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Partial protection and abomasal cytokine expression in sheep experimentally infected with Haemonchus contortus and pre-treated with Taenia hydatigena vesicular concentrate b ˜ J.A. Buendía-Jiménez a , M.A. Munoz-Guzmán , M.A. Vega-López c , C. Cuenca-Verde b , b b J.P. Martínez-Labat , J.A. Cuéllar-Ordaz , F. Alba-Hurtado b,∗ a
Programa de Doctorado en Ciencias de la Producción y de la Salud Animal, Universidad Nacional Autónoma de México, Mexico Departamento de Ciencias Biológicas, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Mexico c Laboratorio de Inmunobiología de las Mucosas, Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Polite´ıcnico Nacional, Mexico b
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 20 April 2015 Accepted 21 April 2015 Keywords: Cytokines Immune protection Ovine haemonchosis Abomasal wall Immunohistochemistry
a b s t r a c t The abomasal expression of IL-2, IL-4, IL-6, IL-10 and IFN␥ in lambs experimentally infected with Haemonchus contortus and its relationship to protection induced by a Taenia hydatigena larvae vesicular concentrate (ThLVC) were evaluated. The lambs that were only infected with H. contortus larvae showed a worm burden greater (p < 0.05) than the lambs that received ThLVC prior to infection. Moreover, the lambs that received ThLVC showed a greater (p < 0.05) number of blood eosinophils than the lambs that did not receive the ThLVC. In general, the lambs that received ThLVC prior to infection had a greater amount of eosinophils and mast cells and higher in situ expression of IFN␥, IL-2, IL-4, IL-6 and IL-10 in the abomasal wall than the lambs that were infected with H. contortus only or that received ThLVC (p < 0.05) only. A higher expression of IL-2 and IFN␥ in the submucosa compared to the abomasal mucosa and a higher expression of IL-4 in the abomasal mucosa compared to the submucosa was observed (p < 0.05). These results suggest that there is a Th1 type response in the abomasal submucosa and a Th2 type response in in the abomasal mucosa. The amount of eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4 and IL-6 in the abomasal walls were negatively correlated with the worm burden (p < 0.05). These results suggest that ThLVC is a non-specific immune stimulator for the abomasal immune response, and it is likely that the protection observed is the result of this effect. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been demonstrated that repeated infections by Haemonchus contortus, simultaneous infections with other parasites, and the application of Taenia hydatigena larvae vesicular concentrate (ThLVC), induce a moderate protection against haemonchosis (Terefe et al., 2005; Yacob et al., 2008; Robinson et al., 2010; Cuenca-Verde et al., 2011). It has been proposed that ThLVC-induced protection is associated with the immune response that is mounted against H. contortus in the abomasum and is regulated by cytokines. However, it has not been determined if there is a specific cytokine pattern associated with protection.
∗ Corresponding author. Tel.: +52 55 56 23 19 99x39411; fax: +52 55 56 23 19 99x39411. E-mail address: [email protected] (F. Alba-Hurtado).
Helminth infections typically induce a Th2 profile, including the induction of IL-4, IL-5 and IL-13 (Meeusen et al., 2005). It has been shown that infections with H. contortus induce a Th2 immune response characterized by the recruitment and/or activation of CD4 T-cells, eosinophils, mast cells and the induction of type-2 cytokines (Balic et al., 2000). Gill et al. (2000) demonstrated in vitro that lymphocytes obtained from sheep resistant to H. contortus infection produced less IFN?? and more IL-5 than lymphocytes obtained from H. contortus susceptible sheep after infection. Shakya et al. (2009) demonstrated the over expression of IL-4 mRNA in lambs resistant to haemonchosis. Helminth parasites or their components are known to alter host immune responses, and the responsible molecules are a potential source of biological immunoadjuvants (Dissanayake and Shahin, 2007). It has been observed that components of metacestodes, such as Taenia solium or Taenia crassiceps, can modulate the immune response of their hosts (Tato et al., 1996; Segura-Velázquez et al.,
http://dx.doi.org/10.1016/j.vetpar.2015.04.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Buendía-Jiménez, J.A., et al., Partial protection and abomasal cytokine expression in sheep experimentally infected with Haemonchus contortus and pre-treated with Taenia hydatigena vesicular concentrate. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.019
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2009). Dissanayake and Shahin (2007) reported the Th-2 type immunomodulatory properties of Taenia crassiceps glycans in mice. Previously, we reported partial protection against the establishment of H. contortus in lambs via inoculation with a ThLVC. The results of this study show the reduction of fecal egg counts (FEC) and number of adult worms (AW) in the abomasum of lambs that received ThLVC prior to infection, and this was associated with eosinophilia and the recruitment of eosinophils and CD4 T-cells in the abomasal wall (Cuenca-Verde et al., 2011), suggesting an increase in cytokine expression by mucosal cells. Several studies quantified the cytokine mRNA in the abomasum, abomasal lymph node and spleen lymphocytes (Lacroux et al., 2006; Terefe et al., 2007), but there are no studies that evaluated in situ cytokine expression. The objective of this study was to evaluate the abomasal expression of IL-2, IL-4, IL-6, IL-10 and IFN␥ in experimentally infected lambs and the relationship of this expression with ThLVCinduced protection.
