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Insights of Leishmania (Viannia) braziliensis infection in golden hamster (Mesocricetus auratus) intestine
Biomedicine & Pharmacotherapy 106 (2018) 1624–1632
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Insights of Leishmania (Viannia) braziliensis infection in golden hamster (Mesocricetus auratus) intestine
T
Amanda Gubert Alves dos Santosa, Lainy Leiny de Limaa, Camila Alves Motaa, Marcelo Biondaro Goisb, Andrea Claudia Bekner Silva Fernandesa, Thaís Gomes Verzignassi Silveiraa, Debora de Mello Gonçales Sant’Anaa, ⁎ Gessilda de Alcântara Nogueira de Meloa, a b
Universidade Estadual de Maringá, Colombo Avenue, 5790, 87020-900, Maringá, Paraná, Brazil Universidade Federal do Recôncavo da Bahia, Viriato Lobo Street, 44571-020, Santo Antônio de Jesus, Bahia, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Leishmaniasis Small intestine Inflammation Histology Real-time polymerase Chain reaction
Aim: The present study compared and evaluated morphological and quantitative alterations in the ileum of hamsters infected by two L. (V.) braziliensis strains isolated from patients with different lesion aspects and treatment responses. Main methods: Hamsters were infected in the left hindpaw with a suspension of promastigotes (2 × 107/100 μl) of two different strains of L. (V.) braziliensis. After 90 or 120 days, the animals were euthanized. Samples of the ileum and mesenteric lymph node were collected for histological examination and quantitative polymerase chain reaction. Key findings: All infected animals developed similar profile of paw lesions. In peripheral blood there was an increase in the number of mononuclear cells which contributed to elevated global leukocytes count. Increases in the width and height of villi and width and depth of crypts were observed. The thickness of the muscular layers, submucosa, and intestinal wall also increased. Histopathological alterations were observed, including inflammatory infiltrate in crypts and a large number of immune cells in the lamina propria, submucosa, and muscular layer. Immune cells were found inside myenteric ganglia, with an increase in the number of intraepithelial lymphocytes. Leishmania DNA was detected in the ileum and mesenteric lymph node at both times of infection. The presence of amastigotes in the ileum was revealed by immunohistochemistry. Significance: The infection with different strains of L. (V.) braziliensis causes morphological and quantitative alterations in the ileum of hamsters and the parasite can migrate to the mesenteric lymph node and intestine.
1. Introduction Leishmaniasis is a disease that is caused by several species of Leishmania. Approximately 1 million new cases and 30,000 deaths are reported every year [1]. In the Americas, the denomination American Tegumentary Leishmaniasis (ATL) usually covers clinical forms with cutaneous and mucous lesions [2], causing visible deformity in the host and psychological, social, and economic impacts [3]. This disease has a wide geographical distribution throughout South America. In Brazil in 2016, more than 12,000 new cases were reported [4]. Leishmania (Viannia) braziliensis is the most important parasite that causes ATL because it exhibits large genetic polymorphisms [5]. It causes metastatic lesions and is difficult to diagnose and treat [2]. In
addition, it has been described in the literature the visceralization this specie to bone marrow of humans [6,7], rodents [8] and dogs which also presented the DNA of the parasite in the peripheral blood, spleen, liver and lymph nodes [9]. The limitations of studies with humans make the use of animal models necessary [10]. Hamsters are one of the best experimental animal models for studies of L. (V.) braziliensis [11–13]. This animal has independent genetic characteristics, leading to the development of immune response [14], skin lesions and histopathologic and clinical manifestations similar to those observed in the human disease [11]. Studies using hamsters demonstrated histopathological changes in the liver [11], spleen and lymph node [11,14] and the migration to spleen and lymph node [11,14].
⁎ Corresponding author at: Department of Clinical Analysis and Biomedicine at the Universidade Estadual de Maringá, Colombo Avenue, 5790, 87020-900, Maringá, Paraná, Brazil. E-mail address: [email protected] (G.d.A. Nogueira de Melo).
https://doi.org/10.1016/j.biopha.2018.07.120 Received 13 March 2018; Received in revised form 20 July 2018; Accepted 24 July 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Despite the findings about the histopathological alterations and the migration of L. (V.) braziliensis to other organs apart from the skin [6–9,11,14–16], no studies have reported the involvement of the intestine, which is both an immune [17] and endocrine [18] organ. Studies with visceral leishmaniasis [19,20] and infections with other protozoa [21–24] have reported alterations of different parameters of the intestine. Therefore, the present study compared and evaluated morphological and quantitative alterations in the ileum of hamsters infected by two L. (V.) braziliensis strains isolated from patients with different lesion aspects and treatment responses and, moreover, we evaluated whether the parasite strains could reach the intestine and mesenteric lymph node. 2. Material and methods
leukocyte counts in a Neubauer chamber and for differential leukocyte counts in blood smears that were stained by the May-Grünwald-Giemsa technique. The hamsters were then euthanized under deep anesthesia. After laparotomy, approximately 2 cm of the ileum and mesenteric lymph node were collected. One centimeter of the ileum was used for histology. Sets of semi-serial 5 μm transverse sections were prepared. Some of the sections were stained with hematoxylin and eosin (HE) for morphometric analyses of the intestinal wall and counts of enterocytes and intraepithelial lymphocytes (IELs). These sections were also used for histopathological evaluation to detect possible morphological alterations in the intestinal layers. To evaluate goblet cells, we used cytochemical periodic acid-Schiff (PAS) staining to detect neutral mucinproducing cells, Alcian blue (AB) staining (pH 1.0) to detect sulphomucins, and Alcian blue (AB) staining (pH 2.5) to detect sialomucinproducing goblet cells.
2.1. Experimental design
2.5. Morphometric analysis
The Ethical Committee on Animal Use of the Universidade Estadual de Maringá (UEM) approved this research (protocol no. 7587260416). We used 36 female hamsters (Mesocricetus auratus) that were housed under controlled temperature and a 12 h/12 h light/dark cycle with food and water available ad libitum. The animals were divided into six groups (n = 6/group): control group (CG) and two groups that were inoculated with different isolates of L. (V.) braziliensis: MHOM/BR/ 2003/2311 (2311) or MHOM/BR/2009/3476 (3476) for each time.
We performed morphometric analyses of images that were captured with a digital camera (Moticam 2000, 2.0 Megapixel) that was coupled to an optical microscope (MOTIC B5). The measurements were made using Motic Images Plus 2.0 software. Images were captured with a 10× objective lens to measure the total thickness of the intestinal wall, muscular tunic, and submucosa, the width and height of villi, and the width and depth of crypts. Sixty-four measurements of each parameter were performed throughout the ileum circumference of each animal. Using a 100× objective lens, we captured images to measure the height and width of 80 enterocytes and their respective nuclei.
