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Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog
Revue vétérinaire clinique (2015) 50, 59—63
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CLINICAL CASE
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog夽 Infection mixte à Dirofilaria repens et Leishmania infantum chez un chien L. Cornegliani a, A. Di Cesare b, G. Aste b, D. Traversa b,∗, E. Di Giulio c, D. Santori b, A. Vercelli a, F. Pampurini d, A. Boari b,∗ a
Associate veterinary practice, Turin, Italy Faculty of veterinary medicine, university of Teramo, Italy c ‘‘Ambulatorio Veterinario Associato’’ veterinary practice, Turin, Italy d Bayer Animal Health, Milan, Italy b
Rec ¸u le 13 juin 2014 ; accepté le 18 f´ evrier 2015 Disponible sur Internet le 24 mars 2015
KEYWORDS Dirofilaria repens; Leishmania infantum; Dog; Nodular dermatitis
Summary The subject. — Dirofilaria repens and Leishmania infantum are agents of skin diseases in dogs. While features of cutaneous lesions in canine leishmaniosis are well-known, the role of D. repens in inducing dermatopathies in infected animals is still unclear. Both parasites are gaining attention, given that their geographic distribution is expanding in different European areas. Topicality and strong points. — The present report describes a case of simultaneous infection by L. infantum and D. repens in a dog with skin lesions. A 6-year-old dog living in Central Italy was referred for a dermatological examination, which showed an erosive-ulcerative nodular dermatitis with erythema on the inner part of left ear. The histological examination of the lesion revealed a Leishmania-induced granuloma. The skin lesion and the blood were also molecularly positive for D. repens. Circulating microfilariae were found with a value of 61 larvae/mL. Perspectives and projects. — The epidemiologic and sanitary importance of co-infections by D. repens and L. infantum and their possible role in causing canine dermatopathies in coinfected dogs is discussed. © 2015 AFVAC. Published by Elsevier Masson SAS. All rights reserved.
夽 Crédits de formation continue. - La lecture de cet article ouvre droit à 0,05 CFC. La déclaration de lecture, individuelle et volontaire, est à effectuer auprès du CNVFCC (cf.sommaire). ∗ Corresponding authors. Adresses e-mail : [email protected] (D. Traversa), [email protected] (A. Boari). http://dx.doi.org/10.1016/j.anicom.2015.02.001 2214-5672/© 2015 AFVAC. Publié par Elsevier Masson SAS. Tous droits réservés.
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L. Cornegliani et al.
MOTS CLÉS Dirofilaria repens ; Leishmania infantum ; Chien ; Dermatite nodulaire
Résumé Le sujet. — Dirofilaria repens et Leishmania infantum sont des agents infectieux cutanés bien connus. Le rôle de D. repens comme inducteur d’affections cutanées reste mal connu, tandis que les lésions cutanées associées à la leishmaniose sont très bien décrites. Points d’intérêts et spécificité. — Un cas clinique de dermatite nodulaire granulomateuse chez un chien avec infection mixte à L. infantum et D. repens est décrit. Le chien âgé de 6 ans et vivant dans la partie centrale de l’Italie a été réferé en dermatologie pour des lésions cutanées nodulaires ulcératives et erythémateuses de la face interne du pavillon auriculaire gauche. L’examen histologique de la lésion a révélé une dermatite ulcérative granulomateuse causée par la leishmaniose. La lésion cutanée était positive pour L. infantum et D. repens. Par ailleurs, des microfilaires ont été mises en évidence dans la circulation sanguine (61 larvae/mL); Prospective et projet. — Le rôle épidémiologique et sanitaire de la présence simultanée de D. repens et L. infantum est ici discuté, ainsi que le rôle de ces agents parasitaires dans le développement des lésions observées. © 2015 AFVAC. Publié par Elsevier Masson SAS. Tous droits réservés.
Introduction Nodular to diffuse granulomatous vs pyogranulomatous skin lesions are often reported in dogs and represent a frequent clinical challenge. They are generally divided in four groups: • infections with detectable agents; • infections with no detectable agents; • not infective lesion with detectable aetiologic agents (e.g. foreign body); • and not infective lesion with an immune-mediated aetiology (e.g. sterile idiopathic dermatitis) [1].