lambs. Those animals were free from other gastrointestinal nematodes. 2.3. ThLVC ThLVC was obtained as described by Cuenca-Verde et al. (2011). Briefly, approximately 100 metacestodes of T. hydatigena were collected from the sheep at a slaughterhouse. Vesicular liquid was obtained by metacestode puncture, followed by the addition of a mixture of protease inhibitors (aprotinin 10 g mL−1 , leupeptin 10 g mL−1 , iodoacetamide 1.8 mg mL−1 and PMSF 1 mM, (Sigma–Aldrich, Germany). The proteins were precipitated with (NH4 )2 SO4 , and the precipitate was resuspended in PBS and preserved at −85 ◦ C until used. Protein integrity was monitored by SDS-PAGE electrophoresis and measured using the Bradford (1976) method. 2.4. Parasitological examinations
2. Materials and methods 2.1. Experimental design Twenty 3- to 4-mo-old, non-grazing, Columbian breed male lambs maintained from birth in nematode-free conditions were used. The feed consisted of a mix of 50% commercial feed for sheep (14% crude protein) and 50% dry ground alfalfa. The lambs were bred in the authors’ institution to assure breed purity. Each lamb received 4% of its live weight daily in feed mix and water ad libitum. Before the experiment, the FEC was zero in all lambs. The Internal Committee for Care of Experimental Animals of the Postgraduate Program of Animal Production and Health (UNAM, México) approved this study. The lambs were divided into 4 groups (n = 5/each). The characteristics and treatments performed in these groups are shown in Table 1. Blood samples were collected directly from the jugular vein, and feces samples were collected directly from the rectum every 48 h between days -14 and 14 post-infection (PI) and on days 21, 28, 35, 42 and 49 PI. Blood eosinophils (BE) were counted in blood samples, and fecal egg counts (FEC) were determined in feces samples. The lambs were humanely killed with a captive bolt stun gun on day 49 PI. After the animals were killed, the abomasum samples were collected and AW were counted and measured. 2.2. H. contortus strain The H. contortus strain was isolated, characterized and used by ˜ our group (Valdez-Ramírez, 2004; Munoz-Guzmán et al., 2006; ˜ et al., 2012); the Cuenca-Verde et al., 2011; Munoz-Guzmán prepatent period of the strain is 18 to 20 days and shows high virulence. The strain has been maintained by successive infections of susceptible lambs and virulence, fertility and morphology have been periodically verified. Larvae for the experimental infections were obtained from fecal larvae cultures derived from the infected
The FEC was determined using the McMaster modified technique and was expressed as the mean eggs per gram of feces (epg). In the experimental lambs, the abomasum was washed and the content was diluted in 2 L of water. A 10% aliquot was separated, the number of AW was determined, and the female/male ratio was determined. The AW size was determined by measuring 100 males and 100 females with a Vernier caliper (Coadwell and Ward, 1981; Stear and Murray, 1994). 2.5. BE count The blood was diluted 1:10 with Carpentier solution (1% eosin Y in 40% formaldehyde). The BE was counted in a Neubauer chamber, and the results were expressed as eosinophils per mm3 of blood (Hohenhaus et al., 1998). 2.6. Tissue eosinophil, mast cell and cytokine producing cell counts Three samples (2 cm2 ) were obtained from the fundic and pyloric abomasal regions of each lamb. One sample was cryopreserved in OCT (Tissue–Tek® ) and stored at −80 ◦ C until further use, one sample was fixed in 4% paraformaldehyde, and the last sample was fixed in Carnoy’s fixative. Five-micrometer-thick sections from each cryopreserved sample were processed by indirect immunohistochemistry using the avidin–biotin complex method. The antibodies used for cytokine producing cell detection are shown in Table 2. Endogenous peroxidase activity was blocked with Peroxo-Block (00-2015, Invitrogen, USA). Endogenous biotin/avidin activity was also blocked with an Avidin/Biotin Blocking Kit (00-4303, Invitrogen, USA). The cells were identified with the Histostain SP Kit (50-209Z, Invitrogen). Paraformaldehyde or Carnoy’s fixed tissues were dehydrated and embedded in melted wax. The embedded samples were
Table 1 Treatments of the experimental Columbia lambs groups. Groups(n = 5)
1 2 3 4
Days before to the infection
Infection day
Days after infection
−6
−2
0
2
– 600 g i.m.a 600 g i.p.a 600 g i.m.a 600 g i.p.a –
– 600 g i.p.a 600 g i.p.a –
– – 5000 L3 of H. contortusb 5000 L3 of H. contortusb
– 600 g i.p.a 600 g i.p.a –
i.m. Intramuscular injection; i.p. Intraperitoneal injection. a Inoculation with Taenia hydatigena larvae vesicular concentrate (ThLVC) b Infection by stomach tube.
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Table 2 Anti-cytokynes antibodies. Primary antibody
Goat-biotinylated secundary antibody
Specificity
Clone
Product code(Serotec labs)
(Invitrogen labs)
Ovine IL-2 Bovine IL-4 Ovine IL-6 Ovine IL-10 Bovine IFN␥
IE10 CC303 Policlonal IgG CC318 CC302
MCA1826 MCA1820 AHP424 MCA2110 MCA1783
anti-mouse IgG anti-mouse IgG anti-rabbit IgG anti-mouse IgG anti-mouse IgG
mounted in small blocks, and 5 m-thick sections were cut. The paraformaldehyde sections were stained with chromotrope 2R (Sigma–Aldrich, Germany), and the Carnoy’s samples were stained with toluidine blue. Counts of the cytokine producing cells, tissue eosinophils (TE) and mast cells were performed at 200 × magnification in eight microscopic fields from the mucosae and eight fields from the submucosae (approx. 2.2 × 106 m2 ) using image analysis software (Image Premier Plus). 2.7. Statistical analysis The number of positive cells for interleukins, eosinophils, mast cells and FEC was transformed by the formula [log10 (HGH + 1)] to stabilize the variances prior to the analysis. The data were analyzed by factorial ANOVA, using the Statistica for Windows software. Pearson’s correlation was used for all variables. 3. Results The lambs in group 4 had the highest FEC (p < 0.05) compared to the group 3 lambs from week 3 PI until the end of the experiment (Fig. 1). At the time of slaughter, the lambs in group 4 had a higher number of AW in the abomasum (p < 0.05) than the lambs in group 3 (1885 ± 253 vs 700 ± 360). There were no differences (p > 0.05) in the size of the AW recovered from the lambs of the groups 3 (females 2.1 ± 0.32 cm and males 1.5 ± 0.17 cm) and 4 (females 2.3 ± 0.19 cm and males 1.5 ± 0.1 cm). There were no AW or eliminated eggs in feces in groups 1 and 2. The lambs in groups 2 and 3 had higher total averages of BE (p < 0.001) (400.5 ± 30.7 and 399 ± 29.3, respectively) than the lambs in groups 1 and 4 (158.5 ± 15.8 and 244 ± 27.1, respectively)
Fig. 1. Weekly mean values (±SE) of fecal egg counts (FEC) in Columbia lambs. Group 3 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC) and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Black arrows indicate the three inoculations with ThLVC (days -6, -2 and 2). The white arrow indicates the infection day with 5000 L3 of H. contortus. *Statistically significant difference between groups (p < 0.05).
