Leishmania infantum in a dog with lymphadenopathy and extensive cutaneous lesions: Hematological, biochemical, cytological and molecular findings

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Dogs are the main reservoirs of the parasites, which play a considerable role in infecting humans and other hosts. This report explains lymph node involvement by amastigotes of the viscerotropic Leishmania species in a dog who suffered from ulcerative nodules and generalized eczematous and exfoliative dermatitis (erythroderma). A 2-year-old male dog with evident acute skin lesions and ulcerative nodules on the face was referred to a small animal hospital in Mazandaran province, Iran. The animal had started showing signs ten days prior and was lethargic and lost appetite. In order to detect Leishmania parasites, the popliteal lymph node was sampled for the cytological examination and also polymerase chain reaction (PCR) based on the ITS-1 region of the ribosomal DNA gene. Also, a blood sample was collected to determine hematological and biochemical parameters. As a result, Leishman bodies were seen both inside and outside of macrophages when light microscopy was performed on the lymph node sample stained with Giemsa. Laboratory findings revealed mild leukocytosis, lymphocytosis, neutrophilia, low hematocrit. hyperglobulinemia, hyperproteinemia, hypoalbuminemia, declined albumin/globulin ratio, and hyperglycemia. The PCR and sequencing results confirmed the presence of Leishmania in the popliteal lymph node. According to the molecular analysis, L. infantum was the causative agent of leishmaniasis in this case. Cutaneous leishmaniasis is prevalent in humans and dogs in Iran. In most cases, L. tropica and L. major are the major causative agents of this form of leishmaniasis, and the lesions are confined. In this report, a generalized skin disease was evident. In addition, hematological and biochemical parameters supported a visceral form of leishmania infection in the animal. Because L. infantum is a more dangerous species than the other types for dogs and specifically humans, our report should be considered to control the infection. This report is significant as this unusual form of visceral leishmaniasis in the dog raises concerns about the possibility of zoonotic transmission and may threaten public health. Dog lymph node Leishmania infantum Hematology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The zoonotic disease known as leishmaniasis is caused by prevalent species belonging to the genus Leishmania . These parasitic protozoa are members of the Trypanosomatidae family and the Kinetoplastidae order (Ikeda et al. 2008) and affect numerous mammals, including humans, cats, dogs, horses, and rodents (Handman and Bullen 2002). The clinical disease is mostly caused by cutaneous and visceral invasions. Dogs and rats act as predominant reservoirs of cutaneous leishmaniasis (CL) (Mohebali et al. 2005), while the primary reservoirs of human visceral leishmaniasis (VL) are wild carnivores such as wolves, foxes, and jackals. Rodents have proven to be the primary reservoirs of mucocutaneous leishmaniasis (MCL) (Dantas-Torres. 2007). There are several species of Leishmania that can infect dogs, including infantum, tropica , major, braziliensis , etc. (de Andrade et al. 2006; Reithinger and Davies 1999) Each species may be related to a range of clinical signs, from cutaneous to visceral. Of those species, the pathogenic potential of L. infantum has been more widely considered because of its systemic implications. The World Health Organization (WHO) reports fatalities in humans due to visceral leishmaniasis (VL) worldwide. (Bi et al. 2018). In canids, Leishmania infantum (from the Donovani complex) may also be characterized by cutaneous manifestations (Saridomichelakis and Koutinas 2014). In the Middle East area, the cutaneous form has mostly been associated with infection with two other species, L. major and L. tropica (Baneth et al. 2022; Fakhar et al. 2022). The latter species has sometimes been reported to be found in blood samples, implying its possibility of causing the visceral type. Also, cross-reactivity has been found between L. tropica and L. infantum (Hajjaran et al. 2007). Therefore, detection of the species involved should be concerned with predicting the pathogenesis of the parasite. The gold standard for laboratory diagnosis for Leishmania is the use of parasitological and serological methods. In addition, hematology, biochemistry, and clinical immunology are examples of nonspecific approaches (Ciaramella and Corona 2003). Conventional PCR (CnPCR) has undeniable advantages over traditional methods, including high sensitivity and specificity and the possibility of direct application to clinical specimens. This technique has also made significant improvements in recent years, employing quantitative real-time PCR (qPCR) (Paiva-Cavalcanti 2010). Many studies are available reporting the Leishmania sp. infection in the Middle East area, both in humans and carnivores. The parasite species are predominantly composed of L. infantum , L. major and L. tropica (Baneth et al. 2022). In Iran, these species have been reported in dogs with an emphasis on the visceral type in recent years. The visceral infection has been described in about half of the provinces; however, many of them were diagnosed by a serologic routine direct antiglobulin test (DAT) test on blood samples. (Shokri et al. 2017), with rare information on its role in forming significant skin lesions. Also, the serologic method is not valid to distinguish at the species level. In this report, we describe the infection of a popliteal lymph node in a dog with considerable cutaneous lesions on the face. Also, the parasite was traced using the PCR method. Methodology Case presentation and sample collection An adult, 2-year-old male Dogo Argentino with extensive skin lesions showing exfoliative dermatitis on the face, around his eyes, and on his nose was referred to a small animal hospital located in Mazandaran province, north of Iran (Fig. 1 ). Ulcerative nodules were also observed in this area (Fig. 1 D). The owner stated that these lesions had started to appear ten days before the visit. One month before the visit, the dog was lethargic and had lost his appetite. A physical examination revealed the presence of generalized eczematous and exfoliative dermatitis (erythroderma) and lymph node enlargement. After a clinical examination, the popliteal lymph node was sampled using the fine needle aspiration technique and underwent direct microscopical investigation. We also drew samples of serum and blood for biochemical and hematological analyses, respectively. Hematological factors were measured by an automated hematology analyzer (Nihon Kohden, MEK-6450 Celltac Alpha, Tokyo, Japan). Moreover, m easurements of biochemical parameters were done using commercial kits supplied by Biorex Co. (Fars, Iran), and an AutoAnalyzer (Alpha ® Classic, Iran). DNA Extraction and Polymerase Chain Reaction (PCR) DNA was extracted from the prepared lymph node smear using a commercial kit (Parstous ® , Iran). The ribosomal DNA gene targeting the internal transcribed spacer 1 gene (ITS1) region was considered to detect Leishmania parasites using primers LSGITS1-F1: CATTTTCCGATGATTACAC and LSGITS1R1: CGTATGTGAGCCGTTATC (de Almeida et al. 2011). The amplification reaction involved a touchdown PCR program as follow: The initial denaturation performed at 95°C for 5 min, followed by 35 cycles including denaturation at 94°C for 1 min, annealing at 58 − 54°C (the temperature was reduced 1°C in each subsequent cycle and fix the final annealing temperature at 54°C) for 1 min, extension 72°C for 45 s. The final extension step was done for 5 min at 72°C following the last cycle. The results were electrophoresed on a 3% agarose gel and stained with Safe DNA Gel Stain (Sinaclone ® ; Iran). PCR products were sequenced (Pishgam Biotech Co., Iran), and the results were compared with other available sequences in the NCBI database using the BLAST search. The MEGA X software was applied to align the sequences and the phylogenetic tree was drawn using the maximum likelihood method (Kumar et al. 2018). Results Light microscopy on the Giemsa-stained lymph node smear revealed the parasite amastigotes, known as Leishman bodies, both inside and outside of the macrophages (Fig. 2 ). The laboratory report disclosed mild leukocytosis: (white blood cell (WBC) = 22.3 ×10 3 /µl), (lymphocytosis = 5.6×10 3 /µl), neutrophilia: (segmented neutrophils = 15.2 ×10 3 /µl), a left shift (band neutrophil = 0.60 ×10 3 /µl), Hematological findings also showed a low hematocrit; however, the thrombocyte counts were normal (Table 1 ). Also, biochemistry analysis indicated hypoalbuminemia (2.3 g/dl), decreased albumin to globulin (A/G) ratio (0.3) and hyperproteinemia (10.1 g/dl) as a result of hyperglobulinemia (7.9 g/dl). Furthermore, hyperglycemia and mild azotemia were also detected as serum biochemical abnormalities (Table 2 ). Table 1 Values of the hematological parameters in the Leishmania infected dog. Hematological parameters Level* Reference Range WBC (×10 3 cells/µL) 22.3 H 6.0–17.0 Lymphocyte (×10 3 cells/µL) 5.6 H 1.0–4.8 Segmented Neutrophil (×10 3 cells/µL) 15.2 H 3.0–11.5 Band Neutrophil (×10 3 cell /µL) 0.6 H 0-0.3 Monocyte (×10 3 cells /µL) 0.20 0.15–1.35 Eosinophil (×10 3 cells /µL) 0.70 0.10–1.25 HCT (%) 27 L 37–55 RBC (×10 6 cells /µL) 4.4 L 5.5–8.5 Hb (g/dl) 9.5 12–18 MCV (fl) 61 60–77 MCH (pg) 21.4 19.5–24.5 MCHC (%) 35.2 32–36 Platelet (×10 3 cells /µL) 577 200–500 *H: High; L: Low Table 2 Values of the biochemical parameters in the Leishmania infected dog. Biochemical parameters Level* Reference Range Blood urea nitrogen (mg/dl) 37 H 10–35 Urea (mg/dl) 79.2 H 15–70 Creatinine (mg/dl) 2 H 0.5–1.7 Triglycerid (mg/dl) 116 22–152 Cholesterol (mg/dl) 151 95–337 Glucose (mg/dl) 153 H 70–120 AST (IU/L) 60 10–66 ALT (IU/L) 34 10–121 ALP (IU/L) 64 35–280 Calcium (mg/dl) 11.0 8.9–11.7 Phosphorus (mg/dl) 3.9 1.8-6.0 Albumin (g/dl) 2.3 L 2.5–4.5 Globulin (g/dl) 7.9 H 2.2–4.2 Total bilirubin (mg/dl) 0.1 ≤ 0.5 Total protein (g/dl) 10.2 H 5.0-7.2 A/G ratio 0.3 L 0.9–1.9 *H: High; L: Low In the molecular test based on the ITS-1 region, a ~ 250 bp band was achieved (Fig. 3 ). According to the sequencing data and the BLAST search, the recovered specimen was L. infantum. According to the phylogenetic analysis, our specimen was grouped with a variety of L. infantum and L. donovani strains reported from dogs, human, sandflies and even cats (Fig. 4 ). These two species were separated well from L. tropica and L. major . The ITS-1 sequence data for the present strain was recorded in the GenBank under the accession number PP848956. Discussion The diagnosis of leishmaniasis has been made after careful consideration of the symptoms and laboratory results. Displaying cutaneous lesions in the animal's face may be a common symptom in affected canines (Saridomichelakis and Koutinas 2014). In this study, we described a case infected with a visceral type of leishmaniasis showing a relatively widespread dermatitis, specifically on the face, which was accompanied by some hematological (anemia, leukocytosis, and neutrophilia with a left shift) and biochemical (hyperglobulinemia, hyperproteinemia, hyperglycemia, and mild azutemia) abnormalities. Previously, the clinical picture of visceral leishmaniasis in dogs was classified into several categories. The occurrence of systemic, reticuloendothelial, cutaneous, ocular, and clinicopathological signs is assumed (Foglia Manzillo et al. 2013). In different studies, variabilities are seen, as some of them are more regular and a group of signs are rare. Conversely, a number of such infections are found in asymptomatic dogs (Ribeiro et al., 2018), and some signs could appear during the progression of the infection (Foglia Manzillo et al. 2013). In this case, reports indicate that signs of lymphadenopathy, ulcerative nodules, and generalized eczematous and exfoliative dermatitis (erythroderma) coincided with clinicopathological features. Based on previous investigations in Iran and the Middle East, the main causative agents of cutaneous leishmaniasis are L. tropica and L. major