2.2. Parasites
2.6. Cell counting
The L. (V.) braziliensis strains were obtained from patients who attended the Laboratório de Ensino e Pesquisa em Análises Clínicas (LEPAC), UEM. The isolates were cultured and cryopreserved in the Leishmaniasis Laboratory of UEM and were sent to Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, for identification. We chose these isolates because the patients presented different lesion aspects and treatment responses [25]. In the human host, infection with the MHOM/BR/2003/2311 strain caused a typical lesion with high and erythematous edges and granular aspect that was moderately resistant to therapy. The patient who was infected with the MHOM/BR/2009/3476 strain developed a lesion with desquamative ulcerated borders and erythematous violaceous center that was considered atypical. In this patient the treatment was difficult and multiple therapeutic interventions were needed to observe healing of the lesion [25].
The quantification of goblet cells and IELs was performed using a Nikon Eclipse E200 optical microscope. We counted the number of these cells among 2560 epithelial cells of each animal, then it was calculated the ratio to 100 cells. 2.7. Immunohistochemistry After deparaffination, the sections were hydrated and incubated for 20 min at 90–95 °C in citrate buffer solution for antigenic recovery. The blockade of endogenous peroxidase activity was performed with methanol and hydrogen peroxide (1:10 dilution) for 10 min, followed by nonspecific immunoglobulin blockade for 10 min with a combination of bovine serum albumin (BSA), donkey serum, and PBS. The primary antibody was produced in mice that were infected with Leishmania (Leishmania) amazonensis and purified with macerated intestine from healthy hamsters. The primary antibody (1:500 dilution) was incubated overnight inside a refrigerator. Incubation with horseradish peroxidase polymer conjugate (Life Technologies Corp., Frederick, MD, USA) was then performed for 30 min, followed by stable DAB (Invitrogen™, Carlsbad, CA, USA) revelation for 5 min. The sections were then counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted with coverslips. The tissue samples were considered positive when there was brown immunostaining of extra- or intramacrophagic amastigote forms, based on the examination of five tissue sections. Sections of popliteal lymph nodes were used as a positive control. Sections without the primary antibody were used as a negative control.
2.3. Experimental infection The promastigotes were cryopreserved in the Leishmaniasis Laboratory of UEM. After thawing and reactivate they were cultured in 199 medium (Gibco Laboratories®, Grand Island, USA) supplemented with 1% L-glutamine, 1% human urine, and 10% fetal bovine serum. We used parasites in the stationary growth phase from the fifth in vitro passage. Before infection, the hamsters were anesthetized with a combination of xylazine (Calmiun Agener-Union Animal Health) and ketamine (Francotar®-Virbac Animal health). The infected groups received an intradermal injection of each isolate (2 × 107/100 μl) in the left hindpaw. The CG received the same volume of phosphate-buffered saline (PBS). The hamsters were kept in individually ventilated cages with filtered air, autoclaved wood shavings and filtered water to avoid external contamination. Once weekly, both paws were measured and analyzed for lesions and edema.
2.8. Real-time polymerase chain reaction The mesenteric lymph node and 1 cm of the ileum were carefully collected to be submitted at Real-Time Polymerase Chain Reaction (qPCR). The organs were weighed, macerated, and stored in Eppendorf vials with 500 μl of STE buffer (10 mM TRIS, 1 mM EDTA, 0.1 M NaCl, pH 8.0) at -20 °C. The DNA was extracted using the guanidine-phenol method [26].
2.4. Tissue collection and histological processing At 90 or 120 days post-infection, blood samples were collected through the retro orbital sinus. Blood samples were used for global 1625
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both infected groups. Increases in the longitudinal layer (2311: 24.89 ± 9.64 μm; 3476: 24.71 ± 9.72 μm) and the height (2311: 308.60 ± 59.83 μm; 3476: 301.70 ± 60.83 μm) and width (2311: 70.26 ± 19.62 μm; 3476: 69.99 ± 19.67 μm) of villi were observed compared with the CG (22.75 ± 9.05 μm, 249.70 ± 56.27 μm, and 67.47 ± 22.69 μm, respectively; Fig. 2). Only the 2311 group exhibited significant increases in some of the parameters, such as the thickness of total wall of the ileum (195.10 ± 50.28 μm), muscularis externa (53.29 ± 18.25 μm), and crypt depth (114.20 ± 26.09 μm), compared with the CG (186.40 ± 50.59 μm, 48.63 ± 18.52 μm, and 102.70 ± 25.94 μm, respectively; Fig. 2). The observations at 120 days post-infection were more evident. Both infected groups exhibited an increase in the intestinal wall thickness (2311: 197.30 ± 47.93 μm; 3476: 194.90 ± 61.57 μm) and submucosa (2311: 23.95 ± 11.26 μm; 3476: 27.15 ± 16.89 μm) compared with the CG (157 ± 44.25 μm and 18.86 ± 9.18 μm, respectively) (Fig. 2). The muscularis externa exhibited a 19.77% increase in the 2311 group and 17.51% increase in the 3476 group. The circular layer increased by approximately 20% in both groups. The longitudinal layer increased by 26.21% in the 2311 group and 20.57% in the 3476 group (Fig. 2). The height of villi increased by 37.31% in the 2311 group and by 33.76% in the 3476 group. The width of villi increased by approximately 29% in both groups. The width of crypts increased by 30%, and the depth of crypts increased by 19% in both infected groups (Fig. 2). At 90 days post-infection, the ileum exhibited reductions of the height of enterocytes and height of their nuclei in both infected groups. The width of enterocytes and their nuclei increased in the 2311 group (Table 2). The height of enterocytes increased in the infected groups at 120 days post-infection compared with the CG, with an increase in the height of nuclei in the 3476 group (Table 2). Fig. 3 shows a morphometric representation of the ileum. We observed increases in villi, crypts, the submucosa, and the muscular layer after 90 and 120 days of infection with the two strains of L. (V.) braziliensis. The migration of immune cells to the intestinal epithelium was observed after infection for 90 and 120 days, which significantly increased the height and width of villi, as previously described, and the number of IELs in both infected groups. The average increase in the proportion of IELs was 41.42%. The 3476 group at 120 days presented the greatest increase (45.32% more cells) compared with the CG. Goblet cells were not significantly different in any of the groups, despite approximately 40% decrease in sialomucin-producing cells at 120 days (Fig. 4). Considering our previous findings, we performed qPCR to confirm whether Leishmania migrates to the intestine and mesenteric lymph node (Table 3). We found L. (V.) braziliensis DNA in the ileum and mesenteric lymph node in the 3476 group at both 90 and 120 days postinfection. The threshold cycle (Ct) in the mesenteric lymph node was 25.4 and 34.1 at 90 days and ranged from 27.9 to 31.3 at 120 days postinfection, while in the ileum ranged from 26.3 to 33.8 at 90 days and from 27.8 to 33.6 at 120 days. In the 2311 group, we detected parasite DNA in the mesenteric lymph node at 90 and 120 post-infection. At 90 days of infection, the Ct values were 30.4 and 33.7 and at 120 days ranged from 20.6 to 31.1. We also detected L. (V.) braziliensis DNA in the ileum at 120 days post-infection with Ct of 34.1. It was considered negative those samples with Ct undetermined or > 35. The melting curve after the reaction showed a single peak, indicating its specificity. All samples were positive for the GAPDH, with the Ct values between 12.4 and 23.1, including those negative for Leishmania (Viannia) kDNA, showing the DNA integrity and absence of PCR inhibitors in these samples.
