Collectins and Galectins in the Abomasum of Goats Susceptible and Resistant T to Gastrointestinal Nematode Infection ⁎ Bárbara M.P.S
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Veterinary Parasitology: Regional Studies and Reports 12 (2018) 99–105 Contents lists available at ScienceDirect Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr Original article Collectins and galectins in the abomasum of goats susceptible and resistant T to gastrointestinal nematode infection ⁎ Bárbara M.P.S. Souzaa, , Sabrina M. Lamberta, Sandra M. Nishia, Gustavo F. Saldañab, Geraldo G.S. Oliveirac, Luis S. Vieirad, Claudio R. Madrugaa, Maria Angela O. Almeidaa a Laboratory of Cellular and Molecular Biology, School of Veterinary Medicine and Animal Science, Federal University of Bahia, Salvador, BA, Brazil b Institute for Research on Genetic Engineering and Molecular Biology (INGEBI-CONICET), Laboratory of Molecular Biology of Chagas Disease, Buenos Aires, Argentina c Laboratory of Cellular and Molecular Immunology, Research Center of Gonçalo Muniz, Fiocruz, BA, Brazil d National Research Center of Goats and Sheep, Embrapa, Sobral, CE, Brazil ARTICLE INFO ABSTRACT Keywords: Originally described in cattle, conglutinin belongs to the collectin family and is involved in innate immune Innate immunity defense. It is thought that conglutinin provides the first line of defense by maintaining a symbiotic relationship Lectins with the microbes in the rumen while inhibiting inflammatory reactions caused by antibodies leaking into the Helminth bloodstream. Due to the lack of information on the similar lectins and sequence detection in goats, we char- Ruminants acterized the goat conglutinin gene using RACE and evaluated the differences in its gene expression profile, as PCR well as in the gene expression profiles for surfactant protein A, galectins 14 and 11, interleukin 4 and interferon- gamma in goats. We used Saanen and Anglo Nubian F2 crossbred goats monitored over a period of four months and characterized them as resistant (R) or susceptible (S) based on the average values of EPG counts. Goat conglutinin was similar to bovine conglutinin, but its gene expression varied among different tissues. However, as with bovine conglutinin, it was most highly expressed in the liver. Variation in conglutinin (R = 24.3 ± 3.9; S = 23.5 ± 2.6, p = 0.059), protein surfactant A (R = 23.8 ± 5.2, S = 24.4 ± 2.3, p = 0.16), galectin 14 (R = 15.9 ± 3.5, S = 14.7 ± 6.2, p = 0.49) and galectin l1 gene expression (R = 25.4 ± 2.6, S = 25.8 ± 3.7, p = 0.53) was not significant between groups. However, there were weak correlations be- tween interleukin 4 and the protein surfactant A gene (r = 0.459, p = 0.02) and between interleukin 4 and galectin 11 (r = 0.498, p = 0.01). Strong correlation between interferon-gamma and galectin 14 (r = 0.744, p = 0.00) was observed. Galectin 14 was negatively correlated with the number of nematodes in the goat (r = −0.416, p = 0.04) as well as the EPG count (r = −0.408, p = 0.04). This is the first study to date that identifies the gene expression of conglutinin, surfactant protein A and galectins 14 and 11 in the goat abomasum. In conclusion, we present evidence that lectin is involved in the immune response to gastrointestinal nematodes, which suggests that collectins and galectins are involved in the molecular recognition of helminths. 1. Introduction gastrointestinal surface, epithelial cells produce molecules that are se- creted in the mucus and contribute to localized immune responses. Gastrointestinal nematode (GIN) infections are one of the main Among these molecules, lectins such as galectins and collectins, which factors that lead to low performance and economic loss in goat herds. are produced by various types of animal cells and may be important in Goats, compared with sheep, have different grazing behaviors and a less controlling parasites, interact with oligosaccharide structures present effective immune response in reducing the number of nematode larvae on the surface of pathogens (Drickamer and Taylor, 1993; Perrigoue and expelling the adults (Hoste et al., 2010). Host immune mechanisms et al., 2008; Van De Wetering et al., 2004a,b). against gastrointestinal nematodes are complex and depend on nu- The galectin family members, galectin 14 and 15, bind glycans on merous genes (Garside et al., 2000). Individuals who are genetically the surface of potentially pathogenic microorganisms and act as re- resistant to Haemonchus contortus present eosinophilia, mucosal mas- cognition and effector factors in innate immunity (Vasta, 2009). These tocitosis and high IgA and IgG levels (Gill et al., 1994, 2000). At the proteins were identified in the abomasal tissue of sheep following ⁎ Corresponding author at: Laboratório de Biologia Celular e Molecular, Escola de Medicina Veterinária e Zootecnia, Universidade Federal da Bahia (UFBA), Av Adhemar de Barros 500 - Ondina, Salvador, BA 40.170-110, Brazil. E-mail address: [email protected] (B.M.P.S. Souza). https://doi.org/10.1016/j.vprsr.2017.12.001 Received 19 January 2017; Received in revised form 4 December 2017; Accepted 7 December 2017 Available online 19 December 2017 2405-9390/ © 2017 Elsevier B.V. All rights reserved. B.M.P.S. Souza et al. Veterinary Parasitology: Regional Studies and Reports 12 (2018) 99–105 exposure to H. contortus (Dunphy et al., 2002). Collectins are a family of transformed into chemically competent E. coli TOP10 cells, and trans- calcium-dependent defense lectins in animals. There have been nine formants were selected with ampicillin (50 μg/ml). After confirmation collectin family members defined so far, of which, mannan binding by sequencing, construct pGEMT-CGN was extracted by alkaline lysis