Diffuse cutaneous granulomatous lesions associated with acid-fast bacilli in a cat

Feline cutaneous granulomas associated with acid-fast bacilli are reviewed briefly. A case of diffuse cutaneous granulomatous lesions associated with acid-fast bacilli and the presence of acid-fast bacilli in the draining lymph nodes and liver is reported and described. The unusual clinical appearance of the skin lesions in this case and the varied clinical presentation of lesions associated with acid-fast bacilli is discussed.     

Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves

Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.     

Comparison of postmortem techniques for the detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus).

A retrospective study of various diagnostic postmortem techniques used in a 4-year surveillance program for detection of Mycobacterium bovis infection in wild white-tailed deer (Odocoileus virginianus) was conducted. The tests evaluated were routine histopathology, acid-fast staining, detection of acid-fast bacilli in culture, and an M. tuberculosis group-specific genetic probe applied to pure cultures. Each of these techniques were compared with a reference or "gold standard" of mycobacterial culture and identification. Histopathology, the most rapid form of testing for M. bovis infection in white-tailed deer samples, had a sensitivity of 98% and a specificity of 87%, resulting in a positive predictive value of 94%. The detection of acid-fast bacilli by staining was less sensitive than histopathology (90%), but its higher specificity (97%) resulted in a positive predictive value of 99%. The detection of acid-fast bacilli on culture was both highly specific (93%) and sensitive (100%). The group-specific genetic probe had the highest sensitivity and specificity and produced results in complete agreement with those of mycobacterial culture, suggesting that this technique could be used as the new "gold standard" for this particular wildlife tuberculosis surveillance program.     

A case of feline leprosy caused by Mycobacterium lepraemurium originating from the island of Kythira (Greece): diagnosis and treatment

A 2-year-old, 4 kg, healthy, domestic shorthair female cat presented with ulcerated subcutaneous nodules on the commissures of its mouth. The cat was negative for feline leukaemia virus and feline immunodeficiency virus. Skin mycobacteriosis was diagnosed after detection of numerous acid-fast bacilli in Ziehl Neelsen-stained smears from the ulcers. Feline leprosy was suspected following preliminary polymerase chain reaction results: positive for Mycobacterium genus but negative for Mycobacterium tuberculosis and Mycobacterium avium complexes. Mycobacterium lepraemurium was later identified following DNA sequence analysis of the 5' end of the 16S rRNA gene and the 16S-23S internal transcribed spacer region. Microscopic lesions consisted of pyogranulomas containing mainly large foamy macrophages with 10-100 intra-cellular acid-fast bacilli per field. The cat was cured after surgery and a 14-week course of clofazimine (30 mg daily) and clarithromycin (50 mg twice daily).     

Outbreak of avian mycobacteriosis in a flock of two-year-old domestic pigeons (Columba livia f. domestica)

This report describes an outbreak of avian mycobacteriosis in a flock of 100 two-yr-old pigeons. Over a 6-mo period, the sick pigeons showed cachexia followed by death. In Columbiformes classic tubercles rarely develop, but in these affected pigeons granulomatous nodular lesions of various sizes, containing numerous acid-fast bacilli, were found in the internal organs. The lesions were observed in the liver, spleen, intestine, bone marrow, ovary, and oviduct. Despite their breeding age, atrophy was also found in the ovary and oviduct. Microorganisms belonging to Mycobacterium avium complex were identified in the affected tissues by polymerase chain reaction.     

Histological observations of bovine tuberculosis in lung and lymph node tissues from British deer

Deer are recognized as hosts of Mycobacterium bovis and assessing the role of wild cervids in perpetuating tuberculosis among cattle has motivated extensive research on several continents. In this paper, the histopathology of lymph node and lung tuberculous granulomas in M. bovis positive British deer is presented. The overall aim was to seek further insights into the potential for onward transmission from infected deer to other species, including cattle. Samples were obtained from an extensive survey of wild mammals in South-West England and from statutory tuberculosis surveillance. M. bovis culture-positive samples were characterised microscopically as to their stage of lesion advancement, number of acid-fast bacilli and granuloma encapsulation. Seventy percent of the deer developed granulomas containing far greater numbers of M. bovis bacilli than typically reported in cattle. Red and fallow deer had the largest number of poorly encapsulated granulomas often containing many hundreds of bacilli. The results are consistent with infected wild British deer being a potential source of environmental contamination and onward transmission to other species. However, further work on levels of bacillary shedding is required before this can be confirmed.     

