared with the detection of acid fast bacilli by conventional Ziehl-Neelsen method. In none of the 10 specimens, acid fast bacilli were demonstrated while mycobacterial antigens were characterised as diffusely staining granular brownish-pink material within the cytoplasm of giant cells and macrophages. In 14 specimens of granulomatous lesions due to non-tuberculous aetiology, immunohistochemical stains were negative for mycobacterial antigen. Thus demonstration of mycobacterial antigen will be not only useful in establishing mycobacterial aetiology of a caseating intracranial granuloma but also can be used as an alternative method to the conventional Ziehl-Neelsen method.     

Brugia pahangi: Histopathological study of golden hamsters

Sixteen male hamsters were inoculated subcutaneously with 95 to 150 infective larvae of B. pahangi and were examined for histopathologic lesions at 39–109 days postinfection. The basic microscopic lesion observed was obstructive granulomatous lymphangitis. Analogous lymph node changes sometimes occurred along with lymphoreticular hyperplasia and increased numbers of eosinophils. Cellular infiltration of perivascular and perinodal tissues was common, with plasma cells and eosinophils predominating. Genital lesions included funiculitis, epididymitis, and mild orchitis. Live and dead worms were found in the testicular parenchyma. Pulmonary changes in hamsters infected more than 105 days included multiple small, granulomatous foci and periarteriolar, peribronchiolar, and subpleural cellular accumulations of plasma cells and eosinophils. Granulomatous obstruction of pulmonary arteries associated with dead worms was observed in two hamsters infected for 39–45 days and in one hamster infected for 109 days. Small liver granulomas were common. Disintegrating microfilariae occurred within giant cells in the lymph nodes, spleen, lungs, and testes.     

Thyroid tuberculosis – role of PCR in diagnosis of a rare entity

N. Gupta, K. Sharma, A. Barwad, M. Sharma, A. Rajwanshi, P. Dutta and A. Sharma
Thyroid tuberculosis – role of PCR in diagnosis of a rare entity Background: Tuberculosis is a rare cause of granulomatous thyroiditis, whose diagnosis may be difficult with routine cytopathology and staining for acid‐fast bacilli (AFB). Study design: Amongst 7962 cases of various thyroid lesions subjected to fine needle aspiration cytology (FNAC) over a period of 12 years, 34 cases (0.43%) were found to have cytological features of granulomatous inflammation with or without necrosis, which could be due to tuberculosis, granulomatous thyroiditis or other causes of granulomatous inflammation such as sarcoidosis or fungal infections. DNA was extracted from the material available on May‐Grünwald–Giemsa‐stained smears from the archival material. PCR for Mycobacterium tuberculosis was performed for insertion sequence IS6110. Results: The age of the patients ranged from 32 to 58 years (median 48 years); 24 were female and 10 male. FNAC from thyroid swellings showed epithelioid granulomas with giant cells and/or necrosis. Although acid‐fast bacilli were only seen in smears in two cases, 19/34 (55.9%) showed the presence of 123 bp DNA band under ultraviolet transillumination. Five control cases were negative. Conclusion: Our study of archival cytological material illustrates the importance of PCR as a potentially useful tool for the detection of M. tuberculosis DNA from FNAC of thyroid lesions, which could provide an alternative for rapid diagnosis of thyroid tuberculosis in AFB‐negative cases.     

Experimental Absidia corymbifera infection in rabbits: Sequential pathological studies

Intravenous inoculation of young rabbits with 1.4 × 105 spores ml-1 of Absidia corymbifera produced gross and microscopic changes principally in the kidneys. Grossly, there was congestion, haemorrhages and greyish-white necrotic foci giving a mottled appearance to the kidneys. Microscopically, besides congestion and haemorrhages, there was a granulomatous reaction characterized by a central necrotic mass containing mycelium surrounded by a reactive zone of neutrophils, lymphocytes, a few macrophages and giant cells. Fibrous tissue proliferation was also present. Characteristic dichotomously branched non-septate fungal hyphae could be demonstrated in the sections of kidneys up to 14 days of infection. In one rabbit, which had died on the 8th day, small granulomatous lesions with fungal hyphae were noticed in the liver's parenchyma. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the pathogenicity of Yersinia enterocolitica. III. Comparative studies between Y. enterocolitica and Y. pseudotuberculosis

