Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report

  • Laura Manna1Email author,

    Affiliated with

    • Orlando Paciello2,

      Affiliated with

      • Rossella Della Morte3 and

        Affiliated with

        • Angelo Elio Gravino1

          Affiliated with

          Parasites & Vectors20125:216

          DOI: 10.1186/1756-3305-5-216

          Received: 10 September 2012

          Accepted: 10 September 2012

          Published: 28 September 2012

          Abstract

          Background

          Transmission of canine leishmaniasis (CanL), a severe infection caused by L. infantum, usually occurs through the sand fly bite to the vertebrate host. A venereal route of transmission has also been suggested, but this issue is still controversial.

          Findings

          Here, we report a case of a dog affected by orchitis showing a clinical profile of L. infantum infection. By exploiting a real-time PCR assay, we detected a significantly higher DNA load of the parasite in the lymph node and testis than in blood and urine samples collected from the dog.

          Conclusions

          Our results suggest that: 1) L. infantum infection can be associated with testicular lesions in naturally infected dogs; 2) genital involvement could result in shedding of the parasites in the semen, favoring venereal transmission of the disease.

          Keywords

          Dog Leishmaniasis Orchitis Real-time PCR Testis

          Findings

          Introduction

          Canine leishmaniasis (CanL) is a severe sandfly-borne infection caused by the protozoan parasite Leishmania infantum (syn. L. chagasi) widely distributed in temperate and subtropical countries. The clinical manifestations of the disease range from unapparent subclinical infections to a systemic disease characterized by weight loss, lymphadenopathy, hemorrhagic diarrhea, ocular lesions, and hyperthermia, frequently associated also with dermatological lesions. Dogs are the main reservoir of L. infantum and they play a central role in the transmission of the disease to humans. The prevalence and the incidence of CanL have been underestimated until now[1, 2]. Transmission of the disease usually occurs through the phlebotomine sand fly bite to the vertebrate host; however, CanL transmission through blood transfusion has been documented[3]. Recent evidence also suggests a venereal route of transmission due to the presence of parasites in the semen of infected dogs[47]. However, the L. infantum tropism to the canine genital system is still a controversial question. Here, we report a case of orchitis in a leishmaniotic dog associated with the presence of Leishmania DNA in the testis.

          Case report

          A 6-yr-old male Husky was hospitalized at the Department of Veterinary Clinical Science of the Federico II University of Naples for the evaluation of a seborrhoeic, pruriginous dermatitis (Figure1) with a history of two months of alopecia and pruritus. The dog had been treated for sarcoptic mange without any improvement. Physical examination showed an enlargement of the right testis (Figure2). No lymphadenopathy was recorded. Routine fine needle aspiration cytology from the enlarged testis was performed by the attending pathologist. Air-dried slides were prepared, and immediately stained with May Grunwald-Giemsa Quick Stain (Bio-Optica, Milan). Clinical laboratory parameters were evaluated. Indirect immunofluorescent antibody test (IFAT) for Leishmania spp. was carried out, and samples from blood, urine, lymph node, and testis were collected, and processed for the evaluation of Leishmania DNA load by real-time PCR assay as previously described[8].
          http://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-216/MediaObjects/13071_2012_Article_701_Fig1_HTML.jpg
          Figure 1

          A 6-yr-old male Husky dog showing clear evidence of dermatitis.

          http://static-content.springer.com/image/art%3A10.1186%2F1756-3305-5-216/MediaObjects/13071_2012_Article_701_Fig2_HTML.jpg
          Figure 2

          Evident enlargement of right testis in the Husky dog.

          Cytological examination of testis samples showed testicular round cells with course nuclear chromatin, single large, prominent nucleoli, and moderate amounts of basophilic cytoplasm. The presence of many inflammatory cells, such as neutrophils, with nuclear degenerative changes, several lymphocytes, plasma cells and a large number of macrophages containing intracellular Leishmania amastigotes was observed. A diagnosis of chronic orchitis due to Leishmania spp. was made. Haematological and biochemical parameters were consistent with CanL (Table1) as demonstrated by the anemia, increased levels of total proteins, and high UP/C ratio.[9, 10] Real time PCR and IFAT results confirmed the infection by L. infantum of the dog (Table2).
          Table 1

          Dog laboratory parameters recorded at time 0 (T0) and after 1 (T1), 2 (T2), 3 (T3), 6 (T6) and 12 (T12) months from the diagnosis

          Substrate

          Parameter

          T0

          T1

          T2

          T3

          T6

          T12

          Normal range

          Serum

          Urea (mg/dl)

          45

          47

          29

          31

          28

          26

          25-50

           

          Creatinine (mg/dl)

          1.65

          1.3

          1.26

          1.47

          1.48

          1.54

          <1.8

           