Primers which amplify a 70-bp fragment of kDNA from parasites belonging to the Leishmania (Viannia) subgenus were used for the parasite detection in the samples. The qPCR was performed in a final volume of 10 μl that contained 1000 nM of forward primer (5′-GAA CGG GGT TTC TGT ATG C-3′) and 1000 nM of reverse primer (5′-TAC TCC CCG ACA TGC CTC TG-3′) [26], 1X SYBR Green Select Master Mix, and 1 μl of DNA sample. The samples were also submitted to an internal control using the GAPDH gene (constitutive gene of golden hamster), which was amplified with the primers Fw 5′-GGT TGC CAA ACC TTA TCA GAA ATG-3′ and Rv 5′-TTC ACC TGT TCC ACA GCC TTG-3′ [14], at the same run and reaction conditions. The DNA samples concentration was 4–60 ng/μl measured by Qubit™ Fluorometer 2.0 (Invitrogen, USA) and the DNA quality indicator (A260/A280 ratio) was around 1.85, measured at Nanodrop Lite 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA). The reaction was run in duplicate using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) with initial heating at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 58 °C for 1 min. To evaluate the specificity of the reaction, a melting curve was generated, with one cycle at 95 °C for 15 s, followed by 60 °C for 1 min and continuous heating to 95 °C at 0.3 °C/s. Positive and negative extraction controls were placed with the samples, and also negative (with ultrapure water) and positive amplification controls, being one high (DNA equivalent to 5 × 104 promastigotes of L. (V.) braziliensis) and another low (DNA equivalent to 5 × 10−3 promastigotes of L. (V.) braziliensis). 2.9. Statistical analysis The statistical analysis was performed using BioEstat 5.3 software to determine the data distribution and GraphPad Prism 5 (San Diego, Ca, USA) software for comparisons between groups. Values of p < 0.05 were considered statistically significant. The analysis of data with a normal distribution was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc tests. Data that presented a non-normal distribution were analyzed using the Friedman test. 3. Results All infected hamsters developed edema in the inoculated paw, which reached double the size of the control paw. The lesions in the infected paw presented elevated borders, with a granular aspect, erythematous and necrotic surface or recovered by crusts. In all animals, the wounds evolved during the first or second month of infection. Likewise, there was a similar profile of paw lesion in both used isolates during the experimental period (Fig. 1). The injury in the paw caused by the infection resulted in locomotor difficulty. No clinical sign of intestinal disease was observed during the experimental period. Global leucocyte counts increased by 41.71% in the 3476 group at 90 days and by 40.62% in the 2311 group and 56.57% in the 3476 group at 120 days. The differential counts of white blood cells revealed an increase in the number of neutrophils at 90 days and lymphocytes at 120 days in the 3476 group. The number of monocytes increased by 91.98% in 2311 group and 95.44% in 3476 group at 90 days and by 94.76% in the 2311 group and 96.97% in the 3476 group at 120 days (Table 1). Infection with both strains of L. (V.) braziliensis caused histopathological alterations in the ileum, including an increase in the number of immune cells in the lamina propria, submucosa, and muscular layer. We observed inflammatory infiltrate in crypts both next to the ganglion and inside the ganglion, which caused periganglionitis and ganglionitis, respectively. We found suggestive forms of Leishmania amastigotes in the ileum in 16 animals. These forms were confirmed by specific Leishmania immunostaining in 12 of these animals (Fig. 1). The morphometry of the ileum 90 days post-infection was altered in 1626
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Fig. 1. Alterations caused by L. (V.) braziliensis infection. (A) Infected paw showing the development of lesions and edema at 90 days post-infection. (B) Large number of immune cells in the lamina propria, inflammatory infiltrate in crypts, and atrophy of crypts. Ganglionitis, periganglionitis, and immune cells were observed in the submucosa and muscular layer (HE staining, 20× magnification, scale bar = 50 μm). (C) Ganglion from the myenteric plexus that presented ganglionitis (HE staining, 100× magnification, scale bar = 5 μm). (D) Suggestive forms of Leishmania amastigotes (arrows) and inflammatory infiltrate in crypts (HE staining, 100× magnification, scale bar = 5 μm). (E) Leishmania amastigotes (arrow) in the popliteal lymph node (immunohistochemistry, 100× magnification, scale bar = 5 μm). (F) Leishmania amastigotes (arrow) in the ileum (immunohistochemistry, 100× magnification, scale bar = 5 μm).
Table 1 Global and differential leucocyte counts in peripheral blood in hamsters infected with L. (V.) braziliensis for 90 and 120 days. Time of infection 90 days
120 days
Groups CG 2311 3476 CG 2311 3476
Global count (cell/μl) 2,550 3,250 4,375 1,900 3,200 4,375
± ± ± ± ± ±
a
387.3 238.0a 963.9b 489.9a 483.0b 899.5b
Neutrophil (cell/μl) 858.8 1,072 1,410 760.8 967.3 1,238
± ± ± ± ± ±
a
265.4 129.2a 140.9b 162.4a 344.1a 512.5a
Lymphocyte (cell/μl) 1,645 1,666 2,076 1,111 1,767 2,332
± ± ± ± ± ±
a
286.2 153.5a 684.8a 372.3a 156.2a 411.7b
Monocyte (cell/μl) a
40.5 ± 33.3 505.5 ± 145.4b 889.3 ± 441.2b 24.0 ± 18.5a 458.5 ± 117.8b 792.5 ± 146.7b
Eosinophil (cell/μl) 12.0 ± 24.0a 15.2 ± 17.6a 0a 4.7 ± 9.5a 7.5 ± 15.0a 12.2 ± 24.5a
Data with different letters in the same column for each time post-infection are significantly different (p < 0.05) compared with the CG. The data are expressed as mean ± SD. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476. Bold column are time of infection and experimental groups. 1627
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Fig. 2. Morphometry of the ileum wall in hamsters infected for 90 or 120 days with different L. (V.) braziliensis strains. The data are expressed as mean ± standard deviation. *p < 0.05, significant difference compared with CG. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476.