Fig. 2. Weekly blood eosinophil count mean (±SE) in Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
(Fig. 2). The lambs of group 4 had a higher BE average than the lambs of group 1 (p < 0.001). The BE kinetics are shown in Fig. 2. The lambs of groups 2 and 3 showed a higher amount of BE than the lambs of group 1 (p < 0.01) between days 4 and 14 PI, and the lambs of group 4 between days -4 to 8 PI. Fig. 3 shows the number of mast cells and eosinophils per cm2 in the different regions of the abomasum wall. The lambs of group 3 had a greater amount of eosinophils and mast cells in the abomasal wall than the lambs in other groups (p < 0.001). The fundic submucosa of group 3 lambs had more eosinophils than the rest of the studied regions (p < 0.001). The pyloric mucosa of the lambs in groups 2–4 had more eosinophils per cm2 (8782 ± 2097, 9079 ± 2002 and 7875 ± 1859, respectively) than the lambs in group 1 (2162 ± 515) (p < 0.05). The fundic mucosa and pyloric submucosa of group 3 lambs had more mast cells per cm2 (21,226 ± 8159 and 18,677 ± 3065, respectively) than the lambs in groups 1 (1490 ± 464 and 3947 ± 1063) and 4 (10,069 ± 3006 and 11,176 ± 3205) (p < 0.01). Fig. 4 shows the number of IFN␥ and IL-2 positive cells per cm2 in the different regions of the abomasal wall. In general, lambs of group 3 had more IFN␥- and IL-2-positive cells in the abomasal wall than lambs in the other groups. In all studied groups, the fundic submucosa had a higher amount of IFN␥-positive cells than the other regions (p < 0.05). The fundic mucosa and submucosa and pyloric mucosa of group 3 lambs had more IFN␥-positive cells than the homologous regions in the remaining groups (p < 0.01). The fundic submucosa had more IL-2-positive cells than the fundic mucosa in all groups (p < 0.05). The fundic submucosa of the group 3 lambs had more IL-2-positive cells than the lambs in groups 1, and 2 (p < 0.05). Fig. 5 shows the number of IL-4-, IL-6- and IL-10-positive cells per cm2 in the different regions of the abomasal wall. The group 3 lambs had more IL-4-, IL-6- and IL-10-positive cells per cm2 of abomasal wall than the lambs in the other groups (p < 0.05). In general, the pyloric and fundic mucosa of the lambs in the study groups had more IL-4-positive cells than the submucosa of their respective
Please cite this article in press as: Buendía-Jiménez, J.A., et al., Partial protection and abomasal cytokine expression in sheep experimentally infected with Haemonchus contortus and pre-treated with Taenia hydatigena vesicular concentrate. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.019
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Fig. 3. Median (±SE) eosinophils and mast cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
groups (p < 0.05). The fundic and pyloric mucosa of group 3 lambs had more IL-4-positive cells than the homologous regions in the lambs in the other groups (p < 0.01). The pyloric mucosa, pyloric submucosa and fundic mucosa of group 3 lambs had more IL-6positive cells than all regions of lambs in the group 1(p < 0.05). The pyloric submucosa and fundic mucosa of group 3 lambs had more
Fig. 5. Mean (±SE) of IL-4, IL-6 and IL-10 positive cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
IL-10-positive cells than the homologous regions of lambs in groups 1, and 2 (p < 0.05). Negative correlations (p < 0.05) were found between the worm burden and pyloric mucosa cells (mastocytes −0.60 and IFN␥ positive cells −0.75), fundic mucosa (mastocytes −0.62, eosinophils −0.55, IL-6-positive cells −0.68 and IFN␥-positive cells −0.62), pyloric submucosa (IL-2-positive cells −0.57 and IL-4-positive cells −0.70) and fundic submucosa (eosinophils −0.75). Additionally, a negative correlation (p < 0.02) was observed between the worm burden and BE (−0.71), and a positive correlation was observed (p < 0.03) between the BE and eosinophils in the fundic submucosa (−0.61) (Fig. 6). 4. Discussion
Fig. 4. Mean (±SE) IL-2 and IFN␥ positive cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
It has been demonstrated that infection with other parasites induces a host response that reduces the implantation by H. contortus in the abomasum (Terefe et al., 2005). In this context, in a previous study performed by our group, we demonstrated that the administration of an extract of T. hydatigena reduced the implantation of H. contortus larvae in lambs, which is associated with the increase of CD4+ and ␥␦+ lymphocytes in the abomasal wall (Cuenca-Verde et al., 2011). In this study, we confirmed the protection induced by administration of the extract, and we associated this protection with the expression of certain types of cytokines. The above results suggests that protection induced by the extract has an immunological basis. The immune system may be stimulated in a non-specific manner by substances, such as glycans, muramyl dipeptides and others, which can improve the immune response against certain pathogens
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Fig. 6. Immunohistochemical detection in wall abomasal section of lambs. (A) IL-2 positive cells in abomasal fundic mucosa. (B) IL-4 positive cells in abomasal piloric mucosa. (C) IL-6 positive cells in abomasal piloric mucosa. (D) IFN␥ positive cells in abomasal fundic mucosa. (E) IL-10 positive cells in abomasal fundic mucosa. (F) control. Arrows show a positive cell in each picture.
