Canine Leproid Granuloma Syndrome Research Articles

PCR-based identification of Mycobacterium murphy causing Canine Leproid Granuloma Syndrome in Niterói, southeast Brazil - case report

ABSTRACT Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.

Micobacteriose cutânea atípica felina autóctone no município do Rio de Janeiro-Brasil

Background : : : Cutaneous mycobacterioses show a worldwide distribution and manifest as three forms: atypical mycobacteriosis, feline leprosy and cutaneous tuberculosis. Atypical cutaneous mycobacteriosis in cats is a rare disease commonly caused by fast-growing mycobacteria belonging group IV of the Runyon classification. This report describes for the first time an autochthonous case of atypical cutaneous mycobacteriosis in a cat from the municipality of Rio de Janeiro. Case : A castrated female mongrel cat presented multiple ulcerated, exudative and alopecic lesions in different regions of the body. After sedation of the animal with 1% acepromazine hydrochloride (0.1 mg/kg) and 10% ketamine hydrochloride (10 mg/kg), clinical examination was performed and an exudate was collected from the ulcerated skin lesion for cytopathological analysis and mycological culture. The slide containing the lesion impression was stained by a rapid panoptic method. For fungal culture, material was collected with a sterile swab, seeded onto Sabouraud dextrose agar containing chloramphenicol and mycobiotic agar, and incubated at 25oC. After antisepsis and asepsis, local anesthesia was performed with 2% lidocaine hydrochloride without vasoconstrictor and a 4 mm punch biopsy was collected from the lesion, fixed in 10% buffered formalin and sent for histopathological analysis. No fungal structures were detected by cytopathological or mycological analysis. Histopathological examination revealed ulcerated skin and the presence of a marked and diffuse pyogranulomatous infiltrate in the superficial and deep dermis, which also involving the subcutaneous tissue and underlying muscle tissue. Grocott’s or PAS staining did not identify fungal structures. The Wade method was positive for acid-fast bacteria suggestive of ycobacterium . Analysis of the hematological and biochemical profiles revealed no noteworthy alterations. After the result of the histopathological exam, exudate material and lesion fragments were collected for bacteriological examination. The clinical specimens were processed and the presence of acid-fast bacteria was confirmed by Ziehl-Neelsen staining. Mycobacterium fortuitum fortuitum was identified based on the Runyon classification and biochemical tests, and was confirmed by molecular tests. Oral administration of enrofloxacin (5 mg/kg) at 24 h intervals was prescribed for 30 days. On the control visit, involution of the skin lesions was observed by physical examination and was also reported by the owner. After 4 months of irregular treatment due to the difficulty in administering the medication because of the cat’s aggressiveness, the animal started to present respiratory difficulties, rapid weight loss, apathy and died. Discussion : The occurrence of atypical mycobacteriosis is rare in Brazil, with isolated reports of mycobacteriosis such as canine leproid granuloma syndrome in Sao Paulo, canine cutaneous mycobacteriosis in Rio Grande do Sul, one case of feline leprosy in Sao Paulo, and one case of feline atypical cutaneous mycobacteriosis in Sao Paulo. The diagnosis and treatment of mycobacterial infections are frequently neglected because they are not included in the differential diagnosis of diverse cutaneous infections. In view of the scarcity of reports and to alert veterinarians that the disease should be included in the differential diagnosis with other feline dermatopathies, this report described a case of atypical feline cutaneous mycobacteriosis that occurred in the state of Rio de Janeiro. In contrast to the previous case report in Sao Paulo, in which was diagnosed the species Mycobacterium fortuitum biovar peregrinum , the species identified here was Mycobacterium fortuitum biovar fortuitum .