4. Discussion
spleen, lymph node [11,15] and bone marrow [6,7]. Hematological alterations are usually indicative of infection [37] and the evaluation of blood cells is important to achieve a systemic view of the diseases [37,38]. In the present study, L. (V.) braziliensis infection in hamsters caused a significant increase in the number of blood monocytes, corroborating the findings of studies of cutaneous leishmaniasis [39,40]. This increase in blood monocytes might be a source of immune cells at the site of infection. These mononuclear cells may be one way by which Leishmania is transported throughout the organism [35]. Once at the site of infection, these cells can transform into macrophages [39], which are the host for the parasite [41]. In our study, the infection with L. (V.) braziliensis induced the formation of inflammatory infiltrates altering the normal morphological
Various infections [27,28] and psychological changes [29–32] can affect the function and morphology of the gastrointestinal tract. Previous studies have reported intestinal involvement in visceral leishmaniasis demonstrating inflammatory infiltrate and structural alterations [20,33] with the presence of amastigote forms [20]. According to Martinez et al. [34], the dissemination of Leishmania can occur through the lymphatic system or hematogenous, since that the parasite DNA has already been found in blood mononuclear cells [35]. Studies demonstrated that L. (V.) braziliensis causes metastatic [2] and disseminated [36] lesions in the skin, histopathological alterations in spleen, liver and lymph node [11] and can migrate to other organs including the
Table 2 Morphometry of enterocytes and their nuclei at 90 and 120 days post-infection. Enterocytes Time 90 days
120 days
Group CG 2311 3476 CG 2311 3476
Nuclei of the enterocytes
Height (μm)
Width (μm) a
5.32 5.70 5.46 5.64 5.59 5.68
11.50 ± 2.97 11.00 ± 3.83b 9.82 ± 2.39b 11.39 ± 2.80a 13.63 ± 4.30b 12.27 ± 2.77b
± ± ± ± ± ±
Height (μm) a
1.23 1.00b 1.04a 1.30a 2.02a 1.19a
5.22 4.62 4.30 4.66 4.65 5.04
± ± ± ± ± ±
Width (μm) a
1.33 1.25b 1.07b 0.97a 1.05a 1.30b
3.65 3.99 3.70 3.57 3.61 3.70
± ± ± ± ± ±
1.14a 0.83b 0.81a 0.78a 0.98a 0.91a
Data with different letters in the same column at each time post-infection indicate significant differences (p < 0.05) compared with the CG. The data are expressed as mean ± SD. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476. Bold column are time of infection and experimental groups. 1628
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Fig. 3. Photomicrograph of cross-sections of the ileum wall. The first column shows animals at 90 days post-infection. The second column shows animals at 120 days post-infection. The images represent the control group (1 st line) and animals infected with MHOM/BR/2003/2311 (2nd line) and MHOM/BR/2009/3476 (3rd line). The sections were stained with HE (10× magnification, scale bar = 100 μm).
increase in the height of villi [24] such as those demonstrated in hamsters infected with the two strains of Leishmania. In addition, the augment in the cellular renewal negatively influences the differentiation of epithelial cells [45]. One of the most important findings of this study was to visualize the presence of immune cells in the ganglia of myenteric plexus in both infected animals characterizing ganglionitis. Inflammatory infiltrate in the ganglia can lead to neuronal degeneration [46,47] and is usually associated with intestinal disorders [46] however, we did not observe intestine clinical signs during the experimental period. Ganglionitis may be related to the increase in the muscular layer of the ileum in infected animals as showed by other authors [48,49]. The increase in the thickness of muscular can also be associated with the cytokines secreted by immune cells present between muscle cells [50]. Furthermore, the reduction of acid-producing mucin cells lead to denser intestinal mucus that is more difficult to expel [24] affecting the contractility of muscle fibers and provoking both functional [51] and morphological [50] alterations. The histopathological alterations in the mucosa, submucosa, and muscular layer supported the increase in the total wall thickness of the ileum.
aspect and inducing ganglionitis which may directly affect intestinal immunity. Some histopathological alterations that were observed in the ileum of hamsters, as inflammatory infiltrate in crypts, and the greater number of immune cells in the lamina propria, submucosa, and muscular layer might signalize tissue damage [23]. The migration of immune cells to the lamina propria may have occurred by the presence of the parasite in the organ, as can be seen in other studies [19,20]. The accumulation of these cells may be responsible for the increase of the thickness of the submucosa and the height and width of villi observed in the groups infected with both strains. The increase in the number of IELs is also related with the inflammatory response [42]. These cells may act as immunoregulators in the intestinal epithelium [43] and promote the flattening of enterocytes in the infected groups at 90 days post-infection. Different alterations in the size of enterocytes were observed at 90 and 120 days post-infection, likely because of the high renewal rate of this cellular type that occurs within 3–4 days [44]. Intestinal cellular proliferation occurs in the crypts and under normal conditions the epithelium is completely renewed within 6–7 days [44]. Increased cellular proliferation results in deeper crypts and contribute to the 1629
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Fig. 4. Cellular alterations in the epithelium of the ileum in hamsters infected for 90 or 120 days with different L. (V.) braziliensis strains. The data are expressed as mean ± standard deviation. *p < 0.05, significant difference compared with CG. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476.
After observing the aforementioned histopathological changes and the suggestive forms of Leishmania amastigotes in the histological sections, we detected DNA of the parasite in the mesenteric lymph node and ileum. Using immunostaining with specific antibodies to Leishmania we confirmed the parasite in the intestine. Thus, we can affirm that L. (V.) braziliensis can visceralize to the intestine and mesenteric lymph node of hamsters, once they are susceptible to this specie as previously reported [11,14]. Additionally, it is interesting to highlight that L. braziliensis [6,7] and L. amazonensis [52] were isolated from bone marrow of human patients with visceral leishmaniasis showing that immunocompromising condition is an important factor for the visceralization of the parasite. Comparing the results of the groups infected with the different strains, the profile of paw lesion was similar in both isolates during the experimental period. The 2311 group presented major alterations at 90 days post-infection and both strains induced similar changes at 120 days post-infection. The MHOM/BR/2009/3476 strain was detected in the ileum and mesenteric lymph node by qPCR at 90 and 120 days. The MHOM/BR/2003/2311 strain was found in the mesenteric lymph node of the infected animals in both infection times and in the ileum at 120 days after infection. This differential behavior between the two strains may be attributable to genetic polymorphisms of Leishmania [53] and the host [13], in addition to the interactions between the parasite and host’s immune response [54].
Table 3 Real-time qualitative PCR assay in samples of the ileum and mesenteric lymph node from hamsters infected with Leishmania (Viannia) braziliensis strains. Groups
Ileum
Control
2311
3476
Mesenteric lymph node
Control
2311
3476
Animal ID
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Ct mean 90 days
120 days
– – – – – – – – – 33.8 26.3 28.4 – – – – 30.4 – – 33.7 34.1 nr 25.4 –
– – – – – 34.1 – – 32.9 33.6 29.0 27.8 – – – – 20.6 – 25.1 31.1 – 30.4 31.3 27.9
5. Conclusion
Real-time PCR results using the MP3H/MP1L primers. Groups: 2311, infected with MHOM/BR/2003/2311; 3476, infected with MHOM/BR/2009/3476. Animal ID, animal identification. Ct, threshold cycle. (Ct ≤ 35): positive; (-): Ct undetermined or > 35; (nr): not realized. Bold column are time of infection and experimental groups.
Chronic infection with the MHOM/BR/2003/2311 and MHOM/BR/ 2009/3476 strains of L. (V.) braziliensis caused similar cellular and morphometric alterations in the ileum of hamsters indicating intestinal inflammation. We also found the parasite DNA in the ileum and 1630
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mesenteric lymph node and amastigotes forms in the ileum showing the visceralization of both strains. Thus, we demonstrated that infection with a specie that usually causes cutaneous lesions in humans can lead to systemic alterations in hamsters. Further studies should be performed to investigate other aspects of L. (V) braziliensis-host interaction in the intestine and other viscera.