(Dissanayake et al., 2002; Dissanayake et al., 2002). In this study, the application of the extract produced eosinophilia with a general increase of eosinophils, mast cells and cells that express IFN␥, IL-2, IL-4, IL-6 and IL-10 in the abomasal wall. This suggests that this extract acts as a non-specific immune stimulator for the abomasal immune response and for the rest of the digestive tract. H. contortus AW are found in the abomasum lumen; therefore, the immunological events that occur in the abomasal mucosa are likely more important than the system events. However, both events may happen simultaneously and may be related to one another. Eosinophilia has frequently been associated with protection against H. contortus (Bricarello et al., 2004; Terefe et al., 2007). In this study, it was observed that the lambs that received ThLVC had increased levels of BE and had a negative correlation with worm burden. Similarly, the amount of BE has a positive correlation with the amount of tissue eosinophils in the abomasal fundic region, such that their presence in the blood may reflect what is occurring in the mucosa. In this study, the AW number in each of the abomasal regions was not established, although in previous studies by our
group, it was observed that the AW are present in greater numbers ˜ in the abomasal fundic region (Munoz-Guzmán et al., 2012). This suggests that BE continually migrates to the site where there is a higher parasite load, increasing the amount of eosinophils present in the abomasal mucosa that is in direct contact with the AW to reduce the amounts. It has also been demonstrated in lambs that infection with H. contortus larvae induces an increase of CD4+ cells in the abomasal wall and the expression of mRNA encoding for certain cytokines ˜ (Meeusen et al., 2005; Robinson et al., 2010; Munoz-Guzmán et al., 2012). Our results show that the infection with H. contortus induced an increase in eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4, IL-6 and IL-10 in a similar manner to ThLVC stimulation. However, the lambs that received ThLVC prior to infection had lower parasite loads, and the expression of cytokines was significantly higher than in the lambs that received ThLVC only or were infected only. Furthermore, the amount of eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4 and IL-6 in the abomasal walls were negatively related to the parasite load. This suggests that the reduction of parasite load in lambs that received
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the extract and infection with H. contortus larvae are the combined result of both stimulating effects. In several animal species, it has been proposed that the number and type of lymphocytes have a characteristic distribution in the histological regions of the small intestine. In the intestinal mucosa, activated CD8+ lymphocytes are mainly observed in the large intestine submucosa, a higher number of CD4+ lymphocytes are found (Murphy, 2012; Williams, 2012). Studies have demonstrated that lambs infected with H. contortus have a higher number of eosinophils in the submucosa than in the abomasal wall, which suggests the existence of a differential response in both histological regions of the abomasum (Amarante et al., 2005; Terefe et al., 2009). In general, our data show that application of the extract produced a higher expression of IL-2 and IFN␥ in the submucosa than in the abomasal mucosa, and by contrast, a higher expression of IL-4 is found in the abomasal mucosa than the submucosa. This suggests a compartmentalized immune response produced by the extract, in which the abomasal submucosa has a Th1 type response and the abomasal mucosa has a Th2 type. Future studies may delineate the role of each compartment in the induction of protection. The majority of studies that evaluated the expression of cytokines in experimental infections with gastroenteric nematodes are based on real-time PCR quantification of the mRNA that encodes the cytokines (Lacroux et al., 2006; Ibelli et al., 2011; Zaros et al., 2014). However, the amount of mRNA present in the tissue may not indicate that these cytokines are produced because there are posttranscriptional regulatory mechanisms that may modify the actual production of cytokines in cells (Alberts et al., 2008). Similarly, this technique does not allow the identification of the histological location of the positive cells or define their morphology. We detected the cells that express the different cytokines using immunohistochemistry with monoclonal or polyclonal antibodies highly specific for IFN␥, IL-2, IL-4, IL-6 and IL-10, which allowed us to measure the density of the cells in the tissue, determine their location in situ and associate their expression with the different histological regions of the abomasum. This provides new information on the local immune response and possibly reduces the error in the evaluation of cytokine expression involved in this immune response. Many researchers have associated protection against haemonchosis with a type Th2 immune response (Meeusen et al., 2005; ˜ et al., 2006 Lacroux et al., 2006; Shakya et al., Munoz-Guzman 2009; Zaros et al., 2014). However, recent research suggests that the dichotomy between the Th1- and Th2-type responses in the digestive tract of sheep are not the same as the systemic immune response observed in murine models (Robinson et al., 2011; Zaros et al., 2010). Our results suggest that in the abomasum, both types of immune response can occur simultaneously in different histological regions. The physiological implications or possible synergic activities remain unknown.
Acknowledgment This study was supported by the PAPIIT/UNAM projects No. IN216913 and IN215314.
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Zaros, L.G., Neves, M.R.M., Benvenuti, C.L., Navarro, A.M.C., Sider, L.H., Coutinho, L.L., Vieira, L.S., 2014. Response of resistant and susceptible Brazilian Somalis crossbreed sheep naturally infected by Haemonchus contortus. Parasitol. Res. 113, 1155–1161.