in dogs (Bamorovat et al. 2015; Baneth et al. 2016; 2017). On the other hand, cases of visceral infection with L. tropica have also been reported in dogs in Iran (Mohebali et al. 2005; Hajjaran et al. 2007), Another species, L . infantum, was reported from endemic regions for VL in Iran, but this parasite has usually not been associated with widespread cutaneous pathogenesis (Shokri et al. 2017). Therefore, our finding represents a rare case of canine leishmaniasis with extensive skin disease, which was confirmed by molecular and cytological methods on the lymph node sample. From the hematological perspective, leukocytosis, neutrophilia, and left shift were seen in the presented case. The leukogram changes may not be as considerable in the symptomatic dogs as described by Ribeiro et al. (2013) But, in line with our data, other available research has linked the Leishmania infection to the observed WBC abnormality (Jafari Shourijeh et al. 2006; Bogdan et al. 2001). Nicolato et al. (2013) reported a high population of myelocytes, metamyelocytes, and band neutrophils among the granulocytic lineage precursors in the bone marrow. Based on the inflammatory response in multiple organs affected by parasitism, these results indicate a high demand for neutrophils in symptomatic dogs. Anemia may be a common symptom of canine leishmaniasis, depending on the parasite load and pathogenesis. Typically, da Costa-Val et al. (2007) observed medullary hypoplasia and normocytic normochromic anemia. Anemia tends to be related to the clinical stage (Dias et al. 2008). But in this instance, the hematocrit was measured at 27%, which was regarded as quite anemic. This finding, however, has been documented before and various factors have been indicated. (Amusategui et al. 2003; Ribeiro et al. 2013). Serum protein analysis may detect anomalies at an early stage in the progression of the disease. (Foglia Manzillo et al. 2013). Fitting with our results, there is a strong correlation between the severity of the clinical score and a rise in total proteins and globulins (Koutinas et al. 1999; Almeida et al., 2005; Solano-Gallego et al. 2009; Paltrinieri 2010; Proverbio et al. 2014). Otherwise, the albumin-to-globulin (A/G) ratio drops for no valid reason other than that albumin is a negative acute phase protein and the proteinuric nephropathy can induce albumin loss as well (Almeida et al. 2005; Ribeiro et al. 2013). Some researchers have argued that a drop in the A/G ratio is the most sensitive indicator of canine leishmaniasis (Almeida et al. 2005; Geisweid et al. 2012; Paltrinieri et al. 2016). These outcomes are comparable to our biochemical findings. According to the literature, various types of samples have been used for molecular diagnosis. The PCR method on bone marrow proposed as a highly sensitive method to find visceral leishmaniasis (Oliva et al., 2006). However, in L. infantum -infected dogs, the popliteal lymph node and conjunctival sampling techniques are less invasive and result in good PCR conclusions (Marcelino et al., 2020). In this study, DNA obtained from a lymph node -as a minimally-invasive approach- was successfully amplified and sequenced using PCR assay. Conclusions This report describes a rare case of visceral leishmaniosis with generalized eczematous and exfoliative dermatitis (erythroderma) and lymph node invasion in a dog from northern Iran. The region has documented cases of visceral Leishmaniasis infection utilizing serologic assays, but there is a lack of information regarding the molecular entity of the parasite. Our study should be taken into account for infection control purposes, as L. infantum is a more dangerous species for canines and may threaten public health in this region of Iran. Abbreviations cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), World Health Organization (WHO), Conventional PCR (CnPCR), quantitative real-time PCR (qPCR), direct antiglobulin test ( DAT ), internal transcribed spacer 1 gene (ITS1), White blood cell (WBC), albumin-to-globulin (A/G), Polymerase Chain Reaction (PCR), Lymph node (LN). Declarations Author contributions Tina Yaghoobpour : Molecular analysis, writing the initial manuscript and editing. Ehsan Rakhshandehroo : writing the manuscript, reviewing and editing. Hassan Sharifi Yazdi : reviewing and editing. Masoud Nematinejad : Hematological and biochemical analysis, sample collection, and providing history and images from the sick animal. Funding This work was partially supported by Shiraz University, Iran, following the projects designed to study the Leishmaniasis in dogs (grant number: PB1403). Data availability Data supporting the reported results are included in the article. In addition, the nucleotide sequence obtained from the parasite was deposited in the GenBank as the accession Number: PP848956. Ethics approval The animal study and sample collection were reviewed and approved by the Research and Ethics Committee of the Faculty of Veterinary Medicine, Shiraz University, Iran (No. MNN402/2024). Consent to participate The consent for research was obtained from the owner before clinical examination and sampling. Consent for publication All images captured and history information from the diseased dog was allowed by the pet owner to be published and available for the scientific community. Competing of interest The authors declare no competing interests. Data availability Data supporting the reported results are included in the article. In addition, the nucleotide sequence obtained from the parasite was deposited in the GenBank as the accession Number: PP848956. 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Shokri A, Fakhar M, Teshnizi SH (2017) Canine visceral leishmaniasis in Iran: A systematic review and meta-analysis. Acta Trop 165:76-89. Solano-Gallego L, Koutinas A, Miró G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, Baneth G (2009) Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol 165:1-18. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4592206","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":323788541,"identity":"6d00b814-e895-4f49-8446-8e556cce3a00","order_by":0,"name":"Tina Yaghoobpour","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Tina","middleName":"","lastName":"Yaghoobpour","suffix":""},{"id":323788542,"identity":"ef8d6863-d629-48f5-ba7b-c41a936e15f2","order_by":1,"name":"Ehsan Rakhshandehroo","email":"data:image/png;base64,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","orcid":"","institution":"Shiraz University","correspondingAuthor":true,"prefix":"","firstName":"Ehsan","middleName":"","lastName":"Rakhshandehroo","suffix":""},{"id":323788543,"identity":"963fc80c-3f7e-490d-aac0-ec357670bd80","order_by":2,"name":"Hassan Sharifiyazdi","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Sharifiyazdi","suffix":""},{"id":323788544,"identity":"103bd9a3-a08e-421b-8710-35edc031b997","order_by":3,"name":"Masoud Nematinejad","email":"","orcid":"","institution":"Shiraz University","correspondingAuthor":false,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Nematinejad","suffix":""}],"badges":[],"createdAt":"2024-06-17 06:44:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4592206/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4592206/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60599179,"identity":"68584163-cbb9-416f-b41c-a1f5d4d950d1","added_by":"auto","created_at":"2024-07-18 15:56:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1101899,"visible":true,"origin":"","legend":"\u003cp\u003eA 2-year-old male \u003cem\u003eDogo Argentino\u003c/em\u003e showing extensive skin lesions with exfoliative dermatitis on the face (A to C) and also ulcerative nodule in this area (D).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4592206/v1/a814b856717fba68a522c7f7.jpg"},{"id":60599178,"identity":"9e8351a0-ceed-42dc-9e2a-73ab5a417a10","added_by":"auto","created_at":"2024-07-18 15:56:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4203668,"visible":true,"origin":"","legend":"\u003cp\u003eGiemsa stained smear rpresented many of Leishman bodies (or amastigotes) of the parasite in a macrophage cell. They are also observed as freely in the field.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4592206/v1/d31201f56d2bc5da2ab64bee.jpg"},{"id":60599182,"identity":"99a59cd5-2ecd-429d-baf0-766997fb82bd","added_by":"auto","created_at":"2024-07-18 15:56:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1794611,"visible":true,"origin":"","legend":"\u003cp\u003eThe agarose gel shows the presence of the expected band of about 250 bp for the sample examined; Lane 1 (S): sample; Lane 2 (PC): Positive control; Lane 3 (L): Ladder (Sina clone\u003csup\u003e®\u003c/sup\u003e, Iran; Cat No., SL7021); Lane 4 (NC): Negative control.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4592206/v1/e5eadfd62a9529de56e4727a.jpg"},{"id":60599181,"identity":"36170b45-81e7-4c91-a9dc-08a21e01a635","added_by":"auto","created_at":"2024-07-18 15:56:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":673643,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree inferred from the analysis on the ITS-1 region using Maximum Likelihood method. The clades belonged to the species of \u003cem\u003eL. infantum\u003c/em\u003e(including the present sample and the other from dogs, human and the sandflies), \u003cem\u003eL. tropica\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e are well seperated.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4592206/v1/ba771d7ca9fcf872703a180b.jpg"},{"id":61653145,"identity":"f4e075d1-7b20-4f07-97af-ab5714d66fa6","added_by":"auto","created_at":"2024-08-02 12:53:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8239030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4592206/v1/5a473996-ee24-4732-83bc-ed32b4223f5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Leishmania infantum in a dog with lymphadenopathy and extensive cutaneous lesions: Hematological, biochemical, cytological and molecular findings","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe zoonotic disease known as leishmaniasis is caused by prevalent species belonging to the genus \u003cem\u003eLeishmania\u003c/em\u003e. These parasitic protozoa are members of the Trypanosomatidae family and the Kinetoplastidae order (Ikeda et al. 2008) and affect numerous mammals, including humans, cats, dogs, horses, and rodents (Handman and Bullen 2002). The clinical disease is mostly caused by cutaneous and visceral invasions. Dogs and rats act as predominant reservoirs of cutaneous leishmaniasis (CL) (Mohebali et al. 2005), while the primary reservoirs of human visceral leishmaniasis (VL) are wild carnivores such as wolves, foxes, and jackals. Rodents have proven to be the primary reservoirs of mucocutaneous leishmaniasis (MCL) (Dantas-Torres. 2007).\u003c/p\u003e \u003cp\u003eThere are several species of \u003cem\u003eLeishmania\u003c/em\u003e that can infect dogs, including \u003cem\u003einfantum, tropica\u003c/em\u003e, \u003cem\u003emajor, braziliensis\u003c/em\u003e, etc. (de Andrade et al. 2006; Reithinger and Davies 1999) Each species may be related to a range of clinical signs, from cutaneous to visceral. Of those species, the pathogenic potential of \u003cem\u003eL. infantum\u003c/em\u003e has been more widely considered because of its systemic implications. The World Health Organization (WHO) reports fatalities in humans due to visceral leishmaniasis (VL) worldwide. (Bi et al. 2018). In canids, \u003cem\u003eLeishmania infantum\u003c/em\u003e (from the \u003cem\u003eDonovani\u003c/em\u003e complex) may also be characterized by cutaneous manifestations (Saridomichelakis and Koutinas 2014). In the Middle East area, the cutaneous form has mostly been associated with infection with two other species, \u003cem\u003eL. major\u003c/em\u003e and \u003cem\u003eL. tropica\u003c/em\u003e (Baneth et al. 2022; Fakhar et al. 2022). The latter species has sometimes been reported to be found in blood samples, implying its possibility of causing the visceral type. Also, cross-reactivity has been found between \u003cem\u003eL. tropica\u003c/em\u003e and \u003cem\u003eL. infantum\u003c/em\u003e (Hajjaran et al. 2007). Therefore, detection of the species involved should be concerned with predicting the pathogenesis of the parasite.