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IntroductionCanine visceral leishmaniosis (CVL), characterized by

both cutaneous and visceral lesions, is an endemic disease ofthe foxes, wild canids, dogs and humans in the Mediterraneancountries, the Middle East, some parts of Africa, India andCentral and South America [8, 13, 18, 24, 31]. CVL andparapoxvirus co-infection has recently been reported in aMediterranean monk seal [36]. The protozoan organismsresponsible for CVL, Leishmania infantum (in Mediterraneanand in Europe) and Leishmania chagasi (in America), aretransmitted through blood-sucking sandflies of the genusLutzomyia in the New World and Phlebotomus in the OldWorld [23, 31] and infest macrophages which are the princi-pal host cells.

Clinical features of leishmaniosis can widely vary in sickdogs. Sick animals with CVL show chronic wasting diseasewith anaemia, intermittent pyrexia, mainly local or generali-zed lymphadenopathy, hepatosplenomegaly, chronic nephri-tis, chronic colitis, epistaxis and skin lesions consisted ofexfoliative dermatitis and alopecia, and ulcerative, nodularand pustular dermatitis [8, 18, 30, 31]. Keratoconjuctivitis,arthritis and colitis as an atypical form of CVL are other rarefindings in endemic areas. The immunosuppressive effect ofCVL may increase susceptibility of co-infections such asNeospora sp., Hepatozoon sp., Rickettsia sp., Ehrlichia sp.,Bartonella sp., Babesia sp., Dirofilaria sp., Sarcoptes sp.,Candida sp. and Demodex sp. [8, 9, 11, 15, 22, 26, 27, 29,33, 34].

SUMMARY

The present study describes pathological alterations and immunohistoche-mical diagnosis observed in 6 visceral leishmaniosis affected dogs withconcomitant hepatozoonosis, toxoplasmosis and canine distemper infections.Marked inflammatory lesions in skin, kidneys and lungs coupled to moderateinfiltration by mononuclear cells (lymphocytes, plasmocytes and macrophages)of lymph nodes, spleen, bone marrow, liver, kidneys, lungs and intestinalsub-mucosa strongly evoked visceral leishmaniosis whose the diagnosticwas confirmed by direct histological and immunochemical evidence ofLeishmania amastigotes in infiltrated macrophages. In 2 dogs (n°1 and 4)inflammatory lesions of brain meninges were also observed and abundantToxoplasma gondii tachyzoites in extracellular position or within macro-phages and tissue cysts were identified by histological and specific immu-nostaining. The presence of the canine distemper virus was confirmed byspecific immunohistochemistry in necrotic areas of lungs and cerebellumfrom a 2 month old puppy (n°6). The histological identification of endogenousdevelopmental stages of Hepatozoon schizontes in lymph nodes, spleen andbone marrow confirmed the co-diagnosis of hepatozoonosis in 4 dogs. Thesecase reports suggest that a primary Leishmania infection could promote theoccurrence of secondary infections such as toxoplasmosis, hepatozoonosisand canine distemper.

Keywords: Visceral leishmaniosis, histology, immunohis-tochemistry, hepatozoonosis, toxoplasmosis, canine dis-temper, dog.

RÉSUMÉ

Leishamaniose viscérale canine et infections concomitantes (hépato-zoonoses, toxoplasmose et maladie de Carré)

Cette étude décrit les altérations anatomopathologiques et le diagnosticimmunohisto-chimique obtenus sur 6 chiens atteints de leishmaniose viscé-rale présentant simultanément une toxoplasmose, une hépatozoonose ou unemaladie de Carré. Des lésions inflammatoires intenses de la peau, des reinset des poumons associées à un important infiltrat de cellules mononucléées(lymphocytes, plasmocytes et macrophages) des noeuds lymphatiques, de larate, de la moelle osseuse, du foie, des reins, des poumons et de la sousmuqueuse intestinale ont conduit à une forte suspicion de leishmaniose dontle diagnostic a été confirmé par la mise en évidence directe des amastigotesdans les macrophages infiltrés par histologie et immunohistochimie. Chez 2chiens (n°1 et 4), des lésions inflammatoires des méninges de la boîte crâ-nienne ont également été observées et de nombreux tachyzoïtes deToxoplasma gondii en position extracellulaire ou au sein des macrophagesou de kystes tissulaires ont été identifiés par histologie et immunomarquagespécifique. La présence du virus de la maladie de Carré a été confirmée parune immunohistochimie spécifique dans les zones nécrotiques des poumonset du cervelet d’un chiot de 2 mois (n°6). L’identification par examen histo-logique des différents stades de développement des schizontes deHepatozoon dans les noeuds lymphatiques, la rate et la moelle osseuse aconfirmé le co-diagnostic d’hépatozoonose chez 4 chiens. L’étude de ces cassuggère qu’une infection primaire par les Leishmania peut promouvoir l’ap-parition d’infections secondaires telles que la toxoplasmose, l’hépatozoonoseet la maladie de Carré.

