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Correlation between chronic inflamation, immunostaining and parasite load in the genital system of female dogs naturally infected with Leishmania infantum/Correlacao entre inflamacao cronica, imunomarcacao e carga parasitaria no sistema genital de cadelas naturalmente infectadas com Leishmania infantum.

INTRODUCTION

Infection by L. infantum causes visceral leishmaniasis (VL), which is a chronic disease with a variety of forms and lesions due to complex host-parasite interactions and immune responses in dogs (NAUCKE & LORENTZ, 2012). Transmission occurs through the bite of sandflies infected with this protozoon (BARATA et al., 2004). However, vertical transmission (DUBEY et al., 2005; ROSYPAL et al., 2005; PANGRAZIO et al., 2009) and venereal transmission (NAUCKE & LORENTZ, 2012) should also be considered.

Lesions related to VL have been described less frequently in the genital system of dogs in comparison to other organs (DUBEY et al., 2005; ROSYPAL et al., 2005; OLIVEIRA et al., 2012). L. infantum amastigotes have been described in the uterus (ROSYPAL et al., 2005), placenta (DUBEY et al., 2005), vulva and vaginal mucosa. The presence of the parasite in the vulva and vaginal mucosa is related to multifocal chronic inflammatory infiltrate within macrophages, lymphocytes and plasma cells (SILVA et al., 2008; OLIVEIRA et al., 2012). However, SILVA et al. (2008) detected no L. infantum amastigotes or lesions related to VL in the cervix, uterine body, uterine horn, uterine tubes and ovaries of naturally infected female dogs.

Various methods are currently used to diagnose infection by L. infantum in dogs. Detection can be performed by parasitological (BARROUIN-MELO et al., 2006) and immunological tests, such as indirect immunofluorescence antibody test or enzyme-linked immunosorbent assay (ELISA). However, both methods are subjected to false-negative results as well as cross-reactions with other pathogens, such as Anaplasma sp. and Ehrlichia sp. (MOREIRA et al., 2007). Immunohistochemistry (IHC) (TAFURI et al., 2004), polymerase chain reaction (PCR) and real-time PCR (qPCR) (QUEIROZ et al., 2010) are currently the most specific tests for the detection of L. infantum.

Therefore, the aim of the present study was to investigate correlations among chronic inflammatory reaction, immunostaining and parasite load in the genital system of female dogs naturally infected with Leishmania infantum.

MATERIALS AND METHODS

Animals and biological samples

All animals were provided by the Department of Vector Control and Animal Surveillance of the municipality of Caruaru, state of Pernambuco, Brazil (Latitude: 08[degrees] 17' 00" S; Longitude: 35[degrees] 58' 34" W). L. infantum infection was confirmed using the indirect fluorescent antibody test (IFAT[greater than or equal to]40), parasitological bone marrow examination, IHC and qPCR of fragments from the liver, spleen, lymph nodes and skin.

Ten stray female dogs positive for L. infantum, as determined by qPCR, were submitted to euthanasia by the Department of Vector Control and Animal Surveillance following the recommendations of the Brazilian Health Ministry for the control of leishmaniasis control. After this procedure, fragments of the vulva, vagina, cervix, uterine body, uterine horns and ovaries were collected. All specimens were submitted to histopathological analysis, immunohistochemistry and qPCR.

Histopathological analysis

Samples were fixed with 10% neutral buffered formalin for 48 hours and routinely processed for embedding in paraffin. Sections (5[micro]m) were mounted on slides and stained with hematoxylin and eosin (HE) (TOLOSA et al., 2003). All tissues were evaluated according to the type of inflammatory infiltrate, intensity of the inflammatory reactions (absent=0; mild=1; moderate=2; and intense=3) as well as structural damage.

Immunohistochemistry

Detection of amastigote forms of L. infantum was performed following the method described by TAFURI et al. (2004). Heterologous hyperimmune serum from a dog naturally infected with L. infantum (positive results of parasitological examination of bone marrow and immunofluorescence antibody test (IFAT)-title [greater than or equal to]1:40), diluted at 1:100 (0.01M PBS) was used as primary antibody. A commercially available streptavidin-peroxidase complex (LSAB+ kit, Dako USA) was employed as the detection system. Reactions were revealed by diaminobenzidine (DAB) and the slides were counter-stained with hematoxylin. IHC was scored based on the number of amastigotes observed in a high magnification microscopic field (0 = no amastigotes; 1 = one to three amastigotes; 2 = four to ten amastigotes; 3 = > ten amastigotes).

