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First report of canine leprosy in Europe: molecular and clinical traits
  1. C. Dedola, DVM,
  2. R. Zobba, PhD,
  3. M. L. Pinna Parpaglia, DVM,
  4. B. Chessa, PhD,
  5. E. Antuofermo, DVM,
  6. M. Polinas, DVM,
  7. M. Pittau, DVM and
  8. A. Alberti, MSc, PhD
  1. Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Via Vienna 2, Sassari 07100, Italy
  1. E-mail for correspondence: alberti{at}uniss.it, carladedola{at}hotmail.com

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Canine leproid granuloma (CLG) is a mycobacterial cutaneous disease characterised by the presence of nodular skin lesions most commonly affecting the head and the dorsal aspect of the pinna (Malik and others 1998, Conceição and others 2011, Smits and others 2012). Affected dogs appear otherwise healthy, and in the majority of cases the lesions tend to heal spontaneously over the course of weeks to months (Malik and others 1998). CLG has a wide distribution and was first reported in Africa (Zimbabwe) in 1973. Since then, all published cases of CLG have originated in Australia, New Zealand, Brazil or the USA (Malik and others 1998, Foley and others 2002, Conceição and others 2011, Smits and others 2012). To date, no case of this disease has been reported in Europe.

The mycobacterium responsible for CLG has not yet been isolated, but molecular investigations based either on 500 bp of the 16S rRNA gene (Hughes and others 2000) or on the ITS1 region (Fyfe and others 2008) identified a mycobacterial species closely related to members of the Mycobacterium simiae clade in nodular skin lesions of dogs. This paper describes the clinical features of the first reported case of CLG in Europe (to our knowledge), and the molecular investigation of the aetiological agent.

A three-year-old female German shorthaired pointer was presented to the dermatology service of the Internal Medicine Department at the University of Sassari for the evaluation of a skin nodule affecting the dorsal area of the right pinna. The skin lesion was previously treated with a topical therapy containing 1 per cent gentamycin (Gentalyn cream; Schering Plough Spa, Milan, Italy) with no signs of improvement. The dog was housed with three other dogs and lived outside all year, received regular vaccination and treatment for parasites. The owner did not report any abnormalities regarding the general health and described an active dog that was performing perfectly during the hunting season. Also, the animal never travelled outside Sardinia/Italy.

On general physical examination the dog did not show any abnormalities. On dermatological examination a 2 cm alopecic, slightly ulcerated cutaneous nodule was detected on the dorsal aspect of the right pinna (Fig 1a). As differential diagnoses the following were considered: neoplasia (histiocytoma and mast cell tumours), cutaneous leishmaniasis, an infectious (fungi, bacteria) granulomatous lesion and canine sterile pyogranuloma syndrome. Skin scrapings and hair plucks collected at the periphery of the lesion were negative for parasites and for fungal spores or hyphae.

FIG 1:

Macroscopic and microscopic features. (a) Nodular ulcerated lesion on the dorsal aspect of the right pinna. (b) A high-power (40}) photomicrograph showing Ziehl–Neelsen-positive rod-shaped bacteria (arrow) within the cytoplasm of macrophages

Cytological samples were obtained by fine needle aspiration of the skin nodule and stained with a modified Wright/Giemsa stain (Dip Quick Stain Kit; Dyaset S.r.l., Portomaggiore, Italy). Microscopic examination revealed the presence of inflammatory cells composed mainly of macrophages, few degenerated neutrophils and small lymphocytes. The macrophages showed phagocytised, negatively staining, rod-shaped bacteria. Thus, a mycobacterial infection was suspected and the nodule was completely excised under sedation with medetomidine hydrochloride 0.05 mg/kg intravenously (Medetor; Virbac S.r.l., Milan, Italy) and local anaesthesia with 2 per cent lidocaine chloridrate (Lidocaina; Pfizer S.r.l., Latina, Italy). Part of the nodule was fixed in 10 per cent formalin for histopathological examination and the remaining tissue was used for PCR. Histopathology confirmed the granulomatous nature of the lesion, and moderate numbers of rod-shaped bacteria were positive to Ziehl–Neelsen stain (Fig 1b). No pharmacological therapy was prescribed and no lesions recurred after a follow-up period of one year.