\u003c/p\u003e \u003cp\u003eThe gold standard for laboratory diagnosis for \u003cem\u003eLeishmania\u003c/em\u003e is the use of parasitological and serological methods. In addition, hematology, biochemistry, and clinical immunology are examples of nonspecific approaches (Ciaramella and Corona 2003). Conventional PCR (CnPCR) has undeniable advantages over traditional methods, including high sensitivity and specificity and the possibility of direct application to clinical specimens. This technique has also made significant improvements in recent years, employing quantitative real-time PCR (qPCR) (Paiva-Cavalcanti 2010).\u003c/p\u003e \u003cp\u003eMany studies are available reporting the \u003cem\u003eLeishmania\u003c/em\u003e sp. infection in the Middle East area, both in humans and carnivores. The parasite species are predominantly composed of \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. major\u003c/em\u003e and \u003cem\u003eL. tropica\u003c/em\u003e (Baneth et al. 2022). In Iran, these species have been reported in dogs with an emphasis on the visceral type in recent years. The visceral infection has been described in about half of the provinces; however, many of them were diagnosed by a serologic routine direct antiglobulin test (DAT) test on blood samples. (Shokri et al. 2017), with rare information on its role in forming significant skin lesions. Also, the serologic method is not valid to distinguish at the species level. In this report, we describe the infection of a popliteal lymph node in a dog with considerable cutaneous lesions on the face. Also, the parasite was traced using the PCR method.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCase presentation and sample collection\u003c/h2\u003e \u003cp\u003eAn adult, 2-year-old male \u003cem\u003eDogo Argentino\u003c/em\u003e with extensive skin lesions showing exfoliative dermatitis on the face, around his eyes, and on his nose was referred to a small animal hospital located in Mazandaran province, north of Iran (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ulcerative nodules were also observed in this area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The owner stated that these lesions had started to appear ten days before the visit. One month before the visit, the dog was lethargic and had lost his appetite. A physical examination revealed the presence of generalized eczematous and exfoliative dermatitis (erythroderma) and lymph node enlargement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter a clinical examination, the popliteal lymph node was sampled using the fine needle aspiration technique and underwent direct microscopical investigation. We also drew samples of serum and blood for biochemical and hematological analyses, respectively. Hematological factors were measured by an automated hematology analyzer (Nihon Kohden, MEK-6450 Celltac Alpha, Tokyo, Japan). Moreover, \u003cem\u003em\u003c/em\u003eeasurements of biochemical parameters were done using commercial kits supplied by Biorex Co. (Fars, Iran), and an AutoAnalyzer (Alpha\u003csup\u003e\u0026reg;\u003c/sup\u003e Classic, Iran).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction and Polymerase Chain Reaction (PCR)\u003c/h2\u003e \u003cp\u003eDNA was extracted from the prepared lymph node smear using a commercial kit (Parstous\u003csup\u003e\u0026reg;\u003c/sup\u003e, Iran). The ribosomal DNA gene targeting the internal transcribed spacer 1 gene (ITS1) region was considered to detect \u003cem\u003eLeishmania\u003c/em\u003e parasites using primers LSGITS1-F1: CATTTTCCGATGATTACAC and LSGITS1R1: CGTATGTGAGCCGTTATC (de Almeida et al. 2011). The amplification reaction involved a touchdown PCR program as follow: The initial denaturation performed at 95\u0026deg;C for 5 min, followed by 35 cycles including denaturation at 94\u0026deg;C for 1 min, annealing at 58\u0026thinsp;\u0026minus;\u0026thinsp;54\u0026deg;C (the temperature was reduced 1\u0026deg;C in each subsequent cycle and fix the final annealing temperature at 54\u0026deg;C) for 1 min, extension 72\u0026deg;C for 45 s. The final extension step was done for 5 min at 72\u0026deg;C following the last cycle. The results were electrophoresed on a 3% agarose gel and stained with Safe DNA Gel Stain (Sinaclone\u003csup\u003e\u0026reg;\u003c/sup\u003e; Iran). PCR products were sequenced (Pishgam Biotech Co., Iran), and the results were compared with other available sequences in the NCBI database using the BLAST search. The MEGA X software was applied to align the sequences and the phylogenetic tree was drawn using the maximum likelihood method (Kumar et al. 2018).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eLight microscopy on the Giemsa-stained lymph node smear revealed the parasite amastigotes, known as Leishman bodies, both inside and outside of the macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The laboratory report disclosed mild leukocytosis: (white blood cell (WBC)\u0026thinsp;=\u0026thinsp;22.3 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l), (lymphocytosis\u0026thinsp;=\u0026thinsp;5.6\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l), neutrophilia: (segmented neutrophils\u0026thinsp;=\u0026thinsp;15.2 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l), a left shift (band neutrophil\u0026thinsp;=\u0026thinsp;0.60 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l), Hematological findings also showed a low hematocrit; however, the thrombocyte counts were normal (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Also, biochemistry analysis indicated hypoalbuminemia (2.3 g/dl), decreased albumin to globulin (A/G) ratio (0.3) and hyperproteinemia (10.1 g/dl) as a result of hyperglobulinemia (7.9 g/dl). Furthermore, hyperglycemia and mild azotemia were also detected as serum biochemical abnormalities (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eValues of the hematological parameters in the \u003cem\u003eLeishmania\u003c/em\u003e infected dog.