Confirmation of canine leproid granuloma syndrome in New Zealand

Canine leproid granuloma syndrome (CLGS) has not been officially reported in New Zealand. The seminal report describing this syndrome is in the Australian Veterinary Journal, 1998, where the results of a questionnaire circulated amongst veterinary pathologists and practitioners in Australia were reported. It included one response of a case seen in New Zealand, but no details of that case were given, despite CLGS being described in the literature as “common in New Zealand”. By injudicious use of references, the international literature has propagated the idea that the condition, including molecular identification, was confirmed in New Zealand, yet none of the articles cited actually confirmed that. An outbreak of skin granulomas in a group of approximately 35 working dogs was investigated, in which skin samples were sent to the Mycobacterium reference laboratory, Victoria, Australia, for PCR testing and molecular characterisation. Results of the clinical presentation, histological features and molecular studies conformed to the published details of CLGS. In particular, the nucleotide sequence of the internal transcribed spacer region, amplified from the mycobacterial DNA present in the clinical specimen provided, was identical to GenBank® Accession Number EF611177. That sequence is representative of the causative agent of CLGS in cases from Australia, the United States of America and Brazil. Although acid-fast organisms are occasionally seen in skin granulomas in dogs in New Zealand, this is the first confirmed identification of CLGS in this country. This is also the first report of an outbreak situation amongst a group of dogs.

Cutaneous sterile granulomas/pyogranulomas, leishmaniasis and mycobacterial infections

Cutaneous "sterile" granulomas represent a group of uncommon skin disorders of unknown aetiopathogenesis. Many diseases are included in this group (for example, sterile granuloma/pyogranuloma syndrome and reactive histiocytosis). The definition of sterile is based on the exclusion of other possible aetiological agents (for example, microorganisms or foreign body). Many techniques are used to rule out a microbial aetiology including cytology, histology, immunohistochemistry and culture. However, some organisms are "fastidious" and difficult to culture or to identify with routine methods, and molecular studies are necessary. This is particularly true for mycobacteria (for example, canine leproid granuloma syndrome) and Leishmania. Recently, studies in human and veterinary medicine have proved the presence of microorganisms (mycobacteria and Leishmania) using a polymerase chain reaction technique in specimens previously diagnosed as sterile. Therefore, it is very important, with the development of new technologies, to use a multidisciplinary diagnostic approach to definitively rule out any microorganism before declaring a disease sterile.

A “down under” lesion on the muzzle of a dog

A 10-year-old, castrated, male Labrador Retriever was presented to a local veterinary practice for investigation of a firm, deeply pigmented, alopecic, subcutaneous mass (8 mm in diameter) on the left side of the muzzle. A fine-needle aspirate of the mass was submitted for cytologic evaluation to the University of Florida. Microscopically, the preparation contained a predominant population of histiocytes that contained variable numbers of intracytoplasmic, negative-staining, filamentous structures consistent with Mycobacterium sp. A presumptive diagnosis of canine leproid granuloma syndrome was based on the cytologic findings and location of the lesion. Acid-fast staining revealed bright pink, acid-fast organisms within the histiocytic cells, supporting the diagnosis. The bacteria were not detected in histopathologic sections or by a polymerase chain reaction (PCR) test 1 week later, however, possibly because of spontaneous remission. Canine leproid granuloma syndrome is a common disease in Australia, but is uncommon in dogs in North America. It is caused by a novel, unnamed Mycobacterium species and usually affects the skin and subcutaneous tissues of the head and ears. A diagnosis usually can be made in Wright's-Giemsa and acid-fast-stained cytologic specimens; however, definitive diagnosis requires PCR testing at a specialized laboratory.

Leproid granulomas: a unique mycobacterial infection of dogs

In 1973, a Rhodesian veterinarian, Richard Smith, documented a mycobacterial skin infection in a Doberman and a Rhodesian Ridgeback dog that was characterised by variably sized subcutaneous nodules. It was thought at first that these dogs may have had tuberculosis and, because of public health considerations, they were euthanased and subjected to postmortem examination. There was no internal organ involvement at necropsy and, although acid-fast bacilli (AFB) were abundant in the lesions, culture was negative for the tubercle bacillus. Similar cases were recorded soon thereafter in an Australian veterinary discussion forum, although the infection was not subjected to systematic investigation until the end of the 20th century. The disease was given the name ?canine leprosy? by some veterinary dermatologists, but the alternative name canine leproid granuloma syndrome (CLGS) was later suggested by an Australian veterinarian David Watson. It is now clear that leproid granulomas are not uncommon and indeed are by far the most frequently encountered mycobacterial disease of dogs in Australia.

Identification by 16S rRNA gene analyses of a potential novel mycobacterial species as an etiological agent of canine leproid granuloma syndrome.

PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separate institutions. PCR products with identical sequences over a 350-bp region encompassing variable regions 2 and 3 of the 16S rRNA gene were obtained from 4 of 37 paraffin-embedded specimens and from all 9 specimens of fresh tissue originating from 12 of the 43 dogs. Identical sequences were determined from amplicons obtained from paraffin-embedded and fresh specimens from one dog. The consensus DNA sequence, amplified from paraffin-embedded tissue and represented by GenBank accession no. AF144747, shared highest nucleotide identity (99.4% over 519 bp) with mycobacterial strain IWGMT 90413 but did not correspond exactly to any EMBL or GenBank database sequence. With a probe derived from the V2 region of the novel canine sequence, reverse cross blot hybridization identified an additional four paraffin-embedded specimens containing the same novel sequence. In total, molecular methodologies identified the proposed novel mycobacterial sequence in 16 of 43 dogs with canine leproid granuloma syndrome, indicating that the species represented by this sequence may be the principal etiological agent of canine leproid granuloma syndrome.

Cytology and histopathology of canine leproid granuloma syndrome.

To describe the histopathology of canine leproid granuloma syndrome. 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. 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. 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. 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.

Mycobacterial Dermatitis

Mycobacterial skin disease in cats associated with atypical mycobacteria is an uncommon disease in small animal practice, and the disease is rarely encountered in dogs. A mycobacterial etiology should be considered in cases of chronic nodular dermatitis, draining tracts, and panniculitis. Cats and dogs affected with atypical mycobacterial infections are usually otherwise healthy, and systemic illness is unusual. In most cases, a diagnosis is made based on histopathological findings and growth of a causative organism. Group IV atypical mycobacteria can usually be grown following submission of affected tissue. Treatment should be based on antibiotic sensitivity test results. Treatment is prolonged and is unsuccessful in many cases. In cases of feline leprosy and canine leproid granuloma syndrome, organisms are difficult if not impossible to grow, and clinical and histopathological findings should be used to make a diagnosis. Spontaneous resolution of disease has been reported in atypical mycobacteriosis, feline leprosy, and canine leproid granuloma syndrome.

Canine leproid granuloma syndrome ‐call for clinical material

Canine leproid granuloma syndrome ‐call for clinical material

Mycobacterial nodular granulomas affecting the subcutis and skin of dogs (canine leproid granuloma syndrome).

To obtain a better understanding of a disease affecting dogs in which nodular mycobacterial granulomas are present in the subcutis or skin. Retrospective survey A diagnosis of an unidentified mycobacterial infection was made in 45 dogs following detection of acid-fast bacilli surrounded by granulomatous or pyogranulomatous inflammation in tissue sections. Eight cases were identified from the records of our diagnostic laboratory. In all cases culture for mycobacteria was unsuccessful. Another 37 cases were identified by practitioners and veterinary pathologists in response to a survey mailed to veterinary clinics throughout Australia. Data from these 45 cases were entered into a data base. The data set was incomplete, as some respondents did not answer all questions. Over 90% of affected dogs had short coats and about half were Boxers or Boxer-cross dogs. A weak seasonal trend for the development of primary lesions in autumn and winter was identified. Cases were recorded from New South Wales (35 dogs), Western Australia (4), Queensland (4), Tasmania (1) and New Zealand (1). The subcutis and skin of the ears and head (including the ears) were involved in 64% and 85% of cases, respectively, for which lesion site was recorded. Culture was attempted but was unsuccessful in 19 cases, including 11 cases where material was submitted to our laboratory and/or a Mycobacteria Reference Laboratory. Lymph node enlargement, internal organ involvement or constitutional signs were not a feature of the syndrome, and most lesions did not worry the dogs. Although many practitioners recorded a favourable response to therapy with doxycycline (response rate 57%) or amoxycillin-clavulanate (63%), spontaneous resolution of infection was thought to have occurred in six of seven dogs (86%) not given systemic antimicrobials, and three dogs where antimicrobials had failed previously. A minority of cases failed to respond to antimicrobial therapy and continued to have chronic lesions. There was no discernible trend for dogs of a particular age or sex to be affected. This syndrome is caused by saprophytic mycobacteria of limited pathogenicity that give rise to lesions restricted principally but not exclusively to the subcutis and skin of body extremities. Fastidious growth requirements have prevented their isolation on synthetic media used for culture of mycobacteria. Organisms presumably enter the subcutis following a breach in integrity of the epidermal barrier and produce self-limiting disease in immunocompetent dogs. Lesions tend to resolve spontaneously. The possibility of a public health threat from affected dogs is highly unlikely.