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Insights of Leishmania (Viannia) braziliensis infection in golden hamster (Mesocricetus auratus) intestine
T
Amanda Gubert Alves dos Santosa, Lainy Leiny de Limaa, Camila Alves Motaa, Marcelo Biondaro Goisb, Andrea Claudia Bekner Silva Fernandesa, Thaís Gomes Verzignassi Silveiraa, Debora de Mello Gonçales Sant’Anaa, ⁎ Gessilda de Alcântara Nogueira de Meloa, a b
Universidade Estadual de Maringá, Colombo Avenue, 5790, 87020-900, Maringá, Paraná, Brazil Universidade Federal do Recôncavo da Bahia, Viriato Lobo Street, 44571-020, Santo Antônio de Jesus, Bahia, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Leishmaniasis Small intestine Inflammation Histology Real-time polymerase Chain reaction
Aim: The present study compared and evaluated morphological and quantitative alterations in the ileum of hamsters infected by two L. (V.) braziliensis strains isolated from patients with different lesion aspects and treatment responses. Main methods: Hamsters were infected in the left hindpaw with a suspension of promastigotes (2 × 107/100 μl) of two different strains of L. (V.) braziliensis. After 90 or 120 days, the animals were euthanized. Samples of the ileum and mesenteric lymph node were collected for histological examination and quantitative polymerase chain reaction. Key findings: All infected animals developed similar profile of paw lesions. In peripheral blood there was an increase in the number of mononuclear cells which contributed to elevated global leukocytes count. Increases in the width and height of villi and width and depth of crypts were observed. The thickness of the muscular layers, submucosa, and intestinal wall also increased. Histopathological alterations were observed, including inflammatory infiltrate in crypts and a large number of immune cells in the lamina propria, submucosa, and muscular layer. Immune cells were found inside myenteric ganglia, with an increase in the number of intraepithelial lymphocytes. Leishmania DNA was detected in the ileum and mesenteric lymph node at both times of infection. The presence of amastigotes in the ileum was revealed by immunohistochemistry. Significance: The infection with different strains of L. (V.) braziliensis causes morphological and quantitative alterations in the ileum of hamsters and the parasite can migrate to the mesenteric lymph node and intestine.
1. Introduction Leishmaniasis is a disease that is caused by several species of Leishmania. Approximately 1 million new cases and 30,000 deaths are reported every year [1]. In the Americas, the denomination American Tegumentary Leishmaniasis (ATL) usually covers clinical forms with cutaneous and mucous lesions [2], causing visible deformity in the host and psychological, social, and economic impacts [3]. This disease has a wide geographical distribution throughout South America. In Brazil in 2016, more than 12,000 new cases were reported [4]. Leishmania (Viannia) braziliensis is the most important parasite that causes ATL because it exhibits large genetic polymorphisms [5]. It causes metastatic lesions and is difficult to diagnose and treat [2]. In
addition, it has been described in the literature the visceralization this specie to bone marrow of humans [6,7], rodents [8] and dogs which also presented the DNA of the parasite in the peripheral blood, spleen, liver and lymph nodes [9]. The limitations of studies with humans make the use of animal models necessary [10]. Hamsters are one of the best experimental animal models for studies of L. (V.) braziliensis [11–13]. This animal has independent genetic characteristics, leading to the development of immune response [14], skin lesions and histopathologic and clinical manifestations similar to those observed in the human disease [11]. Studies using hamsters demonstrated histopathological changes in the liver [11], spleen and lymph node [11,14] and the migration to spleen and lymph node [11,14].
⁎ Corresponding author at: Department of Clinical Analysis and Biomedicine at the Universidade Estadual de Maringá, Colombo Avenue, 5790, 87020-900, Maringá, Paraná, Brazil. E-mail address: [email protected] (G.d.A. Nogueira de Melo).
https://doi.org/10.1016/j.biopha.2018.07.120 Received 13 March 2018; Received in revised form 20 July 2018; Accepted 24 July 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Despite the findings about the histopathological alterations and the migration of L. (V.) braziliensis to other organs apart from the skin [6–9,11,14–16], no studies have reported the involvement of the intestine, which is both an immune [17] and endocrine [18] organ. Studies with visceral leishmaniasis [19,20] and infections with other protozoa [21–24] have reported alterations of different parameters of the intestine. Therefore, the present study compared and evaluated morphological and quantitative alterations in the ileum of hamsters infected by two L. (V.) braziliensis strains isolated from patients with different lesion aspects and treatment responses and, moreover, we evaluated whether the parasite strains could reach the intestine and mesenteric lymph node. 2. Material and methods
leukocyte counts in a Neubauer chamber and for differential leukocyte counts in blood smears that were stained by the May-Grünwald-Giemsa technique. The hamsters were then euthanized under deep anesthesia. After laparotomy, approximately 2 cm of the ileum and mesenteric lymph node were collected. One centimeter of the ileum was used for histology. Sets of semi-serial 5 μm transverse sections were prepared. Some of the sections were stained with hematoxylin and eosin (HE) for morphometric analyses of the intestinal wall and counts of enterocytes and intraepithelial lymphocytes (IELs). These sections were also used for histopathological evaluation to detect possible morphological alterations in the intestinal layers. To evaluate goblet cells, we used cytochemical periodic acid-Schiff (PAS) staining to detect neutral mucinproducing cells, Alcian blue (AB) staining (pH 1.0) to detect sulphomucins, and Alcian blue (AB) staining (pH 2.5) to detect sialomucinproducing goblet cells.
2.1. Experimental design
2.5. Morphometric analysis
The Ethical Committee on Animal Use of the Universidade Estadual de Maringá (UEM) approved this research (protocol no. 7587260416). We used 36 female hamsters (Mesocricetus auratus) that were housed under controlled temperature and a 12 h/12 h light/dark cycle with food and water available ad libitum. The animals were divided into six groups (n = 6/group): control group (CG) and two groups that were inoculated with different isolates of L. (V.) braziliensis: MHOM/BR/ 2003/2311 (2311) or MHOM/BR/2009/3476 (3476) for each time.
We performed morphometric analyses of images that were captured with a digital camera (Moticam 2000, 2.0 Megapixel) that was coupled to an optical microscope (MOTIC B5). The measurements were made using Motic Images Plus 2.0 software. Images were captured with a 10× objective lens to measure the total thickness of the intestinal wall, muscular tunic, and submucosa, the width and height of villi, and the width and depth of crypts. Sixty-four measurements of each parameter were performed throughout the ileum circumference of each animal. Using a 100× objective lens, we captured images to measure the height and width of 80 enterocytes and their respective nuclei.
2.2. Parasites
2.6. Cell counting
The L. (V.) braziliensis strains were obtained from patients who attended the Laboratório de Ensino e Pesquisa em Análises Clínicas (LEPAC), UEM. The isolates were cultured and cryopreserved in the Leishmaniasis Laboratory of UEM and were sent to Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, for identification. We chose these isolates because the patients presented different lesion aspects and treatment responses [25]. In the human host, infection with the MHOM/BR/2003/2311 strain caused a typical lesion with high and erythematous edges and granular aspect that was moderately resistant to therapy. The patient who was infected with the MHOM/BR/2009/3476 strain developed a lesion with desquamative ulcerated borders and erythematous violaceous center that was considered atypical. In this patient the treatment was difficult and multiple therapeutic interventions were needed to observe healing of the lesion [25].