The presence of different pathogens may easily induce pyogranulomatous skin lesions which can be subjected to histopathological examinations to allow a reliable diagnosis based on different assays, e.g. special stains or immunofluorescence [2,3]. Some parasitic agents, as Leishmania infantum, may not be detectable with special stains and, in these cases, PCR and immunohistochemistry are necessary for the diagnosis [1,4]. In more difficult cases, clinical examination and many different laboratory tools should be applied to achieve the diagnosis. The majority of skin lesions with a histological nodular to diffuse granulomatous vs pyogranulomatous infiltrate is mainly represented by papules, nodules, alopecia and erythema [5]. In such scenario, a clinical differentiation is difficult because similar lesions may be shared by different parasitic and non-parasitic diseases. A definitive and aetiological diagnosis is pivotal for a focused therapy in clinical settings and, for parasitic diseases, to interrupt the biological cycle of the parasite(s) involved, towards the control of the disease(s) in dogs and humans as well. Various ectoparasites (e.g. lice, ticks, fleas, mites), some nematodes (e.g. filariae, Ancylostoma caninum) and protozoa (e.g. L. infantum and Neospora caninum) may cause cutaneous lesions in dogs. Some of these parasites are changing their epidemiological distribution and posing new diagnostic challenges in clinical settings. This is particularly the case of the zoonotic vector-borne leishmaniosis and dirofilarioses, which have now an overlapping
geographic distribution and may occur in suitable hosts at the same time [6]. Canine leishmaniosis presents with a plethora of clinical signs, and skin lesions are among the most common. These include typical and atypical presentations, e.g. exfoliative dermatitis, diffuse, non-itching generalized or localized alopecia, dry seborrhoea with scales, cutaneous ulcers, papules and nodules [5,7]. Adult stages and/or circulating microfilariae (mff) of D. repens in dogs may induce subclinical infections or cutaneous signs of varying severity, such as (sub)cutaneous nodules, itching [8], and various allergic reactions [9,10]. Also, this nematode has a zoonotic potential, as the human infection is usually characterized with subcutaneous nodules, pruriginous urticarioid patches, transient swellings, eosinophilia, photophobia, conjunctival irritation and nodules or cysts in eye or in peri-ocular tissues [6]. The present report describes a case of simultaneous infection by both D. repens and L. infantum in a dog with a localized nodular granulomatous dermatitis.
Observations A 6-year-old neutered female mixed breed dog living in Central Italy was referred for a dermatological examination. The clinical history reported a past infection by D. repens two years before. Since then, the dog lived in a shelter located in an area endemic for canine filariae [11] and she was under treatment with allopurinol 10 mg/kg/bid for a leishmaniosis diagnosed 12 months before. The dog was in poor general health conditions (BCS = 3/9) but, at the dermatological examinations, she showed only an erosive-desquamative dermatitis with ulcers, hemorrhagic crusts and erythema on the inner part of left ear (Fig. 1). No other skin lesions were found at the clinical examination. Complementary hematological and dermatological exams were performed. Cell blood counts revealed a moderate neutrophilia, a mild lymphocytopenia and the presence of platelet aggregates. The blood biochemical
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog
Figure 1. Erosive-desquamative dermatitis with ulcers and erythema on the inner part of left ear of a dog co-infected by Leishmania infantum and Dirofilaria repens.
profile showed hypoalbuminemia, hyperglobulinemia, a reduced A/G ratio and notably increased values of blood urea nitrogen (BUN), creatinine, phosphorus and cholesterol, i.e. 268 mg/dL (range 15—45 mg/dL), 4.56 mg/dL (0.75—1.30 mg/dL), 7.7 mg/dL (range 2.5—4.7 mg/dL), 420 mg/dL (range 110—330), respectively. The urinalysis demonstrated a high level of proteinuria, an urine proteincreatinine ratio (P/C) > 3.4 (range 0—< 0.3) with an inactive sediment. The urine culture was negative for any infection while the dog was serologically positive for L. infantum (1:640). Multiple skin scrapings were negative for sarcoptic and demodectic mange, and coat brushing and flea comb were negative for Cheyletiella spp., ticks and fleas. Wood’s lamp examination and fungal culture were negative for dematophytosis, while a Malassezia spp. otitis was detected by cotton swab cytological examination stained with Diff-Quick (Bio-Optica® , Milan, Italy). Impressions smears from ulcers and crusts revealed an inflammatory infiltrate mainly represented by macrophages and neutrophils, red blood cells, keratinocytes and rare extra-cytoplasmic cocci. No L. infantum amastigotes or other pathogens were found in the examined samples. Given that the cytological examination of the skin lesions did not show any parasite or
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infective agent, a biopsy was performed to investigate the aetiology of the skin lesion. An 8 mm punch biopsy of the ear lesion was collected and divided in two aliquots, i.e. one was stored at —20 ◦ C pending molecular analysis (see below) and the other was fixed in 10 % neutral buffered formalin, routinely processed and paraffin embedded. The histological examination demonstrated that the epidermis was partially ulcerated with acanthosis and moderate spongiosis, while the superficial and middle dermis had a nodular to diffuse granulomatous infiltrate (Fig. 2). Numerous L. infantum amastigotes were detectable within macrophages (Fig. 2). No other pathogens were identified in the samples examined at the light microscopy and with the hematoxylin-eosin stain. A sample of whole blood was subjected to a qualitative modified Knott’s technique [12] as on the follow. One mL of blood was pooled with 9 ml of a 2 % formalin solution. The mixture was centrifuged at 1200 rpm for 5 minutes and the supernatant fluid was discarded. One drop of 0.1 % methylene blue was added to the sediment, mixed and the stained sediment was transferred to a microscope slide and observed under light microscopy (Zeiss® Axiocam MrC) at 10 ×, 40 × and 100 × magnifications. Microfilariae were identified on the basis of key morphometric (i.e. length and width) and morphological (i.e. head and tail) features [12,13]. All mff retrieved were identified as D. repens, with a level of microfilariaemia of 61 mff/mL. The remaining blood was stored at —20 ◦ C for further genetic examinations. The blood and the skin biopsy were subjected to PCRs for canine filariae as previously described [11] and scored positive for D. repens. The dog died ten days after the clinical examination due to a worsening of chronic renal failure and no further investigations were possible.
