Introduction

Canine visceral leishmaniasis is usually a chronic disease, which can cause hyperthermia, progressive weight loss and cachexia. Several authors report the presence of myositis and muscular atrophy in infected dogs, suggesting that visceral leishmaniasis may be included as one of the causes for inflammatory myopathy in dogs. However, little is known about the pathogenesis of this disease in the muscular system, often being linked only to its catabolic nature. The immunohistochemical technique has been used for the diagnosis of visceral leishmaniasis and some authors report the presence of amastigote forms of Leishmania sp. in skeletal muscles of infected dogs. Therefore, the objective of this study was to search for the presence of amastigote forms of Leishmania sp, T-lymphocytes (CD3+), macrophages and IgG in the striated skeletal muscle of dogs through immunohistochemistry, in an attempt to obtain a better understanding of the disease's physiopathogenesis in the muscular system.

Materials and Methods

Biopsies of 92 muscles (triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius) of 23 dogs naturally infected by Leishmania chagasi were performed. In all animals, an electromyography of muscles contralateral to the ones submitted to biopsy were performed and, based on electromyographic results, the dogs were divided into two groups: The first was composed of eight dogs that did not show electromyographic signs that could indicate polymyopathy or that showed alterations in only one muscle of the thoracic or pelvic member, and the second group was composed of 15 dogs with polymyopathy signs. For the immunohistochemical technique, the StreptavidineBiotine-Peroxidase's indirect method was employed, using anti-CD3+, anti-Leishmania sp., anti-macrophage and anti-IgG antibodies, in 1:120, 1:1000, 1:600 and 1:2400 dilutions, respectively. The substrate applied was the chromogen 3.3'-diaminobenzidine (DAB) and the counterstaining was performed with Harris's Hematoxylin. The comparison between groups for Leishmania sp., T-lymphocytes (CD3+), macrophages and IgG search was performed using the non-parametrical Mann-Whitney test, at a significance level of 5%.

Results

Among the 92 assessed muscles, only 11 (12%) of them allowed the observation of an antigenic marking for amastigote forms of Leishmania sp, with all of them belonging to the group of animals with chronic lesions and electromyographic and histopathological evidences of polymyopathy. The immune marking for Leishmania sp. were verified especially in degenerative, necrotic areas and/or with a mononuclear inflammatory infiltrate in the endomysial region, varying regarding the intensity. In perimysial region, rare immune markings for Leishmania sp. were found, being absent in perivascular region. T-lymphocytes (CD3+) were identified in 35 (381%) muscles assessed, from which 27 (77.1%) belonged to the group of animals with electromyographic signs of polymyopathy and eight of them (22.8%), without polymyopathy. The marked cells were concentrated in areas with an inflammatory mononuclear infiltrate and/or with the presence of degeneration or necrosis of myofibers, with focal and multifocal distribution in the endomysial region. For macrophages, the antigenic marking was identified in 29 (31.5%) muscles assessed, from which 24 (82.7%) belonged to the group of animals with polymyopathy. The majority of macrophages were spread across the endomysial region, in areas with inflammatory infiltrates and/or degeneration and necrosis; and with a smaller frequency in the perimysial and perivascular region. Out of the 92 muscles assessed, only 14 (12%) had an immune marking for IgG, with 13 muscles belonging to dogs from the group with electromyographic evidences of polymyopathy and with chronic evolution. IgG was spread in endomysial region, without a preferred area, as observed in other immune markings. Statistically significant differences were verified between the muscles pertaining to the group of dogs without and with polymyopathy, regarding Leishmania sp., macrophages and IgG immune markings.

Discussion and Conclusions

In the present study, amastigote forms of Leishmania sp. were observed in the musculature of four dogs (17.4%), diverging from Paciello et al.'s (2008) reports, in which parasites were identified through immunohistochemistry in muscles of 13 (86%) from 15 animals assessed. However, the authors evaluated only infected dogs which showed an evident clinical picture of weakness and muscular atrophy. In regard to T-lymphocytes (CD3+), an immune marking was observed in 38.10% (35/92) of the muscles and in 73.91% (17/23) of the studied dogs. As the immune marking occurred for T-lymphocytes (CD3+) and macrophages in the musculature, it was expected that the animals that showed evidence of the presence of those cells would no longer display the parasite in their muscular tissue, as observed by Marcondes (2008) upon evaluation of the central nervous system of dogs affected by visceral leishmaniasis. The antigenic marking for macrophages was more intense in muscular fragments with the presence of amastigote forms of Leishmania sp. and T lymphocytes (CD3+), probably due to the fact that those cells are the most infected by the parasite, in addition to acting as antigen presenters for T-lymphocytes and still, whenever activated, they aid in the parasite elimination, as described by Engwerda et al. (2004). Out of the 92 skeletal muscles assessed, only 14 (12%) had an antigenic marking for IgG, diverging from the findings of Vamvakidis et al (2000), who observed an immune reactivity in all muscular fragments of 24 dogs with visceral leishmaniasis, both with and without muscular atrophy. According to the authors, the cause for myopathy in infected animals can be the parasite's direct action, an immune mediated mechanism or the formation of circulating antibodies against the parasites and the myofibers, with the deposit of immune complexes in the skeletal muscle. In this research, it was not possible to correlate the presence of IgG with the identification of amastigote forms of the parasite, since only four muscle fragments displayed two immune markings, whereas in other ten dogs IgG was identified without the presence of leishmaniae. These observations suggest that there has happened antibody formation both against the parasite and the myofibrils, confirming the reports of Vamvakidis et al. (2000). Five dogs did not show immune reactivity in any muscle fragment, contradicting the observations of Vamvakidis et al. (2000) and Paciello et al. (2008), who identified immune markings in all muscles of dogs with visceral leishmaniasis, in which histopathological alterations were observed. In regard to antigenic marking for Leishmania sp., macrophages and IgGs, there has been a difference between dogs with general muscular involvement and animals without electromyographic signs of polymyopathy, differently from the observed for CD3+ (p = 0. 0633), which did not show a statistically significant difference among groups. However, it was observed that dogs with polymyopathy showed a number slightly higher of those cells. Proportionally, the amount of T-lymphocytes (CD3+) in muscles was much higher than IgG, suggesting a more intense participation of the cellular immune mechanisms against the parasite, a fact also observed by Torres-Neto et al. (2008), when evaluating the cellular immune and humoral response in skin lesions of dogs with visceral leishmaniasis.

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