The quantification of goblet cells and IELs was performed using a Nikon Eclipse E200 optical microscope. We counted the number of these cells among 2560 epithelial cells of each animal, then it was calculated the ratio to 100 cells. 2.7. Immunohistochemistry After deparaffination, the sections were hydrated and incubated for 20 min at 90–95 °C in citrate buffer solution for antigenic recovery. The blockade of endogenous peroxidase activity was performed with methanol and hydrogen peroxide (1:10 dilution) for 10 min, followed by nonspecific immunoglobulin blockade for 10 min with a combination of bovine serum albumin (BSA), donkey serum, and PBS. The primary antibody was produced in mice that were infected with Leishmania (Leishmania) amazonensis and purified with macerated intestine from healthy hamsters. The primary antibody (1:500 dilution) was incubated overnight inside a refrigerator. Incubation with horseradish peroxidase polymer conjugate (Life Technologies Corp., Frederick, MD, USA) was then performed for 30 min, followed by stable DAB (Invitrogen™, Carlsbad, CA, USA) revelation for 5 min. The sections were then counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted with coverslips. The tissue samples were considered positive when there was brown immunostaining of extra- or intramacrophagic amastigote forms, based on the examination of five tissue sections. Sections of popliteal lymph nodes were used as a positive control. Sections without the primary antibody were used as a negative control.
2.3. Experimental infection The promastigotes were cryopreserved in the Leishmaniasis Laboratory of UEM. After thawing and reactivate they were cultured in 199 medium (Gibco Laboratories®, Grand Island, USA) supplemented with 1% L-glutamine, 1% human urine, and 10% fetal bovine serum. We used parasites in the stationary growth phase from the fifth in vitro passage. Before infection, the hamsters were anesthetized with a combination of xylazine (Calmiun Agener-Union Animal Health) and ketamine (Francotar®-Virbac Animal health). The infected groups received an intradermal injection of each isolate (2 × 107/100 μl) in the left hindpaw. The CG received the same volume of phosphate-buffered saline (PBS). The hamsters were kept in individually ventilated cages with filtered air, autoclaved wood shavings and filtered water to avoid external contamination. Once weekly, both paws were measured and analyzed for lesions and edema.
2.8. Real-time polymerase chain reaction The mesenteric lymph node and 1 cm of the ileum were carefully collected to be submitted at Real-Time Polymerase Chain Reaction (qPCR). The organs were weighed, macerated, and stored in Eppendorf vials with 500 μl of STE buffer (10 mM TRIS, 1 mM EDTA, 0.1 M NaCl, pH 8.0) at -20 °C. The DNA was extracted using the guanidine-phenol method [26].
2.4. Tissue collection and histological processing At 90 or 120 days post-infection, blood samples were collected through the retro orbital sinus. Blood samples were used for global 1625
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both infected groups. Increases in the longitudinal layer (2311: 24.89 ± 9.64 μm; 3476: 24.71 ± 9.72 μm) and the height (2311: 308.60 ± 59.83 μm; 3476: 301.70 ± 60.83 μm) and width (2311: 70.26 ± 19.62 μm; 3476: 69.99 ± 19.67 μm) of villi were observed compared with the CG (22.75 ± 9.05 μm, 249.70 ± 56.27 μm, and 67.47 ± 22.69 μm, respectively; Fig. 2). Only the 2311 group exhibited significant increases in some of the parameters, such as the thickness of total wall of the ileum (195.10 ± 50.28 μm), muscularis externa (53.29 ± 18.25 μm), and crypt depth (114.20 ± 26.09 μm), compared with the CG (186.40 ± 50.59 μm, 48.63 ± 18.52 μm, and 102.70 ± 25.94 μm, respectively; Fig. 2). The observations at 120 days post-infection were more evident. Both infected groups exhibited an increase in the intestinal wall thickness (2311: 197.30 ± 47.93 μm; 3476: 194.90 ± 61.57 μm) and submucosa (2311: 23.95 ± 11.26 μm; 3476: 27.15 ± 16.89 μm) compared with the CG (157 ± 44.25 μm and 18.86 ± 9.18 μm, respectively) (Fig. 2). The muscularis externa exhibited a 19.77% increase in the 2311 group and 17.51% increase in the 3476 group. The circular layer increased by approximately 20% in both groups. The longitudinal layer increased by 26.21% in the 2311 group and 20.57% in the 3476 group (Fig. 2). The height of villi increased by 37.31% in the 2311 group and by 33.76% in the 3476 group. The width of villi increased by approximately 29% in both groups. The width of crypts increased by 30%, and the depth of crypts increased by 19% in both infected groups (Fig. 2). At 90 days post-infection, the ileum exhibited reductions of the height of enterocytes and height of their nuclei in both infected groups. The width of enterocytes and their nuclei increased in the 2311 group (Table 2). The height of enterocytes increased in the infected groups at 120 days post-infection compared with the CG, with an increase in the height of nuclei in the 3476 group (Table 2). Fig. 3 shows a morphometric representation of the ileum. We observed increases in villi, crypts, the submucosa, and the muscular layer after 90 and 120 days of infection with the two strains of L. (V.) braziliensis. The migration of immune cells to the intestinal epithelium was observed after infection for 90 and 120 days, which significantly increased the height and width of villi, as previously described, and the number of IELs in both infected groups. The average increase in the proportion of IELs was 41.42%. The 3476 group at 120 days presented the greatest increase (45.32% more cells) compared with the CG. Goblet cells were not significantly different in any of the groups, despite approximately 40% decrease in sialomucin-producing cells at 120 days (Fig. 4). Considering our previous findings, we performed qPCR to confirm whether Leishmania migrates to the intestine and mesenteric lymph node (Table 3). We found L. (V.) braziliensis DNA in the ileum and mesenteric lymph node in the 3476 group at both 90 and 120 days postinfection. The threshold cycle (Ct) in the mesenteric lymph node was 25.4 and 34.1 at 90 days and ranged from 27.9 to 31.3 at 120 days postinfection, while in the ileum ranged from 26.3 to 33.8 at 90 days and from 27.8 to 33.6 at 120 days. In the 2311 group, we detected parasite DNA in the mesenteric lymph node at 90 and 120 post-infection. At 90 days of infection, the Ct values were 30.4 and 33.7 and at 120 days ranged from 20.6 to 31.1. We also detected L. (V.) braziliensis DNA in the ileum at 120 days post-infection with Ct of 34.1. It was considered negative those samples with Ct undetermined or > 35. The melting curve after the reaction showed a single peak, indicating its specificity. All samples were positive for the GAPDH, with the Ct values between 12.4 and 23.1, including those negative for Leishmania (Viannia) kDNA, showing the DNA integrity and absence of PCR inhibitors in these samples.