Discussion Although both L. infantum and D. repens are causative agents of dermatopathies in dogs, in this particular case
Figure 2. A. Histological examination showing a diffuse pyogranulomatous vs granulomatous inflammatory infiltrate from the superficial to deep dermis. HE stain, 10 ×. B. Magnification of the same section showing Leishmania spp. amastigotes within macrophages. HE stain 40 ×.
62 the clinical history and the diagnostic tests did not allow to identify the primary agent of the skin lesions at the ear of the examined animal. No D. repens mff were visible at the histological examination of the tissue sample from the lesion which scored positive for L. infantum. However the PCR-positivity of the skin sample for D. repens could suggest the presence or a past presence of the parasite in the lesion. In any case, it is hard to understand whether the PCR-amplified DNA of D. repens in the bioptical sample is derived from the skin, from the blood or from both of them. Nodular granulomatous vs pyogranulomatous dermatitis may be induced by infective agents and/or parasites, but the aetiological causes can be difficult to identify in skin samples [4,5,14—16]. Actually, L. infantum can be identified in skin lesions which were previously diagnosed as sterile and it is postulated that this parasite may elicit an inflammatory response and a secondary nodular dermatitis [1,5,16]. Infectious agents, present in small numbers or incomplete cleaning of antigens associated to them, may elicit a persisting inflammatory response [1,16]. In these cases, the detection of pathogens may be impossible by routine examinations. Additionally, it has been shown that skin samples positive upon PCR and immunohistochemistry may result negative for L. infantum amastigotes at the histological examination [1,4]. For the same reasons, it cannot be ruled out that a similar scenario may occur for other (sub)cutaneous parasites, e.g. D. repens, as in the present report. This can explain the lack of detection of mff in the microscopically examined biopsy which, however, resulted PCR-positive. On the other hand, regressed tissue damage induced by mff might have induced macrophages migration into the dermis. Given the presence of systemic leishmaniosis, it can be argued that macrophages with phagocytised amastigotes have reached the inflammatory tissue secondary to vessels migration [17]. In accordance with the present work, no other histological studies have proved the presence of D. repens mff in skin lesions, thus leaving the correlation between the presence of larvae in the skin and clinical signs only conjectural [18]. The only exception is represented by the finding of unidentified mff in a dog with a nodular lesion containing an adult Acanthocheilonema sp. [8]. A recent study has shown the absence of correlation between the inflammatory infiltrate in nodular lesions of subcutaneous dirofilariosis and the presence of the nematode [18]. The results of this examination are compatible with a granulomatous nodular dermatitis secondary to a systemic leishmaniosis due to the presence of macrophages with intracytoplasmatic amastigotes [16]. However, the role of D. repens in contributing in the aetiology of the skin lesion of this infected dog cannot be excluded and further studies are warranted to elucidate the role of the parasite in causing cutaneous lesions. The possibility that the nodular dermatitis was caused by the simultaneous infection by D. repens and L. infantum should be taken into a proper account. In fact, a recent study showed that cutaneous signs in dogs co-infected by L. infantum and D. repens may recover after the administration of a parasiticide with a nematocidal activity [14]. Thus, a possible role of leishmaniosis in promoting skin lesions due to D. repens in co-infected dogs is worth of further investigations.