Mots clés : Leishmaniose viscèrale, histologie, immuno-histochimie, hepatozoonose, toxoplasmose, maladie deCarré, chien.

Canine visceral leishmaniosis and concurrentinfections (hepatozoonosis, toxoplasmosisand canine distemper)

N. TOPLU*, H. AVCI, N. METIN

Department of Pathology, Faculty of Veterinary Medicine, University of Adnan Menderes, 09016 Isikli-Aydin, TURKEY.

*Corresponding author: nihattoplu@hotmail.com

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342 TOPLU (N.) AND COLLABORATORS

The main histological lesions are hypertrophy and hyper-plasia of cells of the mononuclear phagocyte system, granu-lomatous inflammatory reactions of the spleen, lymph nodes,bone marrow and liver, and chronic inflammation of the skin[25]. The aims of the study were to describe histopathologicaland immunohistochemical diagnosis of CVL, and to investigatethe occurrence of co-infections in dogs.

Materials and MethodsANIMALS AND CASE HISTORY

Six dead dogs from the Aegean region (Turkey) were pre-sented to the pathology department of the Faculty ofVeterinary Medicine, University of Adnan Menderes, Isikli-Aydin, Turkey, for investigating the death cause. Among the4 females and the 2 males, 2 months to 5 years old, 3 of ani-mals were mongrels and 3 were purebred (2 pointers and 1Terrier). According to information from practitioners andowners, these animals have generally exhibited a history ofanorexia, weakness, anaemia, depression, weight loss, skinlesions and lymphadenomegaly. The Table I stated the age,breed, case history, and histological and immunohistochemi-cal diagnosis of each dog. Before, the diagnosis of leishma-niosis was confirmed in one dog (dog n°1) by microscopicexamination of Giemsa stained smears from lymph nodeaspirates and detection of Leishmania amastigotes and thisdog was treated for leishmaniosis (allopurinal, oral, 20 mg/kgonce daily for 15 days). The Hepatozoon sp. gametocyteswithin neutrophils in Giemsa-stained peripheral blood smearswere evidenced in 2 animals (dogs n°2 and 5) which weretreated for this infection with a combination of toltrazuril (10mg/kg, orally once daily) and a trimethoprim-sulfamethoxazole(15mg/kg, intravenously twice daily) for 5 days. The dog n°6was not vaccinated against canine distemper and presentednervous signs such as tremors and incoordination. Accordingto anamnesis, this puppy’s mother had usual clinical symp-toms of leishmaniosis such as severe weight loss, exfoliativeand ulcerative dermatitis.

HISTOLOGY AND IMMUNOHISTOCHEMISTRY

Necropsy was performed on all the animals, and the sam-pled tissues including skin, stomach, intestines, liver, spleen,pancreas, kidneys, lymp nodes, lungs, heart, brain andmedulla spinalis were fixed in 10% buffered formalin andembedded in paraffin. Sections cut at 5 µm thickness werestained with haematoxylin and eosin (H&E). Replicate sec-tions were used for the immunohistochemistry. Additionally,touch-impression smears from lymph nodes, spleen and bonemarrow were stained by May-Grunwald-Giemsa.