Experimental disease in young chickens induced by a Mycobacterium paratuberculosis isolate from a patient with Crohn's disease.

The susceptibility of young chickens to infection with an isolate of Mycobacterium paratuberculosis which had been recovered from diseased tissues of a patient with Crohn's disease was determined. Two-week-old Leghorn-Cochin chicks were inoculated with strain Linda. Six birds received 10(7) organisms orally, six intraperitoneally, and five intracardially. Six uninoculated birds served as contact controls. Birds from each group were necropsied at two-week intervals. Focal granulomatous lesions occurred in two of six chickens which were inoculated orally, in six of six inoculated intraperitoneally, in three of five inoculated intracardially, but in none of six controls. Of the 11 birds with lesions, acid-fast bacilli were demonstrated in five. Immunoperoxidase reactivity paralleled the presence of acid-fast bacilli.     

Isolated lepromatous conjunctivo-corneal granuloma in a cat from Italy

Objective  To describe a case of a conjunctivo-corneal mass in a cat associated with acid-fast bacilli.
Methods  A 2-year-old female black European Short-Hair cat, living outdoors in a suburban environment in Italy, was referred for evaluation of a nodular, vascularized mass of 2 weeks duration. The mass involved the dorsal bulbar conjunctiva at the temporal canthus of OS and invaded the sclera and cornea. Routine ophthalmic and systemic examination, serologic testing, cytology and histology of the mass were performed. Mycobacterium specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene was also performed.
Results  Neutrophils, lymphocytes, macrophages and giant cells with intracytoplasmic acid-fast bacilli were seen on cytological examination. The histological examination confirmed the presence of a granulomatous lesion with acid-fast bacilli within macrophages. Bacteriological culture of the material from the lesion was negative for Mycobacterium spp. Mycobacterium 16S rRNA gene specific PCR was positive.
A diagnosis of feline leprosy was made. The owners refused any treatment, and 1 year later the lesion was still present.
Conclusions  Veterinary ophthalmologists should be aware of conjunctivo-corneal leproma as an unusual symptom of leprosy.     

Pathology of naturally occurring paratuberculosis in water buffaloes (Bubalus bubalis)

Gross and histologic lesions of paratuberculosis were studied in water buffaloes. Small intestines and associated mesenteric lymph nodes of 405 water buffaloes were examined. Of these, 20 animals having visible changes of intestinal thickening, mucosal corrugations, and enlargement of mesenteric lymph nodes exhibited histologic alteration characteristics of mild to moderate granulomatous inflammation. The histologic lesions observed in these animals were classified into 3 grades on the basis of type of cellular infiltration, granuloma formation, and presence of acid-fast bacilli. Grade-1 lesions observed in 8 animals were marked by the presence of scattered epithelioid macrophages amid large number of lymphocytes in the intestinal villi and in the paracortical regions of the associated mesenteric lymph nodes. Another 8 animals classified under grade-2 revealed microgranulomas, infiltration with a larger number of epithelioid macrophages besides lymphocytes in the intestinal villi, and granulomas in the mesenteric lymph nodes. Grade-3 lesions observed in 4 animals were characterized by the presence of epithelioid granulomas and giant cells in the intestines and the mesenteric lymph nodes. The Ziehl-Neelsen's stained tissue sections revealed acid-fast bacilli in grade-3 and -2 animals and acid-fast granular debris in grade-1 animals. Among these 20 buffaloes, 14 (70%) were positive in the IS900 specific polymerase chain reaction and 6 (30%) were positive in the bacterial culture.     