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Partial protection and abomasal cytokine expression in sheep experimentally infected with Haemonchus contortus and pre-treated with Taenia hydatigena vesicular concentrate b ˜ J.A. Buendía-Jiménez a , M.A. Munoz-Guzmán , M.A. Vega-López c , C. Cuenca-Verde b , b b J.P. Martínez-Labat , J.A. Cuéllar-Ordaz , F. Alba-Hurtado b,∗ a
Programa de Doctorado en Ciencias de la Producción y de la Salud Animal, Universidad Nacional Autónoma de México, Mexico Departamento de Ciencias Biológicas, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Mexico c Laboratorio de Inmunobiología de las Mucosas, Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Polite´ıcnico Nacional, Mexico b
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 20 April 2015 Accepted 21 April 2015 Keywords: Cytokines Immune protection Ovine haemonchosis Abomasal wall Immunohistochemistry
a b s t r a c t The abomasal expression of IL-2, IL-4, IL-6, IL-10 and IFN␥ in lambs experimentally infected with Haemonchus contortus and its relationship to protection induced by a Taenia hydatigena larvae vesicular concentrate (ThLVC) were evaluated. The lambs that were only infected with H. contortus larvae showed a worm burden greater (p < 0.05) than the lambs that received ThLVC prior to infection. Moreover, the lambs that received ThLVC showed a greater (p < 0.05) number of blood eosinophils than the lambs that did not receive the ThLVC. In general, the lambs that received ThLVC prior to infection had a greater amount of eosinophils and mast cells and higher in situ expression of IFN␥, IL-2, IL-4, IL-6 and IL-10 in the abomasal wall than the lambs that were infected with H. contortus only or that received ThLVC (p < 0.05) only. A higher expression of IL-2 and IFN␥ in the submucosa compared to the abomasal mucosa and a higher expression of IL-4 in the abomasal mucosa compared to the submucosa was observed (p < 0.05). These results suggest that there is a Th1 type response in the abomasal submucosa and a Th2 type response in in the abomasal mucosa. The amount of eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4 and IL-6 in the abomasal walls were negatively correlated with the worm burden (p < 0.05). These results suggest that ThLVC is a non-specific immune stimulator for the abomasal immune response, and it is likely that the protection observed is the result of this effect. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been demonstrated that repeated infections by Haemonchus contortus, simultaneous infections with other parasites, and the application of Taenia hydatigena larvae vesicular concentrate (ThLVC), induce a moderate protection against haemonchosis (Terefe et al., 2005; Yacob et al., 2008; Robinson et al., 2010; Cuenca-Verde et al., 2011). It has been proposed that ThLVC-induced protection is associated with the immune response that is mounted against H. contortus in the abomasum and is regulated by cytokines. However, it has not been determined if there is a specific cytokine pattern associated with protection.
∗ Corresponding author. Tel.: +52 55 56 23 19 99x39411; fax: +52 55 56 23 19 99x39411. E-mail address: [email protected] (F. Alba-Hurtado).
Helminth infections typically induce a Th2 profile, including the induction of IL-4, IL-5 and IL-13 (Meeusen et al., 2005). It has been shown that infections with H. contortus induce a Th2 immune response characterized by the recruitment and/or activation of CD4 T-cells, eosinophils, mast cells and the induction of type-2 cytokines (Balic et al., 2000). Gill et al. (2000) demonstrated in vitro that lymphocytes obtained from sheep resistant to H. contortus infection produced less IFN?? and more IL-5 than lymphocytes obtained from H. contortus susceptible sheep after infection. Shakya et al. (2009) demonstrated the over expression of IL-4 mRNA in lambs resistant to haemonchosis. Helminth parasites or their components are known to alter host immune responses, and the responsible molecules are a potential source of biological immunoadjuvants (Dissanayake and Shahin, 2007). It has been observed that components of metacestodes, such as Taenia solium or Taenia crassiceps, can modulate the immune response of their hosts (Tato et al., 1996; Segura-Velázquez et al.,
http://dx.doi.org/10.1016/j.vetpar.2015.04.019 0304-4017/© 2015 Elsevier B.V. All rights reserved.
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2009). Dissanayake and Shahin (2007) reported the Th-2 type immunomodulatory properties of Taenia crassiceps glycans in mice. Previously, we reported partial protection against the establishment of H. contortus in lambs via inoculation with a ThLVC. The results of this study show the reduction of fecal egg counts (FEC) and number of adult worms (AW) in the abomasum of lambs that received ThLVC prior to infection, and this was associated with eosinophilia and the recruitment of eosinophils and CD4 T-cells in the abomasal wall (Cuenca-Verde et al., 2011), suggesting an increase in cytokine expression by mucosal cells. Several studies quantified the cytokine mRNA in the abomasum, abomasal lymph node and spleen lymphocytes (Lacroux et al., 2006; Terefe et al., 2007), but there are no studies that evaluated in situ cytokine expression. The objective of this study was to evaluate the abomasal expression of IL-2, IL-4, IL-6, IL-10 and IFN␥ in experimentally infected lambs and the relationship of this expression with ThLVCinduced protection.
