Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs
Background – Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions.
Clinical lesions – Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host.
Diagnosis – The most inexpensive, noninvasive means of diagnosis is by submission of methanol-fixed, Romanowsky-stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid-fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers.
Therapy – Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin.
Canine leproid granuloma (CLG)1–8 and the feline leprosy syndromes (FLS)9–13 are relatively uncommon nodular dermatoses caused by species of saprophytic mycobacteria that are extremely fastidious and generally uncultivable in the laboratory. Infection may be self-limiting, particularly in dogs with CLG,2 although it is impossible to predict which cases will have lesions that regress spontaneously. In addition, as differential diagnoses for cutaneous nodules include potentially life-threatening, progressive conditions (e.g. neoplasia and tuberculosis), it is critical to make a definitive diagnosis. These conditions can be diagnosed in general practice (especially with the assistance of a Mycobacterium Reference Laboratory), but definitive diagnosis may be challenging without a high degree of suspicion and experience with the respective entities.
Canine leproid granuloma (canine leprosy)
Canine leproid granuloma is a cutaneous or subcutaneous, typically self-limiting, nodular mycobacteriosis caused by a single, novel mycobacterium yet to be characterized fully. This condition was first described in Rhodesia.1 The causal organism is distributed worldwide and is especially common in Australia and Brazil. Cases have also been reported in the USA, particularly from the central valley of California (Figure S1).5
The route of inoculation is not known, but it is believed that biting flies, midges, mosquitoes or other arthropods may introduce mycobacteria into the patient from an environmental niche. Circumstantial evidence supporting this hypothesis includes the observation of potential vectors in the presence of affected dogs, lesions occurring at sites favoured by such vectors [e.g. the dorsal fold of the ears (Figure 1) and the head] and the prevalence of the condition in short-coated breeds and in dogs housed outdoors.1,2,5,6 Traumatic wounds (e.g. from sharp vegetation) subsequently contaminated with the CLG organism represent a potential, though less likely, route of infection. Occasionally, multiple animals in a kennel develop CLG, possibly due to a combination of seasonal factors and the presence of abundant mechanical vectors. Such case clusters have occurred in New Zealand, Australia (Victoria) and the USA (Georgia).8
Photograph courtesy of Richard Willis. (b) Early leproid granuloma lesion on the ear of a British bulldog. Photograph courtesy of Kim Barrett.
Signalment and clinical findings
The condition occurs almost exclusively in short-coated breeds, with boxer dogs and boxer crossbred dogs remarkably overrepresented. Staffordshire bull terriers, foxhounds and doberman pinchers are also commonly affected.2,5,8 The finding of characteristic lesions in typical locations and in a short-coated breed is strongly suggestive of CLG.
Canine leproid granuloma presents as single or multiple, firm, well-circumscribed nodules in the skin or subcutis (2-50 mm in diameter). Nodules typically are located on the head, particularly the dorsal fold of the ears, but may be located elsewhere, e.g. the trunk, rump or limbs. Nodules tend to occur on anatomical prominences. Larger lesions may ulcerate, generally late in the clinical course (probably as a result of effective cell-mediated immunity, rather than a mycobacterial toxin). Affected dogs are otherwise healthy, with no signs of systemic involvement. Extension of the infection to lymph nodes, nerves and internal organs does not occur. Lesions are typically painless but may be pruritic, particularly when secondary infection with Staphylococcus pseudintermedius occurs.
Haematology and serum biochemistry profiles demonstrate nonspecific changes and are not necessary to support a diagnosis. The differential diagnosis includes infectious, inflammatory and neoplastic diseases of the subcutis and skin, such as actinomycosis, nocardiosis, bacterial pseudomycetoma associated with S. pseudintermedius, cutaneous leishmaniosis, eumycotic mycetoma, phaeohyphomycosis, canine nodular dermatophytosis, cryptococcosis, parasitosis (e.g. neosporosis, cutaneous dirofilariosis), cutaneous foreign body, canine histiocytic disorders, canine sterile pyogranuloma syndrome or neoplasia (e.g. histiocytomas, basal cell tumours and mast cell tumours). Solitary lesions must be distinguished from mast cell tumours and other neoplasms that can have an aggressive clinical course.
When collecting samples, it is important first to wipe the skin surface twice or more with 70% ethanol, because environmental mycobacteria may be present on the epidermis and can cause erroneous results for both culture and PCR studies. When collecting samples for biopsy, some tissue should be preserved in 10% neutral buffered formalin, while fresh tissue should be stored in a sterile specimen jar at approximately 5°C to facilitate microbial culture, molecular studies, or both.