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematological parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLevel*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference Range\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.3 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0\u0026ndash;17.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.6 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026ndash;4.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSegmented Neutrophil (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.2 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026ndash;11.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBand Neutrophil (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecell /\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0-0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocyte (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells /\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u0026ndash;1.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEosinophil (\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells /\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u0026ndash;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCT (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u0026ndash;55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC (\u0026times;10\u003csup\u003e6\u003c/sup\u003ecells /\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.4 L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u0026ndash;8.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHb (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u0026ndash;18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCV (fl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u0026ndash;77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCH (pg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.5\u0026ndash;24.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHC (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u0026ndash;36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelet (\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells /\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u0026ndash;500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*H: High; L: Low\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eValues of the biochemical parameters in the \u003cem\u003eLeishmania\u003c/em\u003e infected dog.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochemical parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLevel*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference Range\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood urea nitrogen (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrea (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.2 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026ndash;70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCreatinine (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026ndash;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerid (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u0026ndash;152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesterol (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95\u0026ndash;337\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u0026ndash;120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALP (IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u0026ndash;280\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.9\u0026ndash;11.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphorus (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8-6.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.3 L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026ndash;4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlobulin (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.9 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2\u0026ndash;4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal bilirubin (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal protein (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.2 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0-7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/G ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3 L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026ndash;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*H: High; L: Low\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the molecular test based on the ITS-1 region, a\u0026thinsp;~\u0026thinsp;250 bp band was achieved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to the sequencing data and the BLAST search, the recovered specimen was \u003cem\u003eL. infantum.\u003c/em\u003e According to the phylogenetic analysis, our specimen was grouped with a variety of \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eL. donovani\u003c/em\u003e strains reported from dogs, human, sandflies and even cats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These two species were separated well from \u003cem\u003eL. tropica\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e. The ITS-1 sequence data for the present strain was recorded in the GenBank under the accession number PP848956.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe diagnosis of leishmaniasis has been made after careful consideration of the symptoms and laboratory results. Displaying cutaneous lesions in the animal's face may be a common symptom in affected canines (Saridomichelakis and Koutinas 2014). In this study, we described a case infected with a visceral type of leishmaniasis showing a relatively widespread dermatitis, specifically on the face, which was accompanied by some hematological (anemia, leukocytosis, and neutrophilia with a left shift) and biochemical (hyperglobulinemia, hyperproteinemia, hyperglycemia, and mild azutemia) abnormalities.