Comparative studies on pathogenicity between Yersinia enterocolitica and Yersinia pseudotuberculosis were performed using experimental infection systems in vivo and in vitro. All of thestra ins of both species successfully produced experimental enterocolitis in rabbits although the severity varied with the strains challenged. The changes were characterized by granulomatous lesions with necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen. These strains uniformly had the ability to penetrate HeLa cells and to survive or multiply within cultured rabbit peritoneal macrophages. In addition, in infections with strain TP-2 or PST-III of Y. pseudotuberculosis, catarrhal inflammation all over the small intestine and/or focal necrosis and parenchymatous degeneration in the liver were observed, along with the granulomatous lesions. These strains, at the same time, exhibited cytotoxic effects on the cultured cells. The pathogenic factors of Y. enterocolitica are discussed in comparison with those of Y. pseudotuberculosis.     

Liver in Leprosy: Histological and Biochemical Findings

The histological findings and their correlation with biochemical functions of the liver in 240 leprosy patients are presented. In 21% with tuberculoid leprosy and in 62% with lepromatous leprosy leprous granulomata were found in the liver. A significant prevalence of granulomatous lesions in the liver among patients with tuberculoid and borderline-tuberculoid leprosy of less than one year''s duration suggests that bacillaemia occurs early in all forms of leprosy.There was a direct correlation between bacterial index and the presence of acid-fast bacilli in the liver. Of 50 patients with negative skin smears seven had acid-fast bacilli at liver biopsy. From none of these liver homogenates were acid-fast bacilli grown on culture in Löwenstein-Jensen medium.The alterations in liver functions were more consistently seen when acid-fast bacilli were associated with the presence of leprous granulomatous lesions. The acid-fast bacilli were found to persist even after one to five years of specific antileprosy therapy and after the bacilli in the skin had cleared up. This may explain the relatively frequent recrudescence or relapse of the bacillated types of leprosy when specific antileprosy therapy is stopped soon after bacterial negativity is attained on skin smears.     

Asteroid bodies in a lymph node aspirate. A case report

BACKGROUND: Sarcoidosis, a multisystem disease with tbe potential to occur at any site, has varied clinical manifestations. Cutaneous lesions, seen in one-third of patients, may precede systemic manifestations. Identification of asteroid bodies in aspirated material may aid early diagnosis in the appropriate clinical setting. CASE: A 37-year-old woman had multiple asymptomatic, buff-colored, sucutaneous nodules, 0.5-1.0 cm in diameter, on the scalp, face, dorsum of the wrist and back of the elbows for 2 months. On examination, firm, nontender right inguinal and epitrochlear lymphadenopathy, 2 x 1 cm each, was detected. The epitrochlear lymph node aspirate showed noncaseating epithelioid granulomas, multinucleate giant cells and asteroid bodies. Subsequent biopsy of 1 of the subcutaneous nodules corroborated the aspiration cytology findings. Following the diagnosis of sarcoidosis, other investigations were done. Erythrocyte sedimentation rate was 66 mm at the end of 1 hour, Mantoux test was negative, and chest radiograph showed bilateral reticulonodular shadows. Angiotensin-converting enzyme level was significantly elevated. CONCLUSION: This case draws attention to 1 of the rare cytologic findings of sarcoidosis. Differentiation from other granulomatous lesions, especially when special stains are negative, is difficult. In our case, a lymph node aspirate showing asteroid bodies in a background of granulomatous inflammation supported the diagnosis.     

Utility of papanicolaou stain-induced fluorescence in the cytodiagnosis of extrapulmonary tuberculosis