Total genomic DNA was extracted from fresh tissue cut from the excised nodule with the ‘DNeasy Blood and tissue Kit (Qiagen, Milan, Italy)’ according to vendor recommendations. To investigate the mycobacterial 16S-ITS1-23S regions, DNA samples were analysed with a PCR strategy developed by using a combination of primers available in the literature with an original primer. In a first PCR round primers 17F (5'-CATGCAAGTCGAACGGAAAG-3'; Davies and others 2006) and Mb23S.44n (5'-TCTCGATGCCAAGGCATCCACC-3'; Fyfe and others 2008) were used to amplify about 1800 bp of the genetic region comprising the partial 16S sequence (1478 bp), the full length ITS1 and a small nucleotide string of the 23S sequence. To facilitate cloning, the PCR product obtained in the first PCR was used as the DNA target in two heminested PCRs, by combining primer 17 F with 525R (5'-TTTCACGAACAACGCGACAA-3'; Davies and others 2006), and the original primer 16SF3 (5'-GGTTTGTCGCGTTGTTCGTG-3') with Mb23S.44n, respectively. PCR reactions were performed in 50 μl reactions containing 100–150 ng DNA, 200 μM dNTPs, 0.3 mM of the appropriate primers and 1.25 U of Taq DNA polymerase (Qiagen). PCR profiles were adapted to primers melting temperatures, length of the target sequence and vendor recommendations for Taq polymerase.

Products obtained from the initial PCR and from the two heminested PCRs were successfully cloned into pCR4-TOPO (Invitrogen, Milan, Italy) and automatically sequenced. Upon submission to standard nucleotide blast, the 1794 bp sequence obtained, named Mycobacterium species CLGS strain Italy (MspCLGS/Italy, genebank accession number KF297336), revealed 100 per cent homology (nucleotides 1–504) with the 16S ribosomal RNA sequence of Mycobacterium species Murphy (AF144747; Hughes and others 2000) and 100 per cent homology (nucleotides 1479–1661) with the ITS1 of Mycobacterium species Metcalfe (EF611177; Fyfe and others 2008).

MspCLGS/Italy 16S and ITS1 regions were aligned with the corresponding regions of 12 other mycobacteria representative of non-tuberculous, tuberculous and leprosy-causing mycobacteria in a single alignment with clustal X 2.0 (Larkin and others 2007). For phylogenetic and molecular evolutionary analyses alignment was edited with CLC Sequence Viewer 6.8 and imported into MEGA 5 (Tamura and others 2011). Maximum likelihood (ML)-based phylogenetic trees were obtained by using the HKY+I substitution model, identified as the best-suited evolutionary model for our data. Trees strength was evaluated by bootstrapping (1000 replicates).

In ML trees (Fig 2), MspCLGS/Italy grouped with other mycobacteria of the M simiae clade. Bootstrapped consensus ML tree was coherent with previous observations (Fyfe and others 2008).

FIG 2:

Molecular phylogenetic analysis by maximum likelihood method. Mycobacterium species CLGS strain Italy (MspCLGS/Italy) clusters together with other species of the Mycobacterium simiae clade. The tree with the highest log likelihood (−3933.0993) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches (1000 bootstrap replicates). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor–Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with superior log likelihood value. The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 25.0246 per cent sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 1619 positions in the final dataset

This is the first case of CLG syndrome described in Europe. It shows clinical features that overlap with what is reported in the literature. As commonly previously observed (Malik and others 1998, Foley and others 2002, Smits and others 2012) the patient was a short-coated hunting dog and the lesion was localised on the dorsal aspect of the ears. Furthermore, there was no recurrence of the lesion despite the incomplete excision of the nodule and the lack of topical and systemic pharmacological treatment after surgery. Other dogs living in close contact with the patient were accurately examined by one of the authors (CD), but no evidence of skin lesions was detected.

A recent study (Smits and others 2012) reports the first occurrence of multiple cases of CLG in short-coated hunting dogs (foxhounds) in New Zealand. Infection was present only in dogs active in the previous hunting season, but not in inactive dogs living in close contact. This supports a possible role for trauma correlated with hunting activity or alternatively the importance of the environment (presence of mycobacteria in the soil, plants, insects). Also, a genetic predisposition for the development of the disease has been suggested in foxhounds, together with boxers, which are breeds that are overrepresented and possibly predisposed to the development of this mycobacterial disease (Malik and others 1998).

Molecular phylogenetic analysis confirms a common causative agent for CLG throughout the world (Fig 2). Comparisons of the 16S and ITS1 regions obtained in our study from a single amplicon of about 1800 bp, indicate that Mycobacterium species Murphy and Metcalfe, together with the European strain described in this study, are closely related strains (no variability in the 16S and ITS1 regions was observed among strains). This is consistent with observations by Fyfe and others (2008). Our work provides a reliable tool for molecular diagnostic studies, as the 16S and ITS1 regions were obtained in a single amplicon of about 1800 bp and were not investigated separately as in previous studies.

In conclusion, our observations support the hypothesis that canine leprosy is associated with conspecific, worldwide distributed mycobacterial strains of the M simiae clade.

  • Accepted January 5, 2014.
View Abstract

Footnotes

  • Provenance not commissioned; externally peer reviewed

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