The avidin-biotin peroxidase complex (ABC) and theimmunofluorescence (IF) methods, essentially described byTOPLU [35], were used for evidencing Leishmania. In bothmethods, mouse anti-Leishmania monoclonal antibodies(Cedarlane Laboratories, Burlington, ON, Canada) wereused as primary antibodies. For the amastigote fluoresceinimmunolabelling, the tissue sections were dewaxed, rehy-drated and digested with 0.1% proteinase K for 10 minutesat 37°C. After washing in PBS (phosphate buffered saline,pH 7.3) for 15 minutes, the sections were incubated for 2 hat 37°C with mouse anti-Leishmania antibody (1:1000), andthen washed again for 15 minutes in PBS. After addition ofgoat anti-mouse gamma globulin serum conjugated withfluorescein isothiocyanate (Sigma, Rehorot, Israel), sectionswere incubated for 30 minutes at 37°C, washed in PBS for20 minutes and mounted in phosphate-buffered glycerol (pH9.0). For control purposes, replicate sections were processed,by substituting the mouse anti-Leishmania antibody withnormal mouse serum. Finally, the tissue sections were exa-mined with a fluorescence microscope (Leica DMLB). Forthe peroxidase immunolabelling, the sections were dewaxedin xylene and hydrated through graded alcohols. Endogenousperoxidase was then blocked with H2O2 3% in 70% methanol.The tissues were digested with 0.1% proteinase K 10 minutesat 37°C and the slides washed for 10 minutes in PBS. Non-specific staining was blocked by treatment with 2% normalhorse serum for 30 minutes. The blocking serum was thenreplaced by mouse anti-Leishmania antibody (1:1000), follo-

Dogs Epidemiological data Clinical history / Breed Sex Age Eventual treatment Diagnosis

N°1 Terrier M 5 year WL, LM, SMTreatment for CVL CVL + HZ + T

N°2 Mongrel F 4 year WL, LM, SM, SLTreatment for HZ CVL + HZ

N°3 Pointer F 4 year WL, LM, SM, SLNo treatment CVL + HZ

N°4 Mongrel F 5 year WL, LMNo treatment CVL + T

N°5 Mongrel M 5 year WL, LM, SLTreatment for HZ CVL + HZ

N°6 Pointer F 2 month Respiratory and nervous signsNo treatment CVL + CD

TABLE I: Epidemiological data, clinical history and histological / immunohistochemical diagnosis of naturally canine visceral leish-maniosis (CVL) and co-infections (hepatozoonosis, toxoplasmosis and canine distemper) in 6 dogs.

M: Male; F: Female; WL: Weight Loss; LM: lymphadenomegaly; SM: Splenomegaly; SL: Skin Lesions; CVL: Canine Visceral Leishmaniosis; HZ:Hepatozoonosis; T: Toxoplasmosis; CD: Canine Distemper.

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wed by overnight incubation at 4°C. After washing for 10minutes, sections were flooded with biotinylated horse anti-mouse immunoglobulin for 30 minutes. After a further wash,the sections were covered with streptavidin-peroxidase andincubated for 30 minutes and treated for 7 minutes with dia-minobenzidine (DAB) containing H2O2 3%. The sectionswere then counterstained with haematoxylin, washed in tapwater, dehydrated in graded alcohols, and mounted. Forcontrol purposes, replicate sections of selected infected tis-sues were processed, by substituting the mouse anti-Leishmania antibody with normal mouse serum. All incuba-tions were performed at room temperature in a humidifiedchamber.

For the Toxoplasma gondii immunolabelling, an indirectfluorescent method was performed as described above (forLeishmania parasite) using a rabbit anti-Toxoplasma gondiipolyclonal antibody (1:2500) as primary antibody. Forcontrol purposes, replicate sections of selected infected tissueswere processed, by substituting the rabbit anti-Toxoplasmagondii antibody with a rabbit anti-Neospora caninum poly-clonal antibody.

An ABC method was used for the canine distemper virusdetection as described above. For control purposes, replicatesections of selected infected tissues were processed, by sub-stituting the rabbit anti-canine distemper virus antibody withnormal rabbit serum.

ResultsCLINICAL AND PATHOLOGICAL FINDINGS

Case history and clinical findings were presented in Table I.A moderate to severe weight loss coupled to lymphadeno-megaly, especially of popliteal and prescapular lymph nodes,was observed in all presented cases except for the dog n°6.Skin lesions were conspicuous gross findings in animals n° 2,3 and 5. They consisted in multifocal hypotrichosis-alopecia,bran and skin xerosis, and ulcers localized on the skin of theears, nose, limbs and hip. Splenomegaly was a marked findingin 3 cases (dogs n° 1, 2 and 3). Hepatomegaly was observedonly in one case (dog n°3) whereas kidney lesions wererecorded in all cases except for the dog n°6. These organswere sclerotic in dogs n°2, 3 and 4 or contained white-greyish foci (dogs n°1 and 5).