Molecular diagnosis

Genomic DNA from all samples collected as well as from an in vitro culture of L. infantum was extracted using the DNeasy Blood & Tissue Kit (Qiagen[R] Hilden-Germany) following the manufacturer's instructions.

The qPCR reaction for the detection of L. infantum DNA was performed using primers Leish-1 [5'AACTTTTCTGGTCCTCCGGGTAG-3']and Leish-2 [5'-ACCCCCAGTTTCCCGCC-3'] and the TaqMan MGB-probe [FAM-5'-AAAAATGGGTGCAGAAAT-3' non-fluorescent quencher-MGB], as described by Francino et al. (2006). qPCR was performed in a CFX96(tm) RealTime System (Bio-Rad Laboratories, Inc., Hercules CA, USA). Reaction mixture (12.5[micro]L) contained 6.25[micro]L of Taqman[R] Universal PCR Master Mix, each primer at a concentration of 900nM, the probe at a concentration of 200nM and 1[micro]L of template DNA. The run consisted of a hot start at 95[degrees]C for 3 minutes and 42 cycles of denaturation (95[degrees]C for 10 seconds) and annealing-extension (60[degrees]C for 30 seconds). All assays were performed in duplicate, with a negative control (DNA of a dog from a non-endemic area) and a positive control (DNA from an in vitro culture of L. infantum) included in each run.

Parasite load was determined using the absolute quantification method. A standard curve was constructed with DNA extracted from serial dilutions of L. infantum cultures containing approximately 5.6 x [10.sup.6] parasites [mL.sup.-1]. Considering an extraction efficiency of 100%, five points were used to construct the curve (0.1, 1, 10, 1000 and 1,000,000 parasites [mL.sup.-1] of culture). As DNA extraction was performed using 300[micro]L of L. infantum culture, the final product of the extractions was diluted in a final volume of 100[micro]L and 1[micro]L of DNA was used per reaction. Points cited above correspond to 0.0003, 0.003, 0.03, 3 and 3000 parasites, respectively. Therefore, based on the same principle and considering that DNA extraction was performed using 25mg of each tissue sample, results were expressed as the number of parasites/250[micro]g of tissue.

Statistical analyses

The parasite load determined by qPCR, the number of amastigotes detected by IHC and the inflammatory score after HE staining were compared using the Kruskal-Wallis test. Spearman's test was used to determine correlations among these values. Fisher's exact test was used for comparisons of the diagnostic techniques. All analyses were performed using the R software version 2.15.2 (R CORE TEAM, 2012). To test the correlation values, qPCR data (parasites 250[micro].sup.g-1] of tissue) were transformed into scores according to the following criteria: 0 = 0; 0.001 to 0.01 = 1; >0.01 to 0.1 = 2; >0.1 to 1 = 3; >1 to 2 = 4; >2 to 5 = 5; >5 to 6 = 6; > 6 to 7 = 7; ...; >100 = 100.

RESULTS

No macroscopic lesions were reported on the genital organs of the dogs examined. Regarding the histopathological findings, the female genital organs exhibited histio-plasmo-lymphocytic inflammatory lesions ranging from mild to severe (Figures 1A-1D; Table 1). During immunohistochemistry, L. infantum amastigotes were detected in 50% (5/10) of the vulva samples (Figure 1E) and 20% (2/10) of the vaginal samples (Figure 1F) (Table 1). No amastigote forms were reported in the cervix, uterine body, uterine horn or ovaries.

The qPCR results revealed that L. infantum DNA was detected in 90% (9/10) of animals in at least one of the samples tested. Parasite load was low in 60% of animals (Table 2). The uterine horn and vulva exhibited 60% positivity (6/10) and the vagina exhibited 70% positivity (7/10). Cervix, uterus and ovaries exhibited 80% positivity for DNA from L. infantum amastigotes (8/10) (Table 1).