lambs. Those animals were free from other gastrointestinal nematodes. 2.3. ThLVC ThLVC was obtained as described by Cuenca-Verde et al. (2011). Briefly, approximately 100 metacestodes of T. hydatigena were collected from the sheep at a slaughterhouse. Vesicular liquid was obtained by metacestode puncture, followed by the addition of a mixture of protease inhibitors (aprotinin 10 g mL−1 , leupeptin 10 g mL−1 , iodoacetamide 1.8 mg mL−1 and PMSF 1 mM, (Sigma–Aldrich, Germany). The proteins were precipitated with (NH4 )2 SO4 , and the precipitate was resuspended in PBS and preserved at −85 ◦ C until used. Protein integrity was monitored by SDS-PAGE electrophoresis and measured using the Bradford (1976) method. 2.4. Parasitological examinations
2. Materials and methods 2.1. Experimental design Twenty 3- to 4-mo-old, non-grazing, Columbian breed male lambs maintained from birth in nematode-free conditions were used. The feed consisted of a mix of 50% commercial feed for sheep (14% crude protein) and 50% dry ground alfalfa. The lambs were bred in the authors’ institution to assure breed purity. Each lamb received 4% of its live weight daily in feed mix and water ad libitum. Before the experiment, the FEC was zero in all lambs. The Internal Committee for Care of Experimental Animals of the Postgraduate Program of Animal Production and Health (UNAM, México) approved this study. The lambs were divided into 4 groups (n = 5/each). The characteristics and treatments performed in these groups are shown in Table 1. Blood samples were collected directly from the jugular vein, and feces samples were collected directly from the rectum every 48 h between days -14 and 14 post-infection (PI) and on days 21, 28, 35, 42 and 49 PI. Blood eosinophils (BE) were counted in blood samples, and fecal egg counts (FEC) were determined in feces samples. The lambs were humanely killed with a captive bolt stun gun on day 49 PI. After the animals were killed, the abomasum samples were collected and AW were counted and measured. 2.2. H. contortus strain The H. contortus strain was isolated, characterized and used by ˜ our group (Valdez-Ramírez, 2004; Munoz-Guzmán et al., 2006; ˜ et al., 2012); the Cuenca-Verde et al., 2011; Munoz-Guzmán prepatent period of the strain is 18 to 20 days and shows high virulence. The strain has been maintained by successive infections of susceptible lambs and virulence, fertility and morphology have been periodically verified. Larvae for the experimental infections were obtained from fecal larvae cultures derived from the infected
The FEC was determined using the McMaster modified technique and was expressed as the mean eggs per gram of feces (epg). In the experimental lambs, the abomasum was washed and the content was diluted in 2 L of water. A 10% aliquot was separated, the number of AW was determined, and the female/male ratio was determined. The AW size was determined by measuring 100 males and 100 females with a Vernier caliper (Coadwell and Ward, 1981; Stear and Murray, 1994). 2.5. BE count The blood was diluted 1:10 with Carpentier solution (1% eosin Y in 40% formaldehyde). The BE was counted in a Neubauer chamber, and the results were expressed as eosinophils per mm3 of blood (Hohenhaus et al., 1998). 2.6. Tissue eosinophil, mast cell and cytokine producing cell counts Three samples (2 cm2 ) were obtained from the fundic and pyloric abomasal regions of each lamb. One sample was cryopreserved in OCT (Tissue–Tek® ) and stored at −80 ◦ C until further use, one sample was fixed in 4% paraformaldehyde, and the last sample was fixed in Carnoy’s fixative. Five-micrometer-thick sections from each cryopreserved sample were processed by indirect immunohistochemistry using the avidin–biotin complex method. The antibodies used for cytokine producing cell detection are shown in Table 2. Endogenous peroxidase activity was blocked with Peroxo-Block (00-2015, Invitrogen, USA). Endogenous biotin/avidin activity was also blocked with an Avidin/Biotin Blocking Kit (00-4303, Invitrogen, USA). The cells were identified with the Histostain SP Kit (50-209Z, Invitrogen). Paraformaldehyde or Carnoy’s fixed tissues were dehydrated and embedded in melted wax. The embedded samples were
Table 1 Treatments of the experimental Columbia lambs groups. Groups(n = 5)
1 2 3 4
Days before to the infection
Infection day
Days after infection
−6
−2
0
2
– 600 g i.m.a 600 g i.p.a 600 g i.m.a 600 g i.p.a –
– 600 g i.p.a 600 g i.p.a –
– – 5000 L3 of H. contortusb 5000 L3 of H. contortusb
– 600 g i.p.a 600 g i.p.a –
i.m. Intramuscular injection; i.p. Intraperitoneal injection. a Inoculation with Taenia hydatigena larvae vesicular concentrate (ThLVC) b Infection by stomach tube.
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Table 2 Anti-cytokynes antibodies. Primary antibody
Goat-biotinylated secundary antibody
Specificity
Clone
Product code(Serotec labs)
(Invitrogen labs)
Ovine IL-2 Bovine IL-4 Ovine IL-6 Ovine IL-10 Bovine IFN␥
IE10 CC303 Policlonal IgG CC318 CC302
MCA1826 MCA1820 AHP424 MCA2110 MCA1783
anti-mouse IgG anti-mouse IgG anti-rabbit IgG anti-mouse IgG anti-mouse IgG
mounted in small blocks, and 5 m-thick sections were cut. The paraformaldehyde sections were stained with chromotrope 2R (Sigma–Aldrich, Germany), and the Carnoy’s samples were stained with toluidine blue. Counts of the cytokine producing cells, tissue eosinophils (TE) and mast cells were performed at 200 × magnification in eight microscopic fields from the mucosae and eight fields from the submucosae (approx. 2.2 × 106 m2 ) using image analysis software (Image Premier Plus). 2.7. Statistical analysis The number of positive cells for interleukins, eosinophils, mast cells and FEC was transformed by the formula [log10 (HGH + 1)] to stabilize the variances prior to the analysis. The data were analyzed by factorial ANOVA, using the Statistica for Windows software. Pearson’s correlation was used for all variables. 3. Results The lambs in group 4 had the highest FEC (p < 0.05) compared to the group 3 lambs from week 3 PI until the end of the experiment (Fig. 1). At the time of slaughter, the lambs in group 4 had a higher number of AW in the abomasum (p < 0.05) than the lambs in group 3 (1885 ± 253 vs 700 ± 360). There were no differences (p > 0.05) in the size of the AW recovered from the lambs of the groups 3 (females 2.1 ± 0.32 cm and males 1.5 ± 0.17 cm) and 4 (females 2.3 ± 0.19 cm and males 1.5 ± 0.1 cm). There were no AW or eliminated eggs in feces in groups 1 and 2. The lambs in groups 2 and 3 had higher total averages of BE (p < 0.001) (400.5 ± 30.7 and 399 ± 29.3, respectively) than the lambs in groups 1 and 4 (158.5 ± 15.8 and 244 ± 27.1, respectively)
Fig. 1. Weekly mean values (±SE) of fecal egg counts (FEC) in Columbia lambs. Group 3 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC) and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Black arrows indicate the three inoculations with ThLVC (days -6, -2 and 2). The white arrow indicates the infection day with 5000 L3 of H. contortus. *Statistically significant difference between groups (p < 0.05).
