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Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonosis. The domestic dog is the primary reservoir in urban areas. This study aimed to evaluate the frequency, active infection and load of L. We studied 45 male and 25 female L. Tissue samples from the testis, epididymis, prostate, vulva, vagina, and uterus were examined by singleplex qPCR and parasitological tests histopathology, immunohistochemistry, and parasitological culture. The latter were performed for the detection of active infection parasites able to multiply and to induce lesions.

Parasite lo were similar in the testis, vulva, epididymis and vagina and lower in the prostate. Only the parasite load in the vagina was ificantly associated with the of clinical s. Granulomatous inflammation predominated in all organs, except for the prostate. Only in the testis and epididymis was the inflammatory infiltrate ificantly more intense among dogs with a higher parasite load in these organs. The high frequency, detection of active infection and similarity of L. Additionally, vertical transmission may be frequent since active L.

This is an open access article distributed under the terms of the Creative Commons Attribution , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study de, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Canine visceral leishmaniasis CVL caused by the protozoan Leishmania infantum is a zoonosis in which the domestic dog Canis familiaris is the primary reservoir in urban areas [ 1 ].

In addition to classical transmission through the bite of infected sandflies, venereal and vertical transmission of the disease is possible [ 2 — 5 ]. In the genital tract of dogs, amastigote forms or DNA of Leishmania have been detected in the testis, epididymis, prostate, glans penis, prepuce, scrotum, smegma, semen, vulva, vagina, vaginal secretion, placenta, uterus, uterine tubes, and ovaries [ 2 , 4 , 6 — 16 ]. An accurate marker of L. In addition to providing a diagnostic result, this technique allows to determine the of copies of the DNA target sequence [ 17 ].

In the male genital tract of dogs naturally infected with L. In the female genital tract, the L. Some authors suggest tropism of L. In addition, venereal transmission of L. However, other studies found a high frequency of parasitism in the genital tract of females, suggesting tropism of L. The detection of vaginal secretion contaminated with L. In an experimental study, one of eight L. Taken together, these findings demonstrate the lack of consensus regarding the role of females in venereal transmission of L. The quantification of L. However, studies on the frequency of L. In addition, these studies have evaluated small populations and only males or females from different endemic areas.

Therefore, this study aims to describe the frequency, presence of active infection and parasite load of L. This was a descriptive study conducted from August to December that used a non-probability sample of 70 dogs 45 males and 25 females. These two serological tests and diagnostic criteria were used because they are accurate [ 24 ] and are recommended by the Brazilian Ministry of Health for considering a dog to be infected with L.

Euthanasia was performed according to the recommendations of the Brazilian Ministry of Health for the control of visceral leishmaniasis [ 1 ], and the owners provided ed consent. The dogs were not housed for any period of time prior to euthanasia. Immediately after arrival at INI-Fiocruz, the dogs were restrained mechanically and first evaluated clinically, including inspection of the skin and mucosae and palpation of superficial lymph nodes and abdominal organs.

The following clinical s of CVL were considered: thinness or cachexia; diffuse or localized hair loss; cutaneous lesions such as ulcers and desquamation; onychogryphosis; enlargement of the superficial lymph nodes, liver or spleen on palpation; pale ocular or oral mucosae, and skeletal muscle hypotrophy [ 26 , 27 ]. The animals were divided into two groups according to the clinical s of CVL: no or few clinical s up to three clinical s and multiple clinical s more than three clinical s.

The euthanasia procedure was performed in accordance with the guidelines of the Federal Council on Veterinary Medicine of Brazil [ 28 ]. During necropsy, three fragments were collected from each organ of the male right and left epididymis, right and left testis, and prostate and female genital tract vulva, caudal vagina adjacent to the vulva, and uterine corpus. One fragment was immersed in sterile saline and cultured for the detection of Leishmania. In the case of double organs, one fragment of the right and left testis and epididymis each was collected and examined separately by HP and IHC.

For parasitological culture and qPCR, a pool of samples containing one fragment of the right and left testis and a pool of samples containing one fragment of the right and left epididymis were examined. The parasitological culture was considered positive if promastigote forms of Leishmania grew in the culture medium up to 30 days and were visualized by conventional optical microscopy [ 30 ]. Parasites isolated in culture were identified as L. Parasite load was quantified by constructing a standard curve with serial dilutions 10 1 to 10 5 parasites of L.