Cytology and histopathology of canine leproid granuloma syndrome

OBJECTIVE: To describe the histopathology of canine leproid granuloma syndrome. DESIGN: Histological examination of biopsy specimens taken from dogs with leproid granuloma syndrome. Biopsies were acquired from four veterinary pathology practices after initial examination showed acid-fast bacilli or lesions suggestive of a mycobacterial aetiology. Tissue from 30 cases formed part of a retrospective survey while a further 7 cases were obtained prospectively. PROCEDURE: Tissue samples were fixed in formalin and paraffin embedded. Sections were stained with haematoxylin and eosin and Ziehl-Neelsen stains. Slides were evaluated for the type of inflammatory response and the number of bacteria. In a few cases smears made from crush preparations and needle aspirates were stained with DiffQuik and acid-fast stains. RESULTS: The common cytological feature seen in DiffQuik stained smears was numerous, often spindle-shaped, macrophages with variable numbers of lymphocytes and plasma cells and lower numbers of neutrophils. Usually few to moderate numbers of medium length bacilli were detected within macrophages or extracellularly. Histologically, lesions were chiefly pyogranulomatous within the subcutis and dermis. Pyogranulomas were composed chiefly of epithelioid macrophages and neutrophils but plasma cells and small lymphocytes were scattered throughout the lesions in which giant cells were seen. Lesions were pyogranulomatous in 36 cases and granulomatous with small suppurative foci in one. The numbers of acid-fast bacilli present were very low to low in 22 cases, moderate in 6 and regionally numerous to numerous in 9. Bacteria were pleomorphic, ranging from long, slender filaments in parallel sheaves to short and variably-beaded bacilli or highly beaded to coccoid organisms. The morphology was more uniform in DiffQuik stained smears than in fixed tissue sections. CONCLUSION: The pathology of canine leproid granuloma syndrome is highly uniform and is suggestive of saprophytic mycobacterial involvement. The morphological diversity of acid-fast bacilli probably results from differences in staining characteristics rather than indicating different species of Mycobacterium. While approximately half of the cases had only few organisms present, the veterinary practitioner using a Romanovsky stain such as DiffQuik on aspirated material might expect to obtain a rapid diagnosis in many cases. This would allow differentiation of the syndrome from neoplastic and other diseases of the skin and direct appropriate treatment at the initial presentation.     

Comparison of the recovery of Mycobacterium bovis isolates using the BACTEC MGIT 960 system, BACTEC 460 system, and Middlebrook 7H10 and 7H11 solid media.

The BACTEC Microbacteria Growth Indicator Tube (MGIT) 960 system was evaluated to determine how it compares with the BACTEC 460 radiometric system and solid media for recovery of Mycobacterium bovis from tissue samples. A total of 506 bovine lymph node samples were collected from abattoirs in the United States and Mexico between November 2003 and September 2004. Processed samples were inoculated into an MGIT 960 tube, BACTEC 460 vial, and Middlebrook 7H10 and Middlebrook 7H11 solid media. Ziehl-Neelsen slides were prepared to check for contaminants and confirm the presence of acid-fast positive bacilli. Samples containing acid-fast bacilli were confirmed as members of the Mycobacterium tuberculosis complex by a nucleic acid assay. Niacin and nitrate biochemical tests were used to distinguish M. bovis from M. tuberculosis isolates. Statistical analyses were performed to compare recovery rate, mean time to detection, contamination rates, as well as pair-wise comparisons in each category. The results showed that the MGIT 960 system had a higher recovery rate of M. bovis (122/129) than did the BACTEC 460 (102/129) and solid media system (96/129). The average time to detection was 15.8 days for the MGIT 960 system, 28.2 days for the BACTEC 460 system, and 43.4 days for solid media. Contamination rates were 6.9% for the MGIT 960 system, 3.4% for the BACTEC 460 system, and 21.7% for solid media. These results indicate the MGIT 960 system can be used as an alternative to the BACTEC 460 system for recovering M. bovis from tissue samples.     

Mycobacterium bovis isolated from a dusky langur with granulomas in the intestine

Tubercles were seen in the spleen of a male dusky langur (Presbytis obscurus) on laparotomy. Subsequently, tuberculous lesions in the intestine, lungs, and a hilar lymph node were observed on necropsy of the monkey. Histologic examinations of these tissues revealed granulomas, and acid-fast bacilli were observed within granulomas in replicate sections that were stained with auramine-O. An acid-fast organism was isolated and identified as Mycobacterium bovis. Guinea pigs and rabbits inoculated intraperitoneally with the organism developed granulomas in the lungs, liver, and spleen. Lesions did not develop in chickens inoculated with the culture.     