A statistically significant difference (Kruskal-Wallis test) was reported in the comparison of the number of parasites detected by IHC (P=0.0019) and the chronic inflammatory reaction score (P=0.0060) were compared. However, no differences among the organs were reported regarding parasite load determined by qPCR (P=0.9387). The vulva had the highest scores using the IHC method in comparison to the other genital organs. The histopathological analysis of the ovaries and cervix revealed the lowest scores for chronic inflammatory infiltrate; whereas, the highest scores were reported in the vulva, vagina and uterine horn (Table 1).

Spearman's test demonstrated a significant negative correlation between the qPCR score and degree of inflammation in the uterine body (P=0.001). However, no correlations were reported for other organs (vulva: P=0.134; vagina: P=0.752; cervix: P=0.617; uterine horn: P=0.305; ovary: P not possible to determined due to a column with all values equal to 0). Fisher's exact test (P<0.001) revealed that qPCR (0.28) was more efficient than IHC (0.90) for detection of tissue infected by amastigote forms of L. infantum.

DISCUSSION

L. infantum amastigotes have wide distribution in the tissues of dogs (TAFURI et al., 2004). In male dogs, several studies have demonstrated the tropism of L. infantum in genital organs (DINIZ et al., 2005). However, few studies previous studies have demonstrated L. infantum tropism in female genital organs of the correlation between parasite load and the onset of lesions in these organs (DUBEY et al., 2005; ROSYPAL et al., 2005; SILVA et al., 2008).

The structural changes in the vulva and vagina observed in the present investigation resemble change reported in previous studies (SILVA et al., 2008), in which chronic inflammatory infiltrate has been considered the only lesion detected in these organs. Lesions were frequently reported next to hair follicles, sebaceous glands and sweat glands. In the present study, besides of these lesions, chronic inflammatory infiltrate was not restricted to the external genitalia and was also reported in the cervix and uterine tissue of the animals analyzed.

[FIGURE 1 OMITTED]

Immunostaining detection for L. infantum amastigotes in the vulva and vagina is in agreement with data described by SILVA et al. (2008), as these were the only organs to have amastigote forms of L. infantum in macrophages. According to the authors cited, previous injuries can attract parasitized macrophages to connective tissue of the vulva and vagina. In the present investigation; however, no macroscopic lesions were reported in the female genital tract. Therefore, the macrophages normally reported in the connective tissue of the vulva and vaginal mucosa made these areas a site of parasitism for L. infantum amastigotes (WATTS et al., 1998). In this context, it was not possible to establish a relationship, at least in the present study, between macrophages infected with L. infantum amastigotes and pre-existing injuries.

No amastigotes were reported in the cervix, uterine body, uterine horn or ovaries. Indeed, few authors have described L. infantum in these organs. Conversely, L. infantum amastigotes have been frequently reported in the uterus (ROSYPAL et al., 2005) and placenta (DUBEY et al., 2005). Although previous studies have detected the presence of amastigote forms of L. infantum in the uterus and placenta, these findings may be underestimated.

In the present study, the detection of L. infantum DNA in all organs of the female genital system was not significantly associated with chronic inflammatory infiltrate in these tissues. In male dogs,L. infantum DNA has been detected in all genital organs. However, no correlation between parasite load and inflammatory lesions has been observed in these organs. Indeed, testicles have been reported to exhibit the highest parasite load as well as the lowest number of inflammatory lesions (OLIVEIRA et al., 2016).

There have been no previous data on the parasite load in the genital organs and the grade of chronic inflammatory reaction in female dogs naturally infected with L. infantum. In the present study, an inverse correlation was only reported in the uterine body. According to TORRES et al. (2013), the parasite load does not influence the characteristic of renal inflammatory lesions in infected dogs. In the present study, the L. infantum parasite load did not influence the intensity of chronic inflammatory lesions in the genital organs of naturally infected female dogs, except the uterine body, as described above. These findings indicated that factors other than L. infantum amastigotes could be responsible for the evolution of the inflammatory reaction and lesion intensity (TORRES et al., 2013).

CONCLUSION

Leishmania infantum has tropism for organs of the reproductive system of female dogs, with the vulva and vagina the major sites of chronic inflammatory lesions and immunostaining for amastigotes. L. infantum DNA and chronic inflammatory lesions were reported in all organs, except the ovaries. However, parasite load did not influence the intensity of the lesions reported in these organs.

http://dx.doi.org/10.1590/0103-8478cr20160137

ACKNOWLEDGMENTS

The authors thank the staff of the Department of Vector Control and Animal Surveillance of the municipality of Caruaru, particularly the veterinarian Claudia Agra.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

The present study was approved by the Ethics Committee for Animal Experimentation of the Universidade Federal Rural de Pernambuco (UFRPE) (protocol ECAE: 23082.015186/2012).