The lungs of the animal n°6 exhibited diffuse consolida-tion, oedema and emphysema, and cranial lobes includeddark red areas of hepatisation. Trachea and bronchi werecongested and contained frothy exudate. The dog n°4 alsopresented pulmonary necrotic brownish-yellow foci ofapproximately 0.3-0.5 cm. The brain meninges from dogsn°1 and 4 were opaque and congested; these lesions wererestricted on the frontal lobes in the dog n°4. Moreover, inthe dog n°1, a softened haemorrhagic area was seen in theleft frontal lobe and in the cut surfaces of the left hemispherecortex. Whilst nervous clinical signs with tremors and inco-ordination were noted in the dog n°6, no macroscopicallesion was observed.

HISTOPATHOLOGICAL FINDINGS

Epidermal hyperkeratosis, interstitial dermatitis, mononu-clear perivascular infiltrations, sebaceous adenitis and per-ifolliculitis were prominent lesions in the skin of dogs.Lymphocytes and plasma cells with scarce macrophages(absent or/with amastigotes) were mainly infiltrating cells insub epidermal, perifollicular and periglandular areas.

Infiltration with lymphocytes and macrophages coupled toproliferation of fibrous tissue thickening the lymph nodecapsules were observed in dogs n°1, 2, 3 and 4. A markedfollicular hyperplasia in cortical and medullar areas wasnoticed in lymph nodes of all dogs with lymphadenomegaly.Plasma cells were predominant especially in medullar cordand proliferation of macrophages especially in the medullarsinuses was intense. Macrophages loaded with amastigotesof Leishmania sp. were frequently observed especially inthese areas and in capsule. Marked lymphoid depletion inparafollicular areas as well as reticuloendothelial cell hyper-plasia were also frequently noticed in lymph nodes.Moreover, splenic follicular structures were strongly alteredand lymphocytes were replaced by plasma cells and macro-phages. Plasma cells and macrophages have also proliferatedin bone marrow. Additionally, Hepatozoon sp. schizonteslocated within the cell in a parasitophorous vacuole werefound in spleen and lymph nodes from the dogs n°1, 2, 3 and5 (figure 1). The touch-impression smears of lymph nodes,spleen and bone marrow revealed the presence ofLeishmania amastigotes in macrophages (in support ofimmunohistochemistry in the tissue sections) andHepatozoon sp. gamontes in neutrophils from the bone mar-row (dogs n°2 and 3) (figure 2).

In all dogs, liver was injured: hepatocyte hydropic degene-ration was observed in 4 animals (dogs n°1, 2, 4 and 5) andwas associated with sinusoid invasion by isolated or clusteredlymphocytes and macrophages. Additionally, mixed cellpopulations constituted of lymphocytes, plasma cells andmacrophages coupled to mild fibrosis mildly to markedlyinvaded the portal areas in 2 dogs (dogs n°1 and 4).Granulomatous foci constituted by macrophages (loadedwith Leishmania amastigotes), lymphocytes and plasmocyteswere scattered within the hepatic parenchyma in the animalsn°3 and 6.

A non suppurative interstitial nephritis located especiallyin the renal cortex was seen in all dogs except for the animaln°6. The inflammation was characterized by foci of lympho-cytes and plasma cells including scarce macrophages, withinthe periglomerular, peritubular, intertubular, and perivascularareas. A fibrous tissue proliferation especially in corticalareas was additionally observed in the dogs n°2, 3 and 4.Glomerular changes consisted of focal segmental glomerulos-clerosis and diffuse mesangial proliferative glomerulonephritis.

The main lesion of the lungs was a chronic interstitialpneumonia; the interalveolar septum was thickened by mixedpopulations of lymphocyte, macrophage and fibrocytes. Inaddition, lungs of the animal n°6 showed acute catarrhalbronchopneumonia whereas focal necrotic areas with macro-phages including T. gondii tachyzoites in the cytoplasm wererecorded in the lungs from the dog n°4.

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Acute non suppurative encephalitis with demyelisationwas evidenced in the dog n°6. Furthermore, in this animal,cerebellar astrocytes contained eosinophilic intranuclearinclusion bodies. Meninges and cerebral hemispheres of thebrain from the dog n°1 showed haemorrhage, necrosis andperivascular macrophage infiltrations and tachyzoites andtissue cysts of Toxoplasma gondii were detected in the injuredareas. Large numbers of tachyzoites were also observed inendothelial cells of blood vessels (figure 3). In the dog n°4,haemorrhages and the infiltrating macrophages with tachy-zoites were restricted only to meninges.