Positive and negative controls were included in each amplification plate and a threshold of 0. The DNA of 1 x 10 5 promastigote forms of L. Samples in which DNA amplification occurred after the 37 th cycle were classified as undetectable.

The L. The are expressed as positive or negative and samples showing amplification were considered free of DNA degradation and PCR inhibitors. For the comparison of parasite load between the different organs of the male and female genital tract, only dogs positive for L. In turn, for the comparison of parasite load between the different organs of the male and female genital tract according to the clinical s of the dogs and intensity of inflammatory infiltration in these organs, only dogs that tested positive for L.

Histological sections of organs intensely parasitized with amastigote forms of Leishmania were incubated with non-immune homologous serum as negative control and with polyclonal rabbit anti- Leishmania serum as positive control. The testis and epididymis were considered positive for Leishmania if amastigote forms of this parasite were detected in at least one fragment of the right or left organ. In addition, the frequencies of positivity for amastigote forms of L. The inflammatory infiltrate in the organs was classified as follows: granulomatous, predominance of cells of the monocyte-macrophage system activated macrophages, epithelioid macrophages, or multinucleate giant cells ; non-granulomatous, predominance of other types of inflammatory cells lymphocytes, plasma cells, and neutrophils.

The intensity of inflammatory infiltration was classified as absent or mild absent cellular infiltrate or mild and dispersed foci and moderate to intense dense and diffuse cellular infiltrate. The frequency of histological alterations in the genital tract was compared between the group of dogs that tested positive and the group of dogs that tested negative for Leishmania by the parasitological methods parasitological culture, HP or IHC in these organs.

This comparison was done to verify the association between lesions with active Leishmania infection. For calculation of the frequency of each histological alteration in the testis and epididymis, the histological alteration was considered present if detected in at least one fragment of the right or left organ. The intensity of inflammatory infiltration was classified in the organ right or left with the most intense inflammatory infiltrate. Data were analyzed using the free R software, version 3. The following variables were reported as simple frequencies: clinical classification no or few clinical s of CVL; multiple clinical s of CVL , positive with the diagnostic techniques, and histological alterations.

Boxplots were used for comparison of log parasite lo in the samples of each organ. The Shapiro-Wilk test rejected the normality assumption of the parasite load data. Thus, the correlation between L. The correlations ranged from -1 to 1, with positive values indicating a positive correlation and negative values an inverse correlation.

In addition, the nonparametric Mann-Whitney test was applied to compare log parasite lo among all organs of the genital tract male and female ones and according to the intensity of clinical s and inflammatory infiltrate. This study was carried out in strict accordance with the recommendations of the Brazilian Ministry of Health and the Federal Council on Veterinary Medicine, with permission of the owners.

Clinical examination of the 70 dogs revealed clinical s in 59 The following clinical s were observed: thinness 26 males and 10 females , skin ulcer 25 males and 9 females , hair loss 23 males and 10 females , onychogryphosis 20 males and 11 females , lymphadenomegaly 21 males and 10 females , furfuraceous desquamation 21 males and 5 females , splenomegaly 11 males and 7 females , cachexia 8 males and 4 females , keratoconjunctivitis 8 males and 3 females , and hepatomegaly 2 males and 2 females.

Thirty-four The only negative animal in all assays was a male without clinical s. In the 23 dogs 14 males and nine females in which the parasitological tests did not detect Leishmania in the genital tract, L. The qPCR technique detected L.

Fig 1 illustrates the L. The colored horizontal lines indicate the median parasite load. The vertical lines indicate the interquartile range. Colored dots represent the parasite load of each dog. The comparison of L. Only parasite load in the vagina was ificantly associated with the of clinical s.

In the genital tract of the 47 dogs that tested positive for Leishmania by the parasitological methods, histological alterations were observed in at least one of the organs examined in all 31 males Fig 2 and 16 females Fig 3. Among the 23 dogs with negative parasitological tests, 10 males and 9 females exhibited histological alterations in at least one of the organs examined. In these 19 dogs, the inflammatory infiltrate observed in the genital tract organs was associated with the detection of L. Table 4 shows the frequencies of histological alterations in the genital tract of dogs that tested positive and negative for Leishmania by the parasitological methods parasitological culture, HP or IHC in these organs.

Hematoxylin-eosin staining a,c,e ; immunohistochemistry b,d,f.

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