          ALT (UI/L)

          27

          23

          25

          24

          26

          33

          10-47

           

          Glucose (mg/dl)

          81

          80

          83

          81

          76

          86

          60-110

           

          Total proteins (g/dl)

          9.4

          8.7

          7.4

          7.4

          7.4

          6.2

          6-7.8

           

          Gamma globulin (g/dl)

          3.63

          2.84

          2.16

          2.16

          2.16

          2.84

          0.9-2.2

           

          Beta globulin (g/dl)

          2.39

          2.43

          2.31

          0.31

          0.31

          0.34

          0.6-1.4

           

          Alpha 2 globulin (g/dl)

          0.41

          1.55

          1.11

          1.11

          1.11

          0.82

          0.3-1.1

           

          Alpha 1 glubulin (g/dl)

          3.50

          1.20

          1.04

          1.04

          1.04

          0.67

          0.2-0.5

           

          Albumin (g/dl)

          2.35

          0.70

          1.12

          1.12

          1.12

          0.70

          2.3-3.4

           

          A/G ratio

          2.25

          2.46

          1.96

          1.96

          1.96

          0.53

          0.7-1.11

          Blood

          RBC (x 106/μL)

          3.99

          6.01

          4.47

          4.71

          5.1

          5.2

          5.6-8.6

           

          Hb (g/dL)

          9.9

          11.3

          11.6

          12.2

          13.5

          16.3

          13.5-18

           

          WBC (x 10 3/μL)

          22.1

          24.2

          15.1

          13.4

          11.3

          8

          6-17

           

          PLT (x 103/μL)

          456

          366

          607

          370

          343

          310

          200-500

          Urine

          Density (g/ml)

          1031

          1021

          1021

          1015

          1010

          1015

          1015-1040

           

          UP/C ratio

          5.5

          2.4

          2.3

          0.3

          0.4

          0.1

          <5

          Table 2

          Leishmania load (parasite DNA/ml) in blood, lymph node aspirates, urine and testis samples at time 0 (T0) and after 1 (T1), 2 (T2), 3 (T3), 6 (T6) and 12 (T12) months from the diagnosis

          Substrate

          T0

          T1

          T2

          T3

          T6

          T12

          Blood

          321.91

          205

          13

          10

          11

          4.05

          Lymph node

          23602.83

          1694.78

          1541.89

          4.95

          10

          2.02

          Urine

          779

          178

          80

          30

          10

          3.0

          Testicle

          14406.57

          116.26

          2.88

          2.18

          3.05

          10.64

          IFAT

          1:1280

          1:640

          1:1280

          1:80

          1:160

          1:320

          The dog was treated with a combination of miltefosine[11] at a dose of 2 mg/kg/day per 28 days and allopurinol at a dose of 10 mg/kg/day for all the observation period (1 year). For follow-up assessment, biological samples were collected at the time of diagnosis (T0), and 1, 2, 3, 6, 12 months after the treatment had started (T1, T2, T3, T6 and T12, respectively).

          Before therapy, a very high parasite DNA load was detected in lymph node aspirates (23602.83 parasite DNA/ml) and testis biopsy (14406.57 parasite DNA/ml), whereas lower load values were observed in blood and urine samples (321.91 and 779 parasite DNA/ml, respectively). These results confirmed previous data on the comparative analysis of different tissues for CanL diagnosis by conventional or real-time PCR[12]. After 1 month of therapy, a progressive clinical improvement was observed, including a moderate decrease of anti-Leishmania antibody titer together with a strong reduction of Leishmania DNA load in all biological samples. However, the complete elimination of Leishmania DNA was never observed in all tissues. After the therapy, at 1 month follow-up, the cytological evaluation of the testis did not show any inflammatory cell.

          Conclusions

          This case report confirms that CanL can be associated with testis lesions (orchitis) in naturally infected dogs. The Leishmania amastigotes in the testis apparently act as a causing factor, triggering the inflammatory response. Experimental infection of hamsters with L. donovani resulted in testicular amyloidosis, degeneration, progressive atrophy, and azoospermy. In this experimental model, the degenerative changes were also associated with infiltration of lymphocytes and macrophages containing amastigotes in the testes[13]. In contrast, testicular amyloidosis was not observed in our case and in other studies[4]. In human visceral leishmaniasis, testicular involvement has not been extensively studied, but there is one study reporting amastigotes in macrophages obtained by fine needle aspiration of the testes from a boy with a severe lymphoblastic leukemia[14].