Whereas no alteration was observed in the animal n°6,there was a mild to a moderate mononuclear cell infiltrationof the submucosa in small and large intestines, sometimesextended to the muscular layer.

IMMUNOHISTOCHEMICAL FINDINGS

Strong immunolabelling of Leishmania amastigotes withthe 2 both IF and ABC methods were evidenced in the cyto-plasm of macrophages from lymph nodes, spleen and bonemarrow (figure 4) in all dogs. The infiltrating macrophagesof the skin lesions and of the intestine mucosa were alsopositively stained. In liver, Kupffer cells, hepatocytes and

macrophages gave a positive reaction. Few macrophages ofthe interstitial inflammatory infiltrate in kidneys as well astubular epithelial cells and glomerular cells were also positivefor Leishmania antigens. Control slides were negative.

Antigens of the canine distemper virus were markedly evi-denced by the specific ABC method in cerebellar astrocytes,in neurons from the cerebral hemispheres (figure 5A) and inalveolar epitheliums in the lungs (figure 5B) in the dog n°6.

The fluorescein labelling of T. gondii tachyzoites was obs-erved especially in endothelial cells and infiltrating macro-phages from the meninges and cerebral hemispheres (dogsn°1 and 4) (figure 6). Extracellular positive T. gondii immu-nostaining was also evidenced. A few tissue cysts showedslight positive reaction, as well. In lungs, the immunostainingwas recognized in macrophages and alveoli especially inareas surrounding necrotic foci. The staining with Neosporacaninum antiserum of replicate sections was negative.

DiscussionClinical diagnosis of CVL may be difficult because clini-

cal signs are not specific and may vary depending on the

FIGURE 1: Developmental stages of Hepatozoon canis schizontes in spleen sections: (A) Schizonte with foamy cytoplasm and oneundifferentiated nucleus (arrow), Haematoxylin – Eosin, bar: 10 µm. (B) Schizonte with numerous nuclei (arrows), Haematoxylin– Eosin, bar: 20 µm. (C) Schizonte with developing merozoites (arrows), Haematoxylin – Eosin, bar: 20 µm. (D) Numerous mero-zoites throughout the schizonte (arrow), Haematoxylin – Eosin, bar: 20 µm.

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FIGURE 4: Lymph nodes. Fluorescein immunolabelling (IF method) (A) and peroxidase immunolabelling (ABC method) (B) ofLeishmania amastigotes (arrow) in the cytoplasm of infiltrating macrophages into capsule, bar: 30 µm.

FIGURE 2: Touch impression smear of bone marrow: macrophages loa-ded with Leishmania amastigotes (arrows) and Hepatozoon sp.gamontes in neutrophil granulocyte (arrowhead), May-Grunwald-Giemsa, bar: 20 µm.

FIGURE 3: Meninges. Infiltrating macrophages (blue arrowheads),endothelial cells (arrow) with Toxoplasma gondii tachyzoites inthe vessel (VL), and tissue cysts (black arrowheads) with protozoaon the vessel wall, Haematoxylin - Eosin, bar: 50 µm.

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organism- or host-specific factors and the state of immunityand previous specific therapies. It is also possible that thedegree and type of manifestations associated with leishma-niosis vary according to individuals, depending on presenceof concomitant vector-borne organisms [11, 27, 29, 32]. Insuch situations, numerous CVL cases, as reported in thisstudy, may be not diagnosed, misdiagnosed or unreported,particularly when leishmaniosis diagnostic methods are notavailable [8, 18, 31].

The main histological lesions of CVL, as described in thedogs of the present study, are the hypertrophy and hyperpla-sia of the mononuclear phagocytic cell system coupled togranulomatous inflammatory reactions (spleen, lymphnodes, bone marrow and liver) and often to a chronic inflam-mation in the skin [17, 25]. The presence of Leishmaniaamastigotes in macrophages from the lymph nodes, spleen,bone marrow and liver, also identified by immunohistoche-mistry, undoubtedly support the CVL diagnostic. Severalreports describe a high prevalence of infection by especiallyLeishmania infantum in dogs throughout the Mediterraneanbasin, as demonstrated by a variety of methods such as sero-logic investigations, immunohistochemistry and the presenceof leishmanial DNA [12, 18, 31, 36]. It is most possible thatagent in the cases presented in the present study may beLeishmania infantum as before identified in dogs of Aegeanregion by ÖZBEL et al. [23].