Primers which amplify a 70-bp fragment of kDNA from parasites belonging to the Leishmania (Viannia) subgenus were used for the parasite detection in the samples. The qPCR was performed in a final volume of 10 μl that contained 1000 nM of forward primer (5′-GAA CGG GGT TTC TGT ATG C-3′) and 1000 nM of reverse primer (5′-TAC TCC CCG ACA TGC CTC TG-3′) [26], 1X SYBR Green Select Master Mix, and 1 μl of DNA sample. The samples were also submitted to an internal control using the GAPDH gene (constitutive gene of golden hamster), which was amplified with the primers Fw 5′-GGT TGC CAA ACC TTA TCA GAA ATG-3′ and Rv 5′-TTC ACC TGT TCC ACA GCC TTG-3′ [14], at the same run and reaction conditions. The DNA samples concentration was 4–60 ng/μl measured by Qubit™ Fluorometer 2.0 (Invitrogen, USA) and the DNA quality indicator (A260/A280 ratio) was around 1.85, measured at Nanodrop Lite 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA). The reaction was run in duplicate using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) with initial heating at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 58 °C for 1 min. To evaluate the specificity of the reaction, a melting curve was generated, with one cycle at 95 °C for 15 s, followed by 60 °C for 1 min and continuous heating to 95 °C at 0.3 °C/s. Positive and negative extraction controls were placed with the samples, and also negative (with ultrapure water) and positive amplification controls, being one high (DNA equivalent to 5 × 104 promastigotes of L. (V.) braziliensis) and another low (DNA equivalent to 5 × 10−3 promastigotes of L. (V.) braziliensis). 2.9. Statistical analysis The statistical analysis was performed using BioEstat 5.3 software to determine the data distribution and GraphPad Prism 5 (San Diego, Ca, USA) software for comparisons between groups. Values of p < 0.05 were considered statistically significant. The analysis of data with a normal distribution was performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison post hoc tests. Data that presented a non-normal distribution were analyzed using the Friedman test. 3. Results All infected hamsters developed edema in the inoculated paw, which reached double the size of the control paw. The lesions in the infected paw presented elevated borders, with a granular aspect, erythematous and necrotic surface or recovered by crusts. In all animals, the wounds evolved during the first or second month of infection. Likewise, there was a similar profile of paw lesion in both used isolates during the experimental period (Fig. 1). The injury in the paw caused by the infection resulted in locomotor difficulty. No clinical sign of intestinal disease was observed during the experimental period. Global leucocyte counts increased by 41.71% in the 3476 group at 90 days and by 40.62% in the 2311 group and 56.57% in the 3476 group at 120 days. The differential counts of white blood cells revealed an increase in the number of neutrophils at 90 days and lymphocytes at 120 days in the 3476 group. The number of monocytes increased by 91.98% in 2311 group and 95.44% in 3476 group at 90 days and by 94.76% in the 2311 group and 96.97% in the 3476 group at 120 days (Table 1). Infection with both strains of L. (V.) braziliensis caused histopathological alterations in the ileum, including an increase in the number of immune cells in the lamina propria, submucosa, and muscular layer. We observed inflammatory infiltrate in crypts both next to the ganglion and inside the ganglion, which caused periganglionitis and ganglionitis, respectively. We found suggestive forms of Leishmania amastigotes in the ileum in 16 animals. These forms were confirmed by specific Leishmania immunostaining in 12 of these animals (Fig. 1). The morphometry of the ileum 90 days post-infection was altered in 1626
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Fig. 1. Alterations caused by L. (V.) braziliensis infection. (A) Infected paw showing the development of lesions and edema at 90 days post-infection. (B) Large number of immune cells in the lamina propria, inflammatory infiltrate in crypts, and atrophy of crypts. Ganglionitis, periganglionitis, and immune cells were observed in the submucosa and muscular layer (HE staining, 20× magnification, scale bar = 50 μm). (C) Ganglion from the myenteric plexus that presented ganglionitis (HE staining, 100× magnification, scale bar = 5 μm). (D) Suggestive forms of Leishmania amastigotes (arrows) and inflammatory infiltrate in crypts (HE staining, 100× magnification, scale bar = 5 μm). (E) Leishmania amastigotes (arrow) in the popliteal lymph node (immunohistochemistry, 100× magnification, scale bar = 5 μm). (F) Leishmania amastigotes (arrow) in the ileum (immunohistochemistry, 100× magnification, scale bar = 5 μm).
Table 1 Global and differential leucocyte counts in peripheral blood in hamsters infected with L. (V.) braziliensis for 90 and 120 days. Time of infection 90 days
120 days
Groups CG 2311 3476 CG 2311 3476
Global count (cell/μl) 2,550 3,250 4,375 1,900 3,200 4,375
± ± ± ± ± ±
a
387.3 238.0a 963.9b 489.9a 483.0b 899.5b
Neutrophil (cell/μl) 858.8 1,072 1,410 760.8 967.3 1,238
± ± ± ± ± ±
a
265.4 129.2a 140.9b 162.4a 344.1a 512.5a
Lymphocyte (cell/μl) 1,645 1,666 2,076 1,111 1,767 2,332
± ± ± ± ± ±
a
286.2 153.5a 684.8a 372.3a 156.2a 411.7b
Monocyte (cell/μl) a
40.5 ± 33.3 505.5 ± 145.4b 889.3 ± 441.2b 24.0 ± 18.5a 458.5 ± 117.8b 792.5 ± 146.7b
Eosinophil (cell/μl) 12.0 ± 24.0a 15.2 ± 17.6a 0a 4.7 ± 9.5a 7.5 ± 15.0a 12.2 ± 24.5a
Data with different letters in the same column for each time post-infection are significantly different (p < 0.05) compared with the CG. The data are expressed as mean ± SD. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476. Bold column are time of infection and experimental groups. 1627
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Fig. 2. Morphometry of the ileum wall in hamsters infected for 90 or 120 days with different L. (V.) braziliensis strains. The data are expressed as mean ± standard deviation. *p < 0.05, significant difference compared with CG. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476.