OBJECTIVE: To study the utility of Papanicolaou stain-induced fluorescence (PIF) in the detection of tubercle bacilli and to compare its diagnostic efficacy with that of the conventional Ziehl-Neelsen (ZN) method. STUDY DESIGN: This prospective study was carried out at a tertiary health care center, over a period of 2 years between January 2001 and December 2002. A total of 500 cases offine needle aspiration cytology from lymph nodes and other extrapulmonary sites were studied. Only cases that were clinically and cytologically suggestive of tuberculosis were included in the study. The smears were stained with ZN and Papanicolaou stain and examined under light and fluorescence microscopes, respectively for detection of acid-fast bacilli (AFB). Mycobacterial culture was used as the gold standard to compare the results. RESULTS: Cytologic smears were categorized into 4 distinct cytomorphologic patterns: epithelioid granulomas without caseous necrosis (101 cases), epithelioid granulomas with caseous necrosis (268 cases), caseation or acute inflammatory exudate only (114 cases), and occasional epithelioid cells without necrosis or giant cells (17 cases). The overall AFB positivity was 30.8% with the ZN method, while it was 40.6% with PIF. Moreover, PIF was more effective in detecting bacilli in group I lesions (18.8% vs. 6.9% with ZN method), in which the bacillary load is very low. CONCLUSION: PIF is superior to the conventional ZN method in detecting tubercle bacilli, particularly when the bacillary load is low. It is a relatively inexpensive and fast technique.     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

Intravesical BCG administration in the guinea pig. A histomorphological study

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10(3) culturable particles (c.p.) to 5 x 10(7) c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10(6) culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by non-caseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual lesion morphology and skin test reaction for Mycobacterium avium complex in macaques

Three rhesus (Macaca mulatta) and one cynomolgus (M. fascicularis) monkey were euthanized because of positive reactions to intradermal tests with mammalian Old Tuberculin (mOT). All the animals had gross or microscopic lesions consisting of unitized macro- or microgranulomas with central necrosis, involving the lung or mesenteric lymph nodes. Small numbers of acid fast organisms were demonstrated in the lesions. Mycobacterium avium complex, serotype 2 was isolated from three of the cases. The cases were unusual because the lesion morphology and mOT hypersensitivity were more typical of mammalian tuberculosis than avian tuberculosis in monkeys.     

AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.     

An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.     

Intravesical BCG administration in the guinea pig

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10³ culturable particles (c.p.) to 5 × 10⁷ c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10₆ culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by noncaseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG. No microorganisms were detected by Ziehl-Neelsen (ZN) staining or culture in L?wenstein-Jensen medium in the first draining (iliac) lymph nodes of the bladder or in the spleen. In this study we found that BCG could induce inflammatory reactions in the bladder wall after its introduction into the previously undamaged bladder. Ulceration of the epithelium covering the mononuclear infiltrates was not observed. Occasionally a generalized inflammatory response to BCG was present in the animals investigated.     

Experimental aerosol inoculation of Mycobacterium bovis in North American opossums (Didelphis virginiana)

The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.     

Mortality in Syrian hamsters infected with Paragonimus kellicotti.

In an attempt to find a small animal model for paragonimiasis, Syrian hamsters were infected with between 1 and 16 metacercariae of Paragonimus kellicotti. A definitive mortality dose-response was observed with 90% of all hamsters given 3 or more parasites succumbing to the infection within 35 days. Hamsters demonstrated acute pleuritis, reactive mesothelial hyperplasia, subpleural accumulations of reactive and mature plasma cells, neovascularization, fibrohistiocytic thickening with and without giant cells, raised fibroconnective tissue lesions, and granulomatous inflammation with hemorrhage. Perivascular plasmacytic (lymphocytic) infiltrate, multifocal bronchopneumonia, and parenchymal necrotizing suppurative granulomatous inflammation, hemorrhagic pneumonia, and diffuse sprinkling of eosinophils, neutrophils, and intraalveolar macrophages also were observed. The response observed here may represent a new small animal mortality model useful in the search for new compounds to treat early trematode infections.     

Studies on the Pathogenicity of Yersinia enterocolitica

Analysis of the pathogenicity of Yersinia enterocolitica was performed with an experimental model successfully produced in rabbits by intraduodenal inoculation with strains isolated from various sources. Pathogenic strains easily penetrated the epithelial linings of the intestinal mucous membrane into the target reticuloendothelial tissues of the intestine, such as the lamina propria and lymph follicles, where they multiplied within mononuclear cells and produced granuloma. Granuloma, in severe infections, underwent necrobiosis and sometimes progressed to ulceration accompanied by colony formation of the organisms. In mild infections, granulomatous lesions were localized in lymph follicles and never progressed to ulceration. Nonpathogenic strains were rapidly excreted without penetration of epithelial linings. Y. enterocolitica should be within the category of invasion type enteropathogenic bacteria such as Shigella and Salmonella. Pathogenic behavior of Y. enterocolitica is discussed in comparison with that of Shigella and Salmonella.