L. Cornegliani et al. Under a practical standpoint, it is important to note that few information are available for the anthelmintic treatment of D. repens-induced or associated to dermatopathies. The off label administrations of melarsomine hydrochloride and of doramectin marketed for large animals have cleared mff in a single dog. Unfortunately, the clinical signs associated with D. repens were not evaluated [19]. The single administration of a spot-formulation containing moxidectin 2.5 % plus imidacloprid 10 % showed a high efficacy in both treating skin lesions and to eliminate D. repens microfilariaemia [14] in nine treated dogs with skin lesions compatible with subcutaneous dirofilariosis. The lesions disappeared in seven out of nine symptomatic animals, but the parasitic origin of the skin lesions in these dogs, although plausible, would have been ultimately corroborated by skin biopsies. The other two animals had a history of leishmaniosis, thus leading to the hypothesis that the lesions were probably (also) related to L. infantum [14]. The persistence of the dermatitis in these animals despite the administration of the antiparasitic treatment and the present report as well may suggest a primary role of L. infantum in causing skin lesions in co-infected dogs. Interestingly, most of the infected dogs with skin lesions had low levels of circulating mff, thus suggesting that presence and number of mff in the bloodstream could not mirror the occurrence and/or severity of dermatopathies [14]. The present results corroborate this hypothesis, given that this dog had a very low level of circulating mff. Hence, an individual susceptibility in developing skin diseases in subcutaneous dirofilariosis can be argued, as also previously indicated [8,9,14]. Unfortunately the dog died before a treatment for D. repens which would have provided more indications on the possible role of the nematode in causing the skin lesion, via an ex juventibus approach, as previously described [11]. In conclusion, the present report and other previous studies [10,14] suggest the hypothesis that D. repens may cause, other than nodules [18], different kinds of dermatopathies. Also, these findings represent a basis towards new studies to investigate more in depth the potential role that L. infantum may have as a predisposition for the onset of skin lesions in dogs infected by D. repens at the same time. In fact, studies have demonstrated that canine vector-borne diseases may nurture and reinforce the pathogenic potential of other pathogens [20]. The reasons for why some dogs are subclinically infected by D. repens and others present relevant skin lesions are also worth investigating. One of the possible causes can be a concomitant infection with another parasite causing skin alterations, i.e. L. infantum. It would be also interesting to prospectively investigate for D. repens leishmaniotic dogs living in certain areas, in order to assess a possible overlapping distribution of these diseases and the concomitant role they may have in symptomatic animals. It will be useful to understand the potential correlation between these two diseases and the severity of clinical signs. Veterinarians working in canine clinical medicine should consider both the possibilities that D. repens may cause various skin lesions, even in cases of co-infections with other parasites, e.g. L. infantum. This is crucial for the unequivocal diagnosis of both single and mixed infections, based on cytological and histological methods, which can be supported by further genetic examinations, and for effective
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog parasiticide treatments. The present expansion in the geographic distribution of vector-borne parasites [6] requires that practitioners are vigilant on these diseases for the risk of co-infections and for the confounding and overlapping clinical pictures they cause. Moreover, an effective treatment is pivotal also to interrupt the biological cycle of these parasites, that have a relevant zoonotic significance.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Références [1] Santoro D, Prisco M, Ciaramella P. Cutaneous sterile granulomas/pyogranulomas, leishmaniasis and mycobacterial infections. J Small An Pract 2008;49:552—61. [2] Roura X, Fondevila D, Sánchez A, Ferrer L. Detection of Leishmania infection in paraffin-embedded skin biopsies of dogs using polymerase chain reaction. J Vet Diagn Invest 1999;11:385—7. [3] Bonenberger TE, Ihrke PJ, Naydan DK, Affolter VK. Rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal BCG antibody. Vet Dermatol 2001;12:41—7. [4] Cornegliani L, Fondevila D, Vercelli A, Fondati A. PCR technique detection of Leishmania spp but not Mycobacterium spp. in canine cutaneous ‘‘sterile’’ pyogranuloma/granuloma syndrome. Vet Dermatol 2005;16:233—8. [5] Miller WJ, Griffin GE, Campbell KL. Miscellaneous skin diseases; viral, rickettsial, and protozoal skin diseases. In: Muller and Kirk’s small animal dermatology. 7th ed. Elsevier Inc; 2013. p. 343—62 [695—723]. [6] Otranto D, Dantas-Torres F, Brianti E, Traversa D, Petri´ c D, Genchi C, et al. Vector-borne helminths of dogs and humans in Europe. Parasit Vectors 2013;6:16. [7] Cavalcanti A, Lobo R, Cupolillo E, Bustamante F, Porrozzi R. Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: a case report. Parasitol Int 2012;61:738—40.