          Genital involvement during visceral leishmaniosis could result in shedding of Leishmania in the semen, favoring venereal transmission of the disease, such as reported in humans[15]. Venereal and vertical transmission of L. infantum in naturally infected dogs in Germany has been reported[7]. Inflammation associated with erosions and/or ulcerations and the presence of amastigotes in the penis and prepuce, as well as the presence of macrophages containing amastigotes migrating through the urethral epithelium might contribute to Leishmania shedding in the semen. Although the biological vector is the most important route of transmission, the possibility of CanL venereal transmission has epidemiological significance, mostly in relation to the implementation of an eradication program. This route of transmission in dogs is particularly relevant in areas where treatment and vaccination of dogs against CanL are frequent, since under those conditions potentially infective dogs may not be readily identified, thus increasing the chance of using these dogs in reproduction, which may favor spread of the disease.

          Declarations

          Acknowledgements

          This work was supported by a grant from University of Naples Federico II (Italy).

          Authors’ Affiliations

          (1)
          Department of Veterinary Clinical Science, University of Naples Federico II
          (2)
          Department of Animal Pathology and Health, University of Naples Federico II
          (3)
          Department of Biological Structures, Functions and Technologies, University of Naples Federico II

          References

          1. Palatnik-de-Sousa CB, Day MJ: One Health: the global challenge of epidemic and endemic leishmaniasis. Parasit Vectors. 2011, 4: 197-10.1186/1756-3305-4-197.PubMed CentralView ArticlePubMed
          2. Wang JY, Ha Y, Gao CH, Wang Y, Yang YT, Chen HT: The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests. Parasit Vectors. 2011, 4: 69-10.1186/1756-3305-4-69.PubMed CentralView ArticlePubMed
          3. Owens SD, Oakley DA, Marryott K, Hatchett W, Walton R, Nolan TJ, Newton A, Steurer F, Schantz P, Giger U: Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. J Am Vet Med Ass. 2001, 219: 1076-1083. 10.2460/javma.2001.219.1076.View Article
          4. Diniz SA, Melo MS, Borges AM, Bueno R, Reis BP, Tafuri WL, Nascimento EF, Santos RL: Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs. Vet Pathol. 2005, 42: 650-658. 10.1354/vp.42-5-650.View ArticlePubMed
          5. Silva F, Rodrigues AAM, Rego IOP, Santos RLH, Oliveira RG, Silva TMA, Xavier MN, Nascimento EF, Santos RL: Genital lesions and distribution of amastigotes in bitches naturally infected with Leishmania chagasi. Vet Parasitol. 2008, 151: 86-90. 10.1016/j.vetpar.2007.09.032.View ArticlePubMed
          6. Silva FL, Oliveira RG, Silva TM, Xavier MN, Nascimento EF, Santos RL: Venereal transmission of canine visceral leishmaniasis. Vet Parasitol. 2009, 160: 55-59. 10.1016/j.vetpar.2008.10.079.View ArticlePubMed
          7. Naucke TJ, Lorentz S: First report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in Germany. Parasit Vectors. 2012, 5: 67-10.1186/1756-3305-5-67.PubMed CentralView ArticlePubMed
          8. Manna L, Reale S, Viola E, Vitale F, Foglia MV, Pavone LM, Caracappa S, Gravino AE: Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs. Vet Parasitol. 2006, 142: 271-280. 10.1016/j.vetpar.2006.06.028.View ArticlePubMed
          9. Manna L, Reale S, Picillo E, Vitale F, Gravino AE: Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis. J Vet Diagn Inv. 2008, 20: 64-67. 10.1177/104063870802000112.View Article
          10. Mugasa CM, Thierry L, Schoone GJ, Basiye FL, Saad AA, el Safi S, Kager PA, Henk DFH S: Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis. Parasites & Vectors. 2010, 3: 13-10.1186/1756-3305-3-13.View Article
          11. Manna L, Reale S, Vitale F, Picillo E, Pavone LM, Gravino AE: Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. Vet J. 2008, 177: 279-282. 10.1016/j.tvjl.2007.04.013.View ArticlePubMed
          12. Manna L, Vitale F, Reale S, Caracappa S, Pavone LM, Della Morte R, Cringoli G, Staiano N, Gravino AE: Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. Vet Parasitol. 2004, 125: 251-262. 10.1016/j.vetpar.2004.07.019.View ArticlePubMed
          13. Gonzalez JL, Gallego E, Castaño M, Rueda A: Testicular amyloidosis in hamsters experimentally infected with Leishmania donovani. Br J Ex Pathol. 1983, 64: 518-523.
          14. Kapila K, Prakash MB, Mehrota R, Vermar K: Testicular leishmaniasis in a boy with acute lymphoblastic leukemia. Acta Cytol. 1994, 38: 878-879.PubMed
          15. Symmers WSC: Leishmaniasis acquired by contagion: a case of marital infection in Britain. Lancet. 1960, 16: 127-132.View Article

          Copyright

          © Manna et al.; licensee BioMed Central Ltd. 2012

          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

          Advertisement