Mycobacteria appear as negatively stained bacilli (NSB) in routine cytology preparations because their lipid cell wall prevents penetration by Romanovsky-type stains (e.g. Diff-Quik® Lab Aids, Sydney, Australia; Figure 2b). Mycobacterial rods can be positively stained using a modified acid-fast procedure, such as the Ziehl-Neelsen (ZN) stain, where the bacilli take up the carbol fuchsin to appear pink, resulting in the name of acid-fast bacilli (AFB; Figure 2a). In smears, CLG is characterized by the presence of numerous, often spindle-shaped, macrophages, variable numbers of lymphocytes and plasma cells, lower numbers of neutrophils and variable numbers of medium-length AFB, either intracellularly (within macrophages or giant cells) or extracellularly.3 While some reports suggest that AFB are easy to locate in lesions, organisms can be extremely difficult to find in some instances, depending on the stage of the infection. Cytological preparations negative for AFB do not rule out CLG and should prompt examination of histological sections or submission of material for PCR. Lesions with scant AFB must be distinguished carefully from sterile granulomatous disease, for which corticosteroids are indicated, because these drugs have the potential to exacerbate CLG lesions.
Photomicrographs courtesy of George Reppas. In (b), note the intracellular negative staining bacilli; the length of these is highly variable in Diff-Quik®-stained smears.
Lesions within the dermis and subcutis consist of pyogranulomas composed of epithelioid macrophages, Langerhans-type giant cells with scattered neutrophils, plasma cells and small lymphocytes. The number and morphology of AFB in ZN-stained tissue sections is highly variable. Bacteria range from long, slender filaments in parallel sheaves to short, variably beaded bacilli or highly beaded to coccoid forms. Morphology may be more uniform in Diff-Quik®-stained smears than in fixed tissue sections. Sections may require a lengthy search, even by experienced pathologists, to locate foci in which AFB are evident.3
Culture of the CLG organism in vitro is not possible because its growth requirements have not been determined. The inability to culture this mycobacterial species has thus far prevented the full gamut of chemotaxonomy-based analyses and next-generation sequencing of the whole mycobacterial genome. Experimental transmission studies have not been attempted to date. Poorly collected specimens may be contaminated by rapidly growing mycobacteria; culture of such organisms on routine media can confound the diagnosis. Other organisms may be cultured, particularly S. pseudintermedius, which represents secondary infection of the lesions.
Polymerase chain reaction
Despite the organism associated with CLG being uncultivable, molecular studies have identified a mycobacterial species closely resembling members of the Mycobacterium simiae-related group, which contains many slow-growing saprophytic species.2,4 A definitive diagnosis is made by amplifying regions of the bacterial 16S rRNA gene,4 16S-23S internal transcribed spacer region or a region within the hsp65 gene,9 using mycobacterium-specific primers. Species identification is made via nucleotide sequence analysis of the amplified DNA fragment and comparison with known sequences in the GenBank database. Although PCR testing is more sensitive when performed on DNA extracted from fresh tissue, published PCR protocols are generally successful even when used on formalin-fixed, paraffin-embedded tissue.4–6 False-negative results may occur, however, due to DNA degradation secondary to fixation, especially when contact time with formalin is over 48 h. Recently, one of the authors (J.F.) has developed a real-time PCR (qPCR) for the CLG organism that is sensitive, specific and improves the availability and accuracy of molecular diagnostics (Janet Fyfe, unpublished data). There is sufficient mycobacterial DNA in methanol-fixed slides stained with Diff-Quik® for this qPCR to be applied to material scraped from glass microscope slides and thus to arrive at a molecular diagnosis noninvasively.
Feline leprosy syndromes
Feline leprosy refers to a group of cutaneous or subcutaneous granulomas caused by mycobacteria that generally do not grow on routine laboratory media.9–13 Cases occur worldwide, although certain mycobacterial species have strong geographical predilections. Critically, FLS lesions cannot be distinguished clinically or microscopically from lesions caused by tuberculosis bacteria (Mycobacterium bovis and Mycobacterium microti) and members of Mycobacterium avium complex (MAC).14–17 Historically, FLS was sometimes included under the umbrella term ‘cutaneous atypical mycobacteriosis’; however, this terminology is confusing and now redundant.
The location of lesions on cats suggests inoculation of organisms through insect bites, rodent bites or (most probably) fight wounds. Fight wounds are ascribed to cats, but may include injuries from prey species, such as rats or possums. Mycobacterial species associated with feline leprosy include Mycobacterium lepraemurium (Figure 3),18,19 Mycobacterium visibile,11 Mycobacterium sp. strain Tarwin (Figure 4)9 and a novel species found in New Zealand and the East coast of Australia.12 Recent work suggests that this novel East coast species is genetically heterogeneous (Janet Fyfe and Carolyn O’Brien, unpublished data), similar to what was suggested for Mycobacterium visibile. Mycobacterium lepraemurium infections have been reported from the UK, the Netherlands, France, Greece, Australia, New Zealand the USA (including the island of Hawaii).18,19
Photograph courtesy of Caroline Laprie.