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[8] Hargis AM, Lewis TP, Duclos DD, Loeffler DG, Raush RL. Dermatitis associated with microfilariae (Filarioidea) in 10 dogs. Vet Dermatol 1999;10:95—107. [9] McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. Adv Parasitol 2008;66:193—285. [10] Rocconi F, Di Tommaso M, Traversa D, Palmieri C, Pampurini F, Boari A. Allergic dermatitis by Dirofilaria repens in a dog: clinical picture and treatment. Parasitol Res 2012;111:493—6. [11] Traversa D, Aste G, Milillo P, Capelli G, Pampurini F, Tunesi C, et al. Autochthonous foci of canine and feline infections by Dirofilaria immitis and Dirofilaria repens in central Italy. Vet Parasitol 2010;169:128—32. [12] Sloss MW, Kemp RL, Zajac AM. Veterinary clinical parasitology. 6th ed. Ames, IA: Iowa State University Press; 1994. p. 107—8. [13] Lindsey LR. Identification of canine microfilariae. J Am Vet Med Assoc 1965;146:1106—14. [14] Traversa D, Aste G, Di Cesare A, Paoletti B, Di Tommaso M, Di Giulio E, et al. Efficacy of a single administration of a spot-on solution containing imidacloprid 10 %/moxidectin 2.5 % in eliminating Dirofilaria repens microfilariae in naturally infected dogs. Vet Parasitol 2011;179:107—12. [15] Panich R, Scott DW, Miller WH. Canine cutaneous sterile pyogranuloma/granuloma syndrome: a retrospective analysis of 29 cases (1976—1988). J Am An Hosp Ass 1999;7:519—28. [16] Gross TL, Ihrke PJ, Walder EJ. Infectious nodular and diffuse granulomatous and pyogranulomatous diseases of the dermis. In: Veterinary dermatopathology: a macroscopic and microscopic evaluation of canine and feline skin diseases. St Louis, MO: Mosby Year Book; 2005. p. 169—71 [186—8,194—8]. [17] Dos-Santos WLC, Badarò JDR, De-Freitas LAR. Association between skin parasitism and granulomatous inflammatory patter in canine visceral leishmaniosis. Parasitol Res 2004;92:89—94. [18] Albanese F, Abramo F, Braglia C, Caporali C, Venco L, Vercelli A, et al. Nodular lesions due to infestation by Dirofilaria repens in dogs from Italy. Vet Dermatol 2013;24:255—6. [19] Baneth G, Volansky Z, Anug Y, Favia G, Bain O, Goldstein RE, et al. Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectin. Vet Parasitol 2002;105:173—8. [20] De Tommasi AS, Otranto D, Dantas-Torres F, Capelli G, Breitschwerdt EB, de Caprariis D. Are vector-borne pathogen co-infections complicating the clinical presentation in dogs? Parasit Vectors 2013;6:97.
Disponible en ligne sur
ScienceDirect www.sciencedirect.com
CLINICAL CASE
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog夽 Infection mixte à Dirofilaria repens et Leishmania infantum chez un chien L. Cornegliani a, A. Di Cesare b, G. Aste b, D. Traversa b,∗, E. Di Giulio c, D. Santori b, A. Vercelli a, F. Pampurini d, A. Boari b,∗ a
Associate veterinary practice, Turin, Italy Faculty of veterinary medicine, university of Teramo, Italy c ‘‘Ambulatorio Veterinario Associato’’ veterinary practice, Turin, Italy d Bayer Animal Health, Milan, Italy b
Rec ¸u le 13 juin 2014 ; accepté le 18 f´ evrier 2015 Disponible sur Internet le 24 mars 2015
KEYWORDS Dirofilaria repens; Leishmania infantum; Dog; Nodular dermatitis
Summary The subject. — Dirofilaria repens and Leishmania infantum are agents of skin diseases in dogs. While features of cutaneous lesions in canine leishmaniosis are well-known, the role of D. repens in inducing dermatopathies in infected animals is still unclear. Both parasites are gaining attention, given that their geographic distribution is expanding in different European areas. Topicality and strong points. — The present report describes a case of simultaneous infection by L. infantum and D. repens in a dog with skin lesions. A 6-year-old dog living in Central Italy was referred for a dermatological examination, which showed an erosive-ulcerative nodular dermatitis with erythema on the inner part of left ear. The histological examination of the lesion revealed a Leishmania-induced granuloma. The skin lesion and the blood were also molecularly positive for D. repens. Circulating microfilariae were found with a value of 61 larvae/mL. Perspectives and projects. — The epidemiologic and sanitary importance of co-infections by D. repens and L. infantum and their possible role in causing canine dermatopathies in coinfected dogs is discussed. © 2015 AFVAC. Published by Elsevier Masson SAS. All rights reserved.
夽 Crédits de formation continue. - La lecture de cet article ouvre droit à 0,05 CFC. La déclaration de lecture, individuelle et volontaire, est à effectuer auprès du CNVFCC (cf.sommaire). ∗ Corresponding authors. Adresses e-mail : [email protected] (D. Traversa), [email protected] (A. Boari). http://dx.doi.org/10.1016/j.anicom.2015.02.001 2214-5672/© 2015 AFVAC. Publié par Elsevier Masson SAS. Tous droits réservés.