Subcutaneous atypical mycobacteriosis in captive tiger quolls (Dasyurus maculatus)

From July 1989 to October 1998, 9/37 (24%) adult captive tiger quolls (Dasyurus maculatus) were diagnosed with atypical mycobacterial infection involving the subcutis and skin. Females were more often affected than males (seven females, two males). Grossly, lesions presented as focal thickenings, plaques, and abscesses within the subcutis, often with fistulous tracts. The subcutis and skin overlying cervical and thoracic regions were the primary sites of infection. Cytology of subcutaneous impression smears from all nine affected tiger quolls revealed pyogranulomatous inflammation admixed with several acid-fast bacilli. Histologically, all tiger quolls had nodular to diffuse pyogranulomatous panniculitis and cellulitis. Small numbers of acid-fast bacilli were noted histologically in 7/9 (78%) animals. Skin cultures from seven tiger quolls were positive for one or more different Runyon group IV mycobacteria. The disease described in these tiger quolls is similar to subcutaneous atypical mycobacteriosis of humans and domestic animals.     

The histopathologic diagnosis of subclinical Johne's disease in North American bison (Bison bison)

The morphologic changes of subclinical Johne's disease in North American Bison (Bison bison) are characterized by microgranulomas composed of epithelioid macrophages and individual multinucleate giant cells of Langhans'-type occasionally containing individual cytoplasmic acid-fast bacilli compatible with Mycobacterium avium paratuberculosis. The microgranulomas are best visualized in the mesenteric lymph nodes of infected subclinical animals. Macrophages that can be confused with infection-associated epithelioid macrophages in the mesenteric lymph nodes are pigment-carrying cells from the intestinal tract. Mesenteric lymph node biopsy may be a useful diagnostic tool for detection of mild subclinical infection in individual ruminants from herds of unknown infection status. The biopsy may also be useful for Johne's disease surveillance during test-and-cull programs.     

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.     

Paratuberculosis in a mandrill (Papio sphinx).

A 2.5-year-old captive female mandrill (Papio sphinx) died following a protracted course of intermittent abdominal bloat, diarrhea, and severe weight loss. Necropsy revealed emaciation and marked gastrointestinal distention with gas and ingesta. Histologic evaluation revealed severe diffuse granulomatous enterocolitis and mesenteric lymphadenitis with massive numbers of 1-2-microm acid-fast bacilli within macrophages. Additionally, there was moderate to severe multifocal myocardial and vascular amyloidosis, moderate multifocal pyogranulomatous interstitial pneumonia with no acid-fast bacteria, and moderate multifocal glossal candidiasis. Samples of feces, ileum, and colon were positive for Mycobacterium avium subsp. paratuberculosis by radiometric culture and a polymerase chain reaction-amplified DNA probe specific for the insertion sequence IS900 of this organism.     

A Disease Resembling Paratuberculosis (Johne’S Disease) in Roe Deer (Capreolus Capreolus L.) an Aetiological and Patho-Anatomical Study

A disease in wild living roe deer (Capreolus capreolus L.) caused by acid-fast bacteria is described.The morphological and cultural properties of these bacteria agree closely with corresponding properties of Mycobact. johnei.Enlarged lymph nodes, especially mesenteric lymph nodes with greyish-yellow necroses, were the most prominent macroscopic lesions. No intestinal lesions were present.The histopathological picture of the lymph nodes resembled mostly lesions which can occur in paratuberculous sheep and goats. The epithelioid cells contained masses of acid-fast bacteria. Such rods were also demonstrated in the intestinal villi.The acid-fast bacteria could be isolated from the mesenteric lymph nodes, spleens, bone marrow, mammary gland and also from the lymph nodes of other organs.The organism did not produce tuberculosis in guinea pigs. Intravenous injections into hens and rabbits resulted in miliary nodules resembling those of tuberculosis in the livers and spleens of the hens, and in joint and tendon sheath lesions in the rabbits. Microscopically the lesions mostly resembled those in paratuberculosis.One of the strains which was inoculated intravenously into two calves caused no lesions. The results of the allergy tests and serological blood tests in one animal indicate that infection with Mycobact. johnei cannot be excluded.A goat which was inoculated in the same manner and with the same strain as the calves died after six weeks. Miliary epithelioid cell granulomas in the liver, spleen, kidneys, bone marrow of long bones and in the submucosa of the ileum, as well as patchy infiltration of epithelioid cells in, among others, the mesenteric lymph nodes were observed on microscopic examinations.By intravenous infection the disease could be reproduced in a roe deer (fawn) (Gapreolus capreolus L.). The animal died nine weeks after the infection, and during the last two days before death it had a profuse diarrhoea. Masses of short acid-fast bacteria in clumps were present in the faeces. Nor in the experimental animal did macroscopic intestinal lesions occur. Enlarged villi infiltrated with epithelioid cells containing acid-fast bacteria were demonstrated by histological examination.The acid-fast bacteria could be recovered, but only on the Taylor/ Finlayson medium, from all experimental animals except the guinea pigs. Concerning the experimental calves, acid-fast bacteria were recovered from only one of them and then nearly three years after the infection.The acid-fast bacteria did not reduce nitrate. They showed positive neutral red reactions and were sensitive to isoniazid in a concentration of 2.5 µg per ml medium after three weeks’ incubation.The possibility that the isolated acid-fast bacteria and the lesions caused by them might be avian tubercle bacilli and avian tuberculosis has been discussed by the author. He does not, however, find any relevant reason for such an assumption. The author considers that the bacteria and the lesions exhibit the greatest similarity to Mycobact. johnei and paratuberculosis (Johne’s disease).On account of the organism’s pathogenicity for hens and rabbits, and necroses in the course of the disease, the author suggests that these bacteria possibly constitute a variety of the classic bovine Mycobact. johnei, different from the pigmented and the Icelandic varieties.     