In CVL, Leishmania ensures its own survival by modulatingthe host immune system either by inducing immunosuppressionor by promoting pro-parasitic host functions. Studies of Leis-hmania infections in mice indicate that resistance or susceptibility

FIGURE 5: Immunoreactivity with ABC method for canine distemper viral antigen in cytoplasm and nucleus of neurons (arrow) fromcerebral hemisphere (A) (bar: 50 µm) and in alveolar lining epithelial cells (arrows) (B) from the dog n°6.

FIGURE 6: Meninges from the dog n°1. Fluorescein immunolabellingof Toxoplasma gondii tachyzoites in the infiltrating macrophages(arrows) in the vessel lumen (VL) and in extracellular position(arrowhead) on the vessel wall, IF method, bar: 20 µm.

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to the disease is associated with the development of Th1 orTh2 cell response, respectively [19] , and lead to an imba-lance between Th1 and Th2 cell responses [5, 14]. A vigorousTh2 immune response, mainly characterized in humans byincreased IL-4 expression, polyclonal B cell activation,intense hypergammaglobulinemia and production of anti-leishmanial IgE antibodies was present during CVL [1, 7, 10,20]. Prolonged latent infection with Leishmania results incell-mediated immunosuppression that could be related tothe expression of IL-10 [28]. On the other hand, phagocyticcapacities of macrophages in animals with CVL are tempo-rarily altered [2, 6]. Thus, impaired T lymphocyte or macro-phage function in CVL predisposed to the development ofinfections caused by intracellular pathogens such asNeospora caninum [3, 9], and Toxoplasma gondii as evidencedin the present study. Toxoplasmosis is usually subclinical indogs, and has been reported as a concurrent infection withcanine distemper or some other underlying immunosuppressiveagent or condition [4]. Similarly, concurrent Toxoplasmagondii and Hepatozoon canis infections were also reportedin a dog [15]. In the present study, toxoplasmosis co-existedwith CVL in 2 dogs (animals n°1 and 4); besides, the dog n°4was also infected with Hepatozoon. It was probable that thesevere necrotic lesions in the brain and lungs caused by toxo-plasmosis have lead to the fatal outcome. According to ourknowledge, the CVL/toxoplasmosis and CVL/hepatozoonosis/toxoplasmosis associations described in these 2 cases havenot been previously documented in the literature.

It is emphasized that possibility of co-infections should beincreased in dogs living in areas that are highly endemic forseveral vector-borne organisms, in dogs that are maintainedpredominantly outdoors (enhanced vector transmission), andin dogs that are not routinely treated with acaricides or otherectoparacidicides [11, 27]. Likewise, canine hepatozoonosis(a protozoon disease transmitted by the tick Rhipicephalussanguineus) was diagnosed based solely on morphologiccharacters during endogenous developmental stages in thelymph nodes, spleen and bone marrow in four dogs withleishmaniosis of the present study. The protozoon speciewould be Hepatozoon canis in these cases, which has beenrecently reported in an epidemiological study, Aegeanregion, Turkey [16].

Previous epidemiological studies indicate that CVL mainlyaffects adult (from 9 month old to 15 year old, with a medianfor 5 years of age) and that the incubation period ranges from3 months to 7 years [18, 30]. Nevertheless, CVL has alsobeen recorded in young immature dogs, throughout verticaltransmission [21]. In the present study, the 2 month oldpuppy with canine distemper (CD) (dog n°6) would probablyhave been infected by its mother since it exhibited clinicalsigns compatible with CVL. It is the first description, to ourknowledge, of a CVL and CD co-infection in a puppy.

As a conclusion, this study demonstrates concurrent infec-tions with toxoplasmosis, hepatozoonosis and even caninedistemper in CVL-affected dogs from the Aegean region ofTurkey. In a clinical perspective, practitioners should beaware of such disease association and should include theirscreening in the routine diagnostic panel.

AcknowledgmentsThis research was supported by Research Fund of Adnan

Menderes University Research Council, Project VTF-07-024. Authors wish to thank Dr. Nalan Kabakci, Dr. TolgaGuvenc and A. Hemphill supplying canine distemper virus,Toxoplasma gondii and Neospora caninum polyclonal anti-bodies, respectively.

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