Fig. 2. Weekly blood eosinophil count mean (±SE) in Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
(Fig. 2). The lambs of group 4 had a higher BE average than the lambs of group 1 (p < 0.001). The BE kinetics are shown in Fig. 2. The lambs of groups 2 and 3 showed a higher amount of BE than the lambs of group 1 (p < 0.01) between days 4 and 14 PI, and the lambs of group 4 between days -4 to 8 PI. Fig. 3 shows the number of mast cells and eosinophils per cm2 in the different regions of the abomasum wall. The lambs of group 3 had a greater amount of eosinophils and mast cells in the abomasal wall than the lambs in other groups (p < 0.001). The fundic submucosa of group 3 lambs had more eosinophils than the rest of the studied regions (p < 0.001). The pyloric mucosa of the lambs in groups 2–4 had more eosinophils per cm2 (8782 ± 2097, 9079 ± 2002 and 7875 ± 1859, respectively) than the lambs in group 1 (2162 ± 515) (p < 0.05). The fundic mucosa and pyloric submucosa of group 3 lambs had more mast cells per cm2 (21,226 ± 8159 and 18,677 ± 3065, respectively) than the lambs in groups 1 (1490 ± 464 and 3947 ± 1063) and 4 (10,069 ± 3006 and 11,176 ± 3205) (p < 0.01). Fig. 4 shows the number of IFN␥ and IL-2 positive cells per cm2 in the different regions of the abomasal wall. In general, lambs of group 3 had more IFN␥- and IL-2-positive cells in the abomasal wall than lambs in the other groups. In all studied groups, the fundic submucosa had a higher amount of IFN␥-positive cells than the other regions (p < 0.05). The fundic mucosa and submucosa and pyloric mucosa of group 3 lambs had more IFN␥-positive cells than the homologous regions in the remaining groups (p < 0.01). The fundic submucosa had more IL-2-positive cells than the fundic mucosa in all groups (p < 0.05). The fundic submucosa of the group 3 lambs had more IL-2-positive cells than the lambs in groups 1, and 2 (p < 0.05). Fig. 5 shows the number of IL-4-, IL-6- and IL-10-positive cells per cm2 in the different regions of the abomasal wall. The group 3 lambs had more IL-4-, IL-6- and IL-10-positive cells per cm2 of abomasal wall than the lambs in the other groups (p < 0.05). In general, the pyloric and fundic mucosa of the lambs in the study groups had more IL-4-positive cells than the submucosa of their respective
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Fig. 3. Median (±SE) eosinophils and mast cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
groups (p < 0.05). The fundic and pyloric mucosa of group 3 lambs had more IL-4-positive cells than the homologous regions in the lambs in the other groups (p < 0.01). The pyloric mucosa, pyloric submucosa and fundic mucosa of group 3 lambs had more IL-6positive cells than all regions of lambs in the group 1(p < 0.05). The pyloric submucosa and fundic mucosa of group 3 lambs had more
Fig. 5. Mean (±SE) of IL-4, IL-6 and IL-10 positive cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
IL-10-positive cells than the homologous regions of lambs in groups 1, and 2 (p < 0.05). Negative correlations (p < 0.05) were found between the worm burden and pyloric mucosa cells (mastocytes −0.60 and IFN␥ positive cells −0.75), fundic mucosa (mastocytes −0.62, eosinophils −0.55, IL-6-positive cells −0.68 and IFN␥-positive cells −0.62), pyloric submucosa (IL-2-positive cells −0.57 and IL-4-positive cells −0.70) and fundic submucosa (eosinophils −0.75). Additionally, a negative correlation (p < 0.02) was observed between the worm burden and BE (−0.71), and a positive correlation was observed (p < 0.03) between the BE and eosinophils in the fundic submucosa (−0.61) (Fig. 6). 4. Discussion
Fig. 4. Mean (±SE) IL-2 and IFN␥ positive cells in two abomasal histological regions of Columbia lambs. Group 1, control lambs (n = 5). Group 2 (n = 5), lambs inoculated with Taenia hydatigena larvae vesicular concentrate (ThLVC). Group 3 (n = 5), lambs inoculated with ThLVC and infected with H. contortus. Group 4 (n = 5), lambs infected with H. contortus. Lambs were killed at day 49 PI.