4. Discussion
spleen, lymph node [11,15] and bone marrow [6,7]. Hematological alterations are usually indicative of infection [37] and the evaluation of blood cells is important to achieve a systemic view of the diseases [37,38]. In the present study, L. (V.) braziliensis infection in hamsters caused a significant increase in the number of blood monocytes, corroborating the findings of studies of cutaneous leishmaniasis [39,40]. This increase in blood monocytes might be a source of immune cells at the site of infection. These mononuclear cells may be one way by which Leishmania is transported throughout the organism [35]. Once at the site of infection, these cells can transform into macrophages [39], which are the host for the parasite [41]. In our study, the infection with L. (V.) braziliensis induced the formation of inflammatory infiltrates altering the normal morphological
Various infections [27,28] and psychological changes [29–32] can affect the function and morphology of the gastrointestinal tract. Previous studies have reported intestinal involvement in visceral leishmaniasis demonstrating inflammatory infiltrate and structural alterations [20,33] with the presence of amastigote forms [20]. According to Martinez et al. [34], the dissemination of Leishmania can occur through the lymphatic system or hematogenous, since that the parasite DNA has already been found in blood mononuclear cells [35]. Studies demonstrated that L. (V.) braziliensis causes metastatic [2] and disseminated [36] lesions in the skin, histopathological alterations in spleen, liver and lymph node [11] and can migrate to other organs including the
Table 2 Morphometry of enterocytes and their nuclei at 90 and 120 days post-infection. Enterocytes Time 90 days
120 days
Group CG 2311 3476 CG 2311 3476
Nuclei of the enterocytes
Height (μm)
Width (μm) a
5.32 5.70 5.46 5.64 5.59 5.68
11.50 ± 2.97 11.00 ± 3.83b 9.82 ± 2.39b 11.39 ± 2.80a 13.63 ± 4.30b 12.27 ± 2.77b
± ± ± ± ± ±
Height (μm) a
1.23 1.00b 1.04a 1.30a 2.02a 1.19a
5.22 4.62 4.30 4.66 4.65 5.04
± ± ± ± ± ±
Width (μm) a
1.33 1.25b 1.07b 0.97a 1.05a 1.30b
3.65 3.99 3.70 3.57 3.61 3.70
± ± ± ± ± ±
1.14a 0.83b 0.81a 0.78a 0.98a 0.91a
Data with different letters in the same column at each time post-infection indicate significant differences (p < 0.05) compared with the CG. The data are expressed as mean ± SD. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476. Bold column are time of infection and experimental groups. 1628
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Fig. 3. Photomicrograph of cross-sections of the ileum wall. The first column shows animals at 90 days post-infection. The second column shows animals at 120 days post-infection. The images represent the control group (1 st line) and animals infected with MHOM/BR/2003/2311 (2nd line) and MHOM/BR/2009/3476 (3rd line). The sections were stained with HE (10× magnification, scale bar = 100 μm).
increase in the height of villi [24] such as those demonstrated in hamsters infected with the two strains of Leishmania. In addition, the augment in the cellular renewal negatively influences the differentiation of epithelial cells [45]. One of the most important findings of this study was to visualize the presence of immune cells in the ganglia of myenteric plexus in both infected animals characterizing ganglionitis. Inflammatory infiltrate in the ganglia can lead to neuronal degeneration [46,47] and is usually associated with intestinal disorders [46] however, we did not observe intestine clinical signs during the experimental period. Ganglionitis may be related to the increase in the muscular layer of the ileum in infected animals as showed by other authors [48,49]. The increase in the thickness of muscular can also be associated with the cytokines secreted by immune cells present between muscle cells [50]. Furthermore, the reduction of acid-producing mucin cells lead to denser intestinal mucus that is more difficult to expel [24] affecting the contractility of muscle fibers and provoking both functional [51] and morphological [50] alterations. The histopathological alterations in the mucosa, submucosa, and muscular layer supported the increase in the total wall thickness of the ileum.
aspect and inducing ganglionitis which may directly affect intestinal immunity. Some histopathological alterations that were observed in the ileum of hamsters, as inflammatory infiltrate in crypts, and the greater number of immune cells in the lamina propria, submucosa, and muscular layer might signalize tissue damage [23]. The migration of immune cells to the lamina propria may have occurred by the presence of the parasite in the organ, as can be seen in other studies [19,20]. The accumulation of these cells may be responsible for the increase of the thickness of the submucosa and the height and width of villi observed in the groups infected with both strains. The increase in the number of IELs is also related with the inflammatory response [42]. These cells may act as immunoregulators in the intestinal epithelium [43] and promote the flattening of enterocytes in the infected groups at 90 days post-infection. Different alterations in the size of enterocytes were observed at 90 and 120 days post-infection, likely because of the high renewal rate of this cellular type that occurs within 3–4 days [44]. Intestinal cellular proliferation occurs in the crypts and under normal conditions the epithelium is completely renewed within 6–7 days [44]. Increased cellular proliferation results in deeper crypts and contribute to the 1629
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Fig. 4. Cellular alterations in the epithelium of the ileum in hamsters infected for 90 or 120 days with different L. (V.) braziliensis strains. The data are expressed as mean ± standard deviation. *p < 0.05, significant difference compared with CG. CG, control group; 2311, group infected with MHOM/BR/2003/2311; 3476, group infected with MHOM/BR/2009/3476.
After observing the aforementioned histopathological changes and the suggestive forms of Leishmania amastigotes in the histological sections, we detected DNA of the parasite in the mesenteric lymph node and ileum. Using immunostaining with specific antibodies to Leishmania we confirmed the parasite in the intestine. Thus, we can affirm that L. (V.) braziliensis can visceralize to the intestine and mesenteric lymph node of hamsters, once they are susceptible to this specie as previously reported [11,14]. Additionally, it is interesting to highlight that L. braziliensis [6,7] and L. amazonensis [52] were isolated from bone marrow of human patients with visceral leishmaniasis showing that immunocompromising condition is an important factor for the visceralization of the parasite. Comparing the results of the groups infected with the different strains, the profile of paw lesion was similar in both isolates during the experimental period. The 2311 group presented major alterations at 90 days post-infection and both strains induced similar changes at 120 days post-infection. The MHOM/BR/2009/3476 strain was detected in the ileum and mesenteric lymph node by qPCR at 90 and 120 days. The MHOM/BR/2003/2311 strain was found in the mesenteric lymph node of the infected animals in both infection times and in the ileum at 120 days after infection. This differential behavior between the two strains may be attributable to genetic polymorphisms of Leishmania [53] and the host [13], in addition to the interactions between the parasite and host’s immune response [54].
Table 3 Real-time qualitative PCR assay in samples of the ileum and mesenteric lymph node from hamsters infected with Leishmania (Viannia) braziliensis strains. Groups
Ileum
Control
2311
3476
Mesenteric lymph node
Control
2311
3476
Animal ID
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Ct mean 90 days
120 days
– – – – – – – – – 33.8 26.3 28.4 – – – – 30.4 – – 33.7 34.1 nr 25.4 –
– – – – – 34.1 – – 32.9 33.6 29.0 27.8 – – – – 20.6 – 25.1 31.1 – 30.4 31.3 27.9
5. Conclusion
Real-time PCR results using the MP3H/MP1L primers. Groups: 2311, infected with MHOM/BR/2003/2311; 3476, infected with MHOM/BR/2009/3476. Animal ID, animal identification. Ct, threshold cycle. (Ct ≤ 35): positive; (-): Ct undetermined or > 35; (nr): not realized. Bold column are time of infection and experimental groups.
Chronic infection with the MHOM/BR/2003/2311 and MHOM/BR/ 2009/3476 strains of L. (V.) braziliensis caused similar cellular and morphometric alterations in the ileum of hamsters indicating intestinal inflammation. We also found the parasite DNA in the ileum and 1630
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mesenteric lymph node and amastigotes forms in the ileum showing the visceralization of both strains. Thus, we demonstrated that infection with a specie that usually causes cutaneous lesions in humans can lead to systemic alterations in hamsters. Further studies should be performed to investigate other aspects of L. (V) braziliensis-host interaction in the intestine and other viscera.
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