\u003c/p\u003e \u003cp\u003ePreviously, the clinical picture of visceral leishmaniasis in dogs was classified into several categories. The occurrence of systemic, reticuloendothelial, cutaneous, ocular, and clinicopathological signs is assumed (Foglia Manzillo et al. 2013). In different studies, variabilities are seen, as some of them are more regular and a group of signs are rare. Conversely, a number of such infections are found in asymptomatic dogs (Ribeiro et al., 2018), and some signs could appear during the progression of the infection (Foglia Manzillo et al. 2013). In this case, reports indicate that signs of lymphadenopathy, ulcerative nodules, and generalized eczematous and exfoliative dermatitis (erythroderma) coincided with clinicopathological features.\u003c/p\u003e \u003cp\u003eBased on previous investigations in Iran and the Middle East, the main causative agents of cutaneous leishmaniasis are \u003cem\u003eL. tropica\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e in dogs (Bamorovat et al. 2015; Baneth et al. 2016; 2017). On the other hand, cases of visceral infection with \u003cem\u003eL. tropica\u003c/em\u003e have also been reported in dogs in Iran (Mohebali et al. 2005; Hajjaran et al. 2007), Another species, \u003cem\u003eL\u003c/em\u003e. infantum, was reported from endemic regions for VL in Iran, but this parasite has usually not been associated with widespread cutaneous pathogenesis (Shokri et al. 2017). Therefore, our finding represents a rare case of canine leishmaniasis with extensive skin disease, which was confirmed by molecular and cytological methods on the lymph node sample.\u003c/p\u003e \u003cp\u003eFrom the hematological perspective, leukocytosis, neutrophilia, and left shift were seen in the presented case. The leukogram changes may not be as considerable in the symptomatic dogs as described by Ribeiro et al. (2013) But, in line with our data, other available research has linked the \u003cem\u003eLeishmania\u003c/em\u003e infection to the observed WBC abnormality (Jafari Shourijeh et al. 2006; Bogdan et al. 2001). Nicolato et al. (2013) reported a high population of myelocytes, metamyelocytes, and band neutrophils among the granulocytic lineage precursors in the bone marrow. Based on the inflammatory response in multiple organs affected by parasitism, these results indicate a high demand for neutrophils in symptomatic dogs.\u003c/p\u003e \u003cp\u003eAnemia may be a common symptom of canine leishmaniasis, depending on the parasite load and pathogenesis. Typically, da Costa-Val et al. (2007) observed medullary hypoplasia and normocytic normochromic anemia. Anemia tends to be related to the clinical stage (Dias et al. 2008). But in this instance, the hematocrit was measured at 27%, which was regarded as quite anemic. This finding, however, has been documented before and various factors have been indicated. (Amusategui et al. 2003; Ribeiro et al. 2013). Serum protein analysis may detect anomalies at an early stage in the progression of the disease. (Foglia Manzillo et al. 2013). Fitting with our results, there is a strong correlation between the severity of the clinical score and a rise in total proteins and globulins (Koutinas et al. 1999; Almeida et al., 2005; Solano-Gallego et al. 2009; Paltrinieri 2010; Proverbio et al. 2014). Otherwise, the albumin-to-globulin (A/G) ratio drops for no valid reason other than that albumin is a negative acute phase protein and the proteinuric nephropathy can induce albumin loss as well (Almeida et al. 2005; Ribeiro et al. 2013). Some researchers have argued that a drop in the A/G ratio is the most sensitive indicator of canine leishmaniasis (Almeida et al. 2005; Geisweid et al. 2012; Paltrinieri et al. 2016). These outcomes are comparable to our biochemical findings. According to the literature, various types of samples have been used for molecular diagnosis. The PCR method on bone marrow proposed as a highly sensitive method to find visceral leishmaniasis (Oliva et al., 2006). However, in \u003cem\u003eL. infantum\u003c/em\u003e-infected dogs, the popliteal lymph node and conjunctival sampling techniques are less invasive and result in good PCR conclusions (Marcelino et al., 2020). In this study, DNA obtained from a lymph node -as a minimally-invasive approach- was successfully amplified and sequenced using PCR assay.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis report describes a rare case of visceral leishmaniosis with generalized eczematous and exfoliative dermatitis (erythroderma) and lymph node invasion in a dog from northern Iran. The region has documented cases of visceral Leishmaniasis infection utilizing serologic assays, but there is a lack of information regarding the molecular entity of the parasite. Our study should be taken into account for infection control purposes, as \u003cem\u003eL. infantum\u003c/em\u003e is a more dangerous species for canines and may threaten public health in this region of Iran.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ecutaneous leishmaniasis (CL), visceral leishmaniasis (VL), World Health Organization (WHO), Conventional PCR (CnPCR), quantitative real-time PCR (qPCR),\u003cem\u003e\u0026nbsp;direct\u003c/em\u003e antiglobulin test (\u003cem\u003eDAT\u003c/em\u003e), internal transcribed spacer 1 gene (ITS1), White blood cell (WBC), albumin-to-globulin (A/G), Polymerase Chain Reaction (PCR), Lymph node (LN).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTina Yaghoobpour\u003c/strong\u003e: Molecular analysis, writing the initial manuscript and editing. \u003cstrong\u003eEhsan Rakhshandehroo\u003c/strong\u003e: writing the manuscript, reviewing and editing. \u003cstrong\u003eHassan Sharifi Yazdi\u003c/strong\u003e: reviewing and editing. \u003cstrong\u003eMasoud Nematinejad\u003c/strong\u003e: Hematological and biochemical analysis, sample collection, and providing history and images from the sick animal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by Shiraz University, Iran, following the projects designed to study the Leishmaniasis in dogs (grant number: PB1403).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data supporting the reported results are included in the article. In addition, the nucleotide sequence obtained from the parasite was deposited in the GenBank as the accession Number: PP848956.