60
L. Cornegliani et al.
MOTS CLÉS Dirofilaria repens ; Leishmania infantum ; Chien ; Dermatite nodulaire
Résumé Le sujet. — Dirofilaria repens et Leishmania infantum sont des agents infectieux cutanés bien connus. Le rôle de D. repens comme inducteur d’affections cutanées reste mal connu, tandis que les lésions cutanées associées à la leishmaniose sont très bien décrites. Points d’intérêts et spécificité. — Un cas clinique de dermatite nodulaire granulomateuse chez un chien avec infection mixte à L. infantum et D. repens est décrit. Le chien âgé de 6 ans et vivant dans la partie centrale de l’Italie a été réferé en dermatologie pour des lésions cutanées nodulaires ulcératives et erythémateuses de la face interne du pavillon auriculaire gauche. L’examen histologique de la lésion a révélé une dermatite ulcérative granulomateuse causée par la leishmaniose. La lésion cutanée était positive pour L. infantum et D. repens. Par ailleurs, des microfilaires ont été mises en évidence dans la circulation sanguine (61 larvae/mL); Prospective et projet. — Le rôle épidémiologique et sanitaire de la présence simultanée de D. repens et L. infantum est ici discuté, ainsi que le rôle de ces agents parasitaires dans le développement des lésions observées. © 2015 AFVAC. Publié par Elsevier Masson SAS. Tous droits réservés.
Introduction Nodular to diffuse granulomatous vs pyogranulomatous skin lesions are often reported in dogs and represent a frequent clinical challenge. They are generally divided in four groups: • infections with detectable agents; • infections with no detectable agents; • not infective lesion with detectable aetiologic agents (e.g. foreign body); • and not infective lesion with an immune-mediated aetiology (e.g. sterile idiopathic dermatitis) [1].
The presence of different pathogens may easily induce pyogranulomatous skin lesions which can be subjected to histopathological examinations to allow a reliable diagnosis based on different assays, e.g. special stains or immunofluorescence [2,3]. Some parasitic agents, as Leishmania infantum, may not be detectable with special stains and, in these cases, PCR and immunohistochemistry are necessary for the diagnosis [1,4]. In more difficult cases, clinical examination and many different laboratory tools should be applied to achieve the diagnosis. The majority of skin lesions with a histological nodular to diffuse granulomatous vs pyogranulomatous infiltrate is mainly represented by papules, nodules, alopecia and erythema [5]. In such scenario, a clinical differentiation is difficult because similar lesions may be shared by different parasitic and non-parasitic diseases. A definitive and aetiological diagnosis is pivotal for a focused therapy in clinical settings and, for parasitic diseases, to interrupt the biological cycle of the parasite(s) involved, towards the control of the disease(s) in dogs and humans as well. Various ectoparasites (e.g. lice, ticks, fleas, mites), some nematodes (e.g. filariae, Ancylostoma caninum) and protozoa (e.g. L. infantum and Neospora caninum) may cause cutaneous lesions in dogs. Some of these parasites are changing their epidemiological distribution and posing new diagnostic challenges in clinical settings. This is particularly the case of the zoonotic vector-borne leishmaniosis and dirofilarioses, which have now an overlapping
geographic distribution and may occur in suitable hosts at the same time [6]. Canine leishmaniosis presents with a plethora of clinical signs, and skin lesions are among the most common. These include typical and atypical presentations, e.g. exfoliative dermatitis, diffuse, non-itching generalized or localized alopecia, dry seborrhoea with scales, cutaneous ulcers, papules and nodules [5,7]. Adult stages and/or circulating microfilariae (mff) of D. repens in dogs may induce subclinical infections or cutaneous signs of varying severity, such as (sub)cutaneous nodules, itching [8], and various allergic reactions [9,10]. Also, this nematode has a zoonotic potential, as the human infection is usually characterized with subcutaneous nodules, pruriginous urticarioid patches, transient swellings, eosinophilia, photophobia, conjunctival irritation and nodules or cysts in eye or in peri-ocular tissues [6]. The present report describes a case of simultaneous infection by both D. repens and L. infantum in a dog with a localized nodular granulomatous dermatitis.
Observations A 6-year-old neutered female mixed breed dog living in Central Italy was referred for a dermatological examination. The clinical history reported a past infection by D. repens two years before. Since then, the dog lived in a shelter located in an area endemic for canine filariae [11] and she was under treatment with allopurinol 10 mg/kg/bid for a leishmaniosis diagnosed 12 months before. The dog was in poor general health conditions (BCS = 3/9) but, at the dermatological examinations, she showed only an erosive-desquamative dermatitis with ulcers, hemorrhagic crusts and erythema on the inner part of left ear (Fig. 1). No other skin lesions were found at the clinical examination. Complementary hematological and dermatological exams were performed. Cell blood counts revealed a moderate neutrophilia, a mild lymphocytopenia and the presence of platelet aggregates. The blood biochemical
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog
Figure 1. Erosive-desquamative dermatitis with ulcers and erythema on the inner part of left ear of a dog co-infected by Leishmania infantum and Dirofilaria repens.