It has been demonstrated that infection with other parasites induces a host response that reduces the implantation by H. contortus in the abomasum (Terefe et al., 2005). In this context, in a previous study performed by our group, we demonstrated that the administration of an extract of T. hydatigena reduced the implantation of H. contortus larvae in lambs, which is associated with the increase of CD4+ and ␥␦+ lymphocytes in the abomasal wall (Cuenca-Verde et al., 2011). In this study, we confirmed the protection induced by administration of the extract, and we associated this protection with the expression of certain types of cytokines. The above results suggests that protection induced by the extract has an immunological basis. The immune system may be stimulated in a non-specific manner by substances, such as glycans, muramyl dipeptides and others, which can improve the immune response against certain pathogens
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Fig. 6. Immunohistochemical detection in wall abomasal section of lambs. (A) IL-2 positive cells in abomasal fundic mucosa. (B) IL-4 positive cells in abomasal piloric mucosa. (C) IL-6 positive cells in abomasal piloric mucosa. (D) IFN␥ positive cells in abomasal fundic mucosa. (E) IL-10 positive cells in abomasal fundic mucosa. (F) control. Arrows show a positive cell in each picture.
(Dissanayake et al., 2002; Dissanayake et al., 2002). In this study, the application of the extract produced eosinophilia with a general increase of eosinophils, mast cells and cells that express IFN␥, IL-2, IL-4, IL-6 and IL-10 in the abomasal wall. This suggests that this extract acts as a non-specific immune stimulator for the abomasal immune response and for the rest of the digestive tract. H. contortus AW are found in the abomasum lumen; therefore, the immunological events that occur in the abomasal mucosa are likely more important than the system events. However, both events may happen simultaneously and may be related to one another. Eosinophilia has frequently been associated with protection against H. contortus (Bricarello et al., 2004; Terefe et al., 2007). In this study, it was observed that the lambs that received ThLVC had increased levels of BE and had a negative correlation with worm burden. Similarly, the amount of BE has a positive correlation with the amount of tissue eosinophils in the abomasal fundic region, such that their presence in the blood may reflect what is occurring in the mucosa. In this study, the AW number in each of the abomasal regions was not established, although in previous studies by our
group, it was observed that the AW are present in greater numbers ˜ in the abomasal fundic region (Munoz-Guzmán et al., 2012). This suggests that BE continually migrates to the site where there is a higher parasite load, increasing the amount of eosinophils present in the abomasal mucosa that is in direct contact with the AW to reduce the amounts. It has also been demonstrated in lambs that infection with H. contortus larvae induces an increase of CD4+ cells in the abomasal wall and the expression of mRNA encoding for certain cytokines ˜ (Meeusen et al., 2005; Robinson et al., 2010; Munoz-Guzmán et al., 2012). Our results show that the infection with H. contortus induced an increase in eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4, IL-6 and IL-10 in a similar manner to ThLVC stimulation. However, the lambs that received ThLVC prior to infection had lower parasite loads, and the expression of cytokines was significantly higher than in the lambs that received ThLVC only or were infected only. Furthermore, the amount of eosinophils and mast cells and the in situ expression of IFN␥, IL-2, IL-4 and IL-6 in the abomasal walls were negatively related to the parasite load. This suggests that the reduction of parasite load in lambs that received
Please cite this article in press as: Buendía-Jiménez, J.A., et al., Partial protection and abomasal cytokine expression in sheep experimentally infected with Haemonchus contortus and pre-treated with Taenia hydatigena vesicular concentrate. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.019
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the extract and infection with H. contortus larvae are the combined result of both stimulating effects. In several animal species, it has been proposed that the number and type of lymphocytes have a characteristic distribution in the histological regions of the small intestine. In the intestinal mucosa, activated CD8+ lymphocytes are mainly observed in the large intestine submucosa, a higher number of CD4+ lymphocytes are found (Murphy, 2012; Williams, 2012). Studies have demonstrated that lambs infected with H. contortus have a higher number of eosinophils in the submucosa than in the abomasal wall, which suggests the existence of a differential response in both histological regions of the abomasum (Amarante et al., 2005; Terefe et al., 2009). In general, our data show that application of the extract produced a higher expression of IL-2 and IFN␥ in the submucosa than in the abomasal mucosa, and by contrast, a higher expression of IL-4 is found in the abomasal mucosa than the submucosa. This suggests a compartmentalized immune response produced by the extract, in which the abomasal submucosa has a Th1 type response and the abomasal mucosa has a Th2 type. Future studies may delineate the role of each compartment in the induction of protection. The majority of studies that evaluated the expression of cytokines in experimental infections with gastroenteric nematodes are based on real-time PCR quantification of the mRNA that encodes the cytokines (Lacroux et al., 2006; Ibelli et al., 2011; Zaros et al., 2014). However, the amount of mRNA present in the tissue may not indicate that these cytokines are produced because there are posttranscriptional regulatory mechanisms that may modify the actual production of cytokines in cells (Alberts et al., 2008). Similarly, this technique does not allow the identification of the histological location of the positive cells or define their morphology. We detected the cells that express the different cytokines using immunohistochemistry with monoclonal or polyclonal antibodies highly specific for IFN␥, IL-2, IL-4, IL-6 and IL-10, which allowed us to measure the density of the cells in the tissue, determine their location in situ and associate their expression with the different histological regions of the abomasum. This provides new information on the local immune response and possibly reduces the error in the evaluation of cytokine expression involved in this immune response. Many researchers have associated protection against haemonchosis with a type Th2 immune response (Meeusen et al., 2005; ˜ et al., 2006 Lacroux et al., 2006; Shakya et al., Munoz-Guzman 2009; Zaros et al., 2014). However, recent research suggests that the dichotomy between the Th1- and Th2-type responses in the digestive tract of sheep are not the same as the systemic immune response observed in murine models (Robinson et al., 2011; Zaros et al., 2010). Our results suggest that in the abomasum, both types of immune response can occur simultaneously in different histological regions. The physiological implications or possible synergic activities remain unknown.
Acknowledgment This study was supported by the PAPIIT/UNAM projects No. IN216913 and IN215314.
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