Disseminated Mycobacterium avium-intracellulare Infection in a Persian cat

This report describes a disseminated infection with mycobacteria of the avium/intracellulare complex in a Persian cat in the absence of cutaneous lesions. Postmortem examination revealed severe granulomatous inflammation with numerous intrahistiocytic acid-fast bacilli in multiple organs. A gastrointestinal port of entry seems likely, as the ileocaecal lymph node was most severely affected.     

Feline leprosy: two different clinical syndromes

Feline leprosy refers to a condition in which cats develop granulomas of the subcutis and skin in association with intracellular acid-fast bacilli that do not grow on routine laboratory media. In this study, the definition was extended to include cases not cultured, but in which the polymerase chain reaction (PCR) identified amplicons characteristic of mycobacteria. Tissue specimens from 13 such cases from eastern Australia were obtained between 1988 and 2000. This cohort of cats could be divided into two groups on the basis of the patients' age, histology of lesions, clinical course and the sequence of 16S rRNA PCR amplicons.One group consisted of four young cats (less than 4 years) which initially developed localised nodular disease affecting the limbs. Lesions progressed rapidly and sometimes ulcerated. Sparse to moderate numbers of acid-fast bacilli were identified using cytology and/or histology, typically in areas of caseous necrosis and surrounded by pyogranulomatous inflammation. Organisms did not stain with haematoxylin and ranged from 2 to 6 microm (usually 2 to 4 microm). Mycobacterium lepraemurium was diagnosed in two cases based on the sequence of a 446 bp fragment encompassing the V2 and V3 hypervariable regions of the 16S rRNA gene a different sequence was obtained from one additional case, while no PCR product could be obtained from the remaining case. The clinical course was considered aggressive, with a tendency towards local spread, recurrence following surgery and development of widespread lesions over several weeks. The cats resided in suburban or rural environments. A second group consisted of nine old cats (greater than 9 years) with generalised skin involvement, multibacillary histology and a slowly progressive clinical course. Seven cats initially had localised disease which subsequently became widespread, while two cats allegedly had generalised disease from the outset. Disease progression was protracted (compared to the first group of cats), typically taking months to years, and skin nodules did not ulcerate. Microscopically, lesions consisted of sheets of epithelioid cells containing large to enormous numbers of acid-fast bacilli 2 to 8 microm (mostly 4 to 6 microm) which stained also with haematoxylin. A single unique sequence spanning a 557 bp fragment of the 16S rRNA gene was identified in six of seven cases in which it was attempted. Formalin-fixed paraffin-embedded material was utilised by one laboratory, while fresh tissue was used in another. The same unique sequence was identified despite the use of different primers and PCR methodologies in the two laboratories. A very slow, pure growth of a mycobacteria species was observed on Lowenstein-Jensen medium (supplemented with iron) and semi-solid agar in one of three cases in which culture was attempted at a reference laboratory. Affected cats were domicile in rural or semi-rural environments. These infections could generally be cured using two or three of rifampicin (10-15 mg/kg once a day), clofazimine (25 to 50 mg once a day or 50 mg every other day) and clarithromycin (62.5 mg per cat every 12 h).These findings suggest that feline leprosy comprises two different clinical syndromes, one tending to occur in young cats and caused typically by M lepraemurium and another in old cats caused by a single novel mycobacterial species.