profile showed hypoalbuminemia, hyperglobulinemia, a reduced A/G ratio and notably increased values of blood urea nitrogen (BUN), creatinine, phosphorus and cholesterol, i.e. 268 mg/dL (range 15—45 mg/dL), 4.56 mg/dL (0.75—1.30 mg/dL), 7.7 mg/dL (range 2.5—4.7 mg/dL), 420 mg/dL (range 110—330), respectively. The urinalysis demonstrated a high level of proteinuria, an urine proteincreatinine ratio (P/C) > 3.4 (range 0—< 0.3) with an inactive sediment. The urine culture was negative for any infection while the dog was serologically positive for L. infantum (1:640). Multiple skin scrapings were negative for sarcoptic and demodectic mange, and coat brushing and flea comb were negative for Cheyletiella spp., ticks and fleas. Wood’s lamp examination and fungal culture were negative for dematophytosis, while a Malassezia spp. otitis was detected by cotton swab cytological examination stained with Diff-Quick (Bio-Optica® , Milan, Italy). Impressions smears from ulcers and crusts revealed an inflammatory infiltrate mainly represented by macrophages and neutrophils, red blood cells, keratinocytes and rare extra-cytoplasmic cocci. No L. infantum amastigotes or other pathogens were found in the examined samples. Given that the cytological examination of the skin lesions did not show any parasite or
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infective agent, a biopsy was performed to investigate the aetiology of the skin lesion. An 8 mm punch biopsy of the ear lesion was collected and divided in two aliquots, i.e. one was stored at —20 ◦ C pending molecular analysis (see below) and the other was fixed in 10 % neutral buffered formalin, routinely processed and paraffin embedded. The histological examination demonstrated that the epidermis was partially ulcerated with acanthosis and moderate spongiosis, while the superficial and middle dermis had a nodular to diffuse granulomatous infiltrate (Fig. 2). Numerous L. infantum amastigotes were detectable within macrophages (Fig. 2). No other pathogens were identified in the samples examined at the light microscopy and with the hematoxylin-eosin stain. A sample of whole blood was subjected to a qualitative modified Knott’s technique [12] as on the follow. One mL of blood was pooled with 9 ml of a 2 % formalin solution. The mixture was centrifuged at 1200 rpm for 5 minutes and the supernatant fluid was discarded. One drop of 0.1 % methylene blue was added to the sediment, mixed and the stained sediment was transferred to a microscope slide and observed under light microscopy (Zeiss® Axiocam MrC) at 10 ×, 40 × and 100 × magnifications. Microfilariae were identified on the basis of key morphometric (i.e. length and width) and morphological (i.e. head and tail) features [12,13]. All mff retrieved were identified as D. repens, with a level of microfilariaemia of 61 mff/mL. The remaining blood was stored at —20 ◦ C for further genetic examinations. The blood and the skin biopsy were subjected to PCRs for canine filariae as previously described [11] and scored positive for D. repens. The dog died ten days after the clinical examination due to a worsening of chronic renal failure and no further investigations were possible.
Discussion Although both L. infantum and D. repens are causative agents of dermatopathies in dogs, in this particular case
Figure 2. A. Histological examination showing a diffuse pyogranulomatous vs granulomatous inflammatory infiltrate from the superficial to deep dermis. HE stain, 10 ×. B. Magnification of the same section showing Leishmania spp. amastigotes within macrophages. HE stain 40 ×.