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Vinicius Vasconcelos Gomes de Oliveira (I) *, (IV) Simone Regina Barros de Macedo (I) Flabio Ribeiro de Araujo (II) Carlos Alberto do Nascimento Ramos (II) Rafael Antonio Nascimento Ramos (III) Frederico Celso Lyra Maia (III) Leucio Camara Alves (III) Valdemiro Amaro da Silva Junior (III)

(I) Departamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco (UFRPE), Rua Dom Manoel de Medeiros s/n, Dois Irmaos, 52171-900, Recife, PE, Brasil. E-mail: vinicius-vasconcelos@hotmail.com. * Corresponding author.

(II) Area de Saude Animal, Embrapa Gado de Corte, Avenida Raio Maia 830-Zona Rural, Campo Grande, MS, Brasil.

(III) Departamento de Medicina Veterinaria, Universidade Federal Rural de Pernambuco (UFRPE), Rua Dom Manoel de Medeiros s/n, Dois Irmaos, Recife, PE, Brasil.

(IV) Centro Acad[ecaret]mico de Vitoria, Universidade Federal de Pernambuco (UFPE), Rua Alto do Reservatorio s/n, Bela Vista, Vitoria de Santo Antao, PE, Brasil.

Received 02.12.16

Approved 05.22.16

Returned by the author 08.30.16

CR-2016-0137.R2
Table 1--Absolute and relative values of histopathological
lesions (HE), IHC for amastigotes, qPCR positivity and
comparisons between mean ranks for each organ evaluated in
the genital system of female dogs naturally infected with
L. infantum.

HE

Organ     Mild           Moderate    Severe      IHC       qPCR
(n=12)

Cervix    2 (16.6%)      0           2 (16.6%)   0         8 (61.5%)

Uterine   1 (8.3%)       4 (33.3%)   1 (8.3%)    0         6 (50.0%)
horn

Uterine   0              1 (8.3%)    3 (25.0%)   0         8 (61.5%)
body

Ovary     0              0           0           0         8 (61.5%)

Vagina    1 (8.3%)       5 (41.6%)   1 (8.3%)    2         7 (58.3%)
                                                 (16.6%)

Vulva     0              9 (75.0%)   1 (8.3%)    5         6 (50.0%)
                                                 (41.6%)
Comparisons of mean ranks

                         qPCR        IHC                   HE

Organs    Cervix         35.20 a     33.00 b               32.66 bc

          Uterine horn   26.80 a     33.00 b               38.16 ab

          Uterine body   30.90 a     33.00 b               35.70 b

          Ovary          30.40 a     33.00 b               21.00 c

          Vagina         30.50 a     39.25 b               41.00 ab

          Vulva          29.20 a     47.75 a               50.45 a

Means followed by same letter in column do not differ
significantly (P>0.05).

Table 2--Parasite load of each biological sample from
female dogs naturally infected by L. Infantum.

Animal   Cervix     Uterine horn   Uterine body

A1       8.56       6.43           1.455
A2       533.232    259.503        1,615.506
A3       0.322      0              0.062
A4       0          0              0.013
A5       0.72       3.625          0.503
A6       543.452    0              0.383
A7       1,274.93   290.803        3,801.012
A8       0          0              0
A9       0.178      0.041          0
A10      667.393    72.015         36.335

Animal   Ovary     Vagina      Vulva

A1       0.57      28.991      0.003
A2       519.225   34.254      84,702.805
A3       0.163     0           0
A4       0         0           0.204
A5       0.466     0.049       0.744
A6       8.022     1,345.295   26,447.793
A7       116.321   575.396     0
A8       0         0           0
A9       0.092     0.144       0
A10      57.047    115.126     272,211.562

Results expressed as number of parasite/250[micro]g of tissue.
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Title Annotation:texto en ingles
Author:Gomes de Oliveira, Vinicius Vasconcelos; Barros de Macedo, Simone Regina; de Araujo, Flabio Ribeiro;
Publication:Ciencia Rural
Date:Nov 1, 2016
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