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study and sample collection were reviewed and approved by the Research and Ethics Committee of the Faculty of Veterinary Medicine, Shiraz University, Iran (No. MNN402/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe consent for research was obtained from the owner before clinical examination and sampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll images captured and history information from the diseased dog was allowed by the pet owner to be published and available for the scientific community.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData supporting the reported results are included in the article. In addition, the nucleotide sequence obtained from the parasite was deposited in the GenBank as the accession Number: PP848956.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlmeida MAOD, Jesus EEV, Sousa-Atta, MLB, Alves LC, Berne MEA, Atta AM (2005) Clinical and serological aspects of visceral leishmaniasis in northeast Brazili dogs naturally infected with \u003cem\u003eLeishmania chagasi\u003c/em\u003e. Vet Parasitol 127:227-232. \u003c/li\u003e\n\u003cli\u003eAmusategui I, Sainz A, Rodriguez F, Tesouro MA (2003) Distribution and relationships betweenclinical and biopathological parameters in canine leishmaniasis. Eur J Epidemiol 18:147-156.\u003c/li\u003e\n\u003cli\u003eBamorovat M, Sharifi I, Dabiri S, Mohammadi MA, Harandi MF, Mohebali M, Aflatoonian MR, Keyhani A (2015) Leishmania tropica in stray dogs in southeast Iran. 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Am J Trop Med Hyg\u003cem\u003e \u003c/em\u003e61:530-541. \u003c/li\u003e\n\u003cli\u003eRibeiro RR, Silva SMD, Fulg\u0026ecirc;ncio GDO, Michalick MSM, Fr\u0026eacute;zard FJG (2013) Relationship between clinical and pathological signs and severity of canine leishmaniasis. Rev Bras Parasitol 22:373-378.\u003c/li\u003e\n\u003cli\u003eRibeiro RR, Michalick MSM, da Silva ME, Dos Santos CCP, Fr\u0026eacute;zard FJG, da Silva SM (2018). Canine Leishmaniasis: An Overview of the Current Status and Strategies for Control. Biomed Res Int 2018:3296893.\u003c/li\u003e\n\u003cli\u003eSaridomichelakis MN, Koutinas AF (2014) Cutaneous involvement in canine leishmaniosis due to L eishmania infantum (syn. \u003cem\u003eL. chagasi\u003c/em\u003e). Vet Dermatol 25:61-e22. \u003c/li\u003e\n\u003cli\u003eShokri A, Fakhar M, Teshnizi SH (2017) Canine visceral leishmaniasis in Iran: A systematic review and meta-analysis. Acta Trop 165:76-89.\u003c/li\u003e\n\u003cli\u003eSolano-Gallego L, Koutinas A, Mir\u0026oacute; G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, Baneth G (2009) Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol 165:1-18.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dog, lymph node, Leishmania infantum, Hematology","lastPublishedDoi":"10.21203/rs.3.rs-4592206/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4592206/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eLeishmania\u003c/em\u003e spp., protozoal parasites belonging to the Trypanosomatidae family, are the cause of a common zoonotic illness. Dogs are the main reservoirs of the parasites, which play a considerable role in infecting humans and other hosts. This report explains lymph node involvement by amastigotes of the viscerotropic \u003cem\u003eLeishmania\u003c/em\u003e species in a dog who suffered from ulcerative nodules and generalized eczematous and exfoliative dermatitis (erythroderma).\u003c/p\u003e\n\u003cp\u003eA 2-year-old male dog with evident acute skin lesions and ulcerative nodules on the face was referred to a small animal hospital in Mazandaran province, Iran. The animal had started showing signs ten days prior and was lethargic and lost appetite. In order to detect \u003cem\u003eLeishmania\u003c/em\u003e parasites, the popliteal lymph node was sampled for the cytological examination and also polymerase chain reaction (PCR) based on the ITS-1 region of the ribosomal DNA gene. Also, a blood sample was collected to determine hematological and biochemical parameters. As a result, Leishman bodies were seen both inside and outside of macrophages when light microscopy was performed on the lymph node sample stained with Giemsa. Laboratory findings revealed mild leukocytosis, lymphocytosis, neutrophilia, low hematocrit. hyperglobulinemia, hyperproteinemia, hypoalbuminemia, declined albumin/globulin ratio, and hyperglycemia. The PCR and sequencing results confirmed the presence of \u003cem\u003eLeishmania\u0026nbsp;\u003c/em\u003ein the popliteal lymph node. According to the molecular analysis, \u003cem\u003eL. infantum\u003c/em\u003e was the causative agent of leishmaniasis in this case.\u003c/p\u003e\n\u003cp\u003eCutaneous leishmaniasis is prevalent in humans and dogs in Iran. In most cases, \u003cem\u003eL. tropica\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e are the major causative agents of this form of leishmaniasis, and the lesions are confined. In this report, a generalized skin disease was evident. In addition, hematological and biochemical parameters supported a visceral form of leishmania infection in the animal. Because \u003cem\u003eL. infantum\u003c/em\u003e is a more dangerous species than the other types for dogs and specifically humans, our report should be considered to control the infection. This report is significant as this unusual form of visceral leishmaniasis \u0026nbsp;in the dog raises concerns about the possibility of zoonotic transmission and may threaten public health.\u003c/p\u003e","manuscriptTitle":"Leishmania infantum in a dog with lymphadenopathy and extensive cutaneous lesions: Hematological, biochemical, cytological and molecular findings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 15:56:31","doi":"10.21203/rs.3.rs-4592206/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9ab5874-d604-4242-b5da-663717513c4a","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-02T12:45:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 15:56:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4592206","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4592206","identity":"rs-4592206","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}