62 the clinical history and the diagnostic tests did not allow to identify the primary agent of the skin lesions at the ear of the examined animal. No D. repens mff were visible at the histological examination of the tissue sample from the lesion which scored positive for L. infantum. However the PCR-positivity of the skin sample for D. repens could suggest the presence or a past presence of the parasite in the lesion. In any case, it is hard to understand whether the PCR-amplified DNA of D. repens in the bioptical sample is derived from the skin, from the blood or from both of them. Nodular granulomatous vs pyogranulomatous dermatitis may be induced by infective agents and/or parasites, but the aetiological causes can be difficult to identify in skin samples [4,5,14—16]. Actually, L. infantum can be identified in skin lesions which were previously diagnosed as sterile and it is postulated that this parasite may elicit an inflammatory response and a secondary nodular dermatitis [1,5,16]. Infectious agents, present in small numbers or incomplete cleaning of antigens associated to them, may elicit a persisting inflammatory response [1,16]. In these cases, the detection of pathogens may be impossible by routine examinations. Additionally, it has been shown that skin samples positive upon PCR and immunohistochemistry may result negative for L. infantum amastigotes at the histological examination [1,4]. For the same reasons, it cannot be ruled out that a similar scenario may occur for other (sub)cutaneous parasites, e.g. D. repens, as in the present report. This can explain the lack of detection of mff in the microscopically examined biopsy which, however, resulted PCR-positive. On the other hand, regressed tissue damage induced by mff might have induced macrophages migration into the dermis. Given the presence of systemic leishmaniosis, it can be argued that macrophages with phagocytised amastigotes have reached the inflammatory tissue secondary to vessels migration [17]. In accordance with the present work, no other histological studies have proved the presence of D. repens mff in skin lesions, thus leaving the correlation between the presence of larvae in the skin and clinical signs only conjectural [18]. The only exception is represented by the finding of unidentified mff in a dog with a nodular lesion containing an adult Acanthocheilonema sp. [8]. A recent study has shown the absence of correlation between the inflammatory infiltrate in nodular lesions of subcutaneous dirofilariosis and the presence of the nematode [18]. The results of this examination are compatible with a granulomatous nodular dermatitis secondary to a systemic leishmaniosis due to the presence of macrophages with intracytoplasmatic amastigotes [16]. However, the role of D. repens in contributing in the aetiology of the skin lesion of this infected dog cannot be excluded and further studies are warranted to elucidate the role of the parasite in causing cutaneous lesions. The possibility that the nodular dermatitis was caused by the simultaneous infection by D. repens and L. infantum should be taken into a proper account. In fact, a recent study showed that cutaneous signs in dogs co-infected by L. infantum and D. repens may recover after the administration of a parasiticide with a nematocidal activity [14]. Thus, a possible role of leishmaniosis in promoting skin lesions due to D. repens in co-infected dogs is worth of further investigations.
L. Cornegliani et al. Under a practical standpoint, it is important to note that few information are available for the anthelmintic treatment of D. repens-induced or associated to dermatopathies. The off label administrations of melarsomine hydrochloride and of doramectin marketed for large animals have cleared mff in a single dog. Unfortunately, the clinical signs associated with D. repens were not evaluated [19]. The single administration of a spot-formulation containing moxidectin 2.5 % plus imidacloprid 10 % showed a high efficacy in both treating skin lesions and to eliminate D. repens microfilariaemia [14] in nine treated dogs with skin lesions compatible with subcutaneous dirofilariosis. The lesions disappeared in seven out of nine symptomatic animals, but the parasitic origin of the skin lesions in these dogs, although plausible, would have been ultimately corroborated by skin biopsies. The other two animals had a history of leishmaniosis, thus leading to the hypothesis that the lesions were probably (also) related to L. infantum [14]. The persistence of the dermatitis in these animals despite the administration of the antiparasitic treatment and the present report as well may suggest a primary role of L. infantum in causing skin lesions in co-infected dogs. Interestingly, most of the infected dogs with skin lesions had low levels of circulating mff, thus suggesting that presence and number of mff in the bloodstream could not mirror the occurrence and/or severity of dermatopathies [14]. The present results corroborate this hypothesis, given that this dog had a very low level of circulating mff. Hence, an individual susceptibility in developing skin diseases in subcutaneous dirofilariosis can be argued, as also previously indicated [8,9,14]. Unfortunately the dog died before a treatment for D. repens which would have provided more indications on the possible role of the nematode in causing the skin lesion, via an ex juventibus approach, as previously described [11]. In conclusion, the present report and other previous studies [10,14] suggest the hypothesis that D. repens may cause, other than nodules [18], different kinds of dermatopathies. Also, these findings represent a basis towards new studies to investigate more in depth the potential role that L. infantum may have as a predisposition for the onset of skin lesions in dogs infected by D. repens at the same time. In fact, studies have demonstrated that canine vector-borne diseases may nurture and reinforce the pathogenic potential of other pathogens [20]. The reasons for why some dogs are subclinically infected by D. repens and others present relevant skin lesions are also worth investigating. One of the possible causes can be a concomitant infection with another parasite causing skin alterations, i.e. L. infantum. It would be also interesting to prospectively investigate for D. repens leishmaniotic dogs living in certain areas, in order to assess a possible overlapping distribution of these diseases and the concomitant role they may have in symptomatic animals. It will be useful to understand the potential correlation between these two diseases and the severity of clinical signs. Veterinarians working in canine clinical medicine should consider both the possibilities that D. repens may cause various skin lesions, even in cases of co-infections with other parasites, e.g. L. infantum. This is crucial for the unequivocal diagnosis of both single and mixed infections, based on cytological and histological methods, which can be supported by further genetic examinations, and for effective
Simultaneous infection by Dirofilaria repens and Leishmania infantum in a dog parasiticide treatments. The present expansion in the geographic distribution of vector-borne parasites [6] requires that practitioners are vigilant on these diseases for the risk of co-infections and for the confounding and overlapping clinical pictures they cause. Moreover, an effective treatment is pivotal also to interrupt the biological cycle of these parasites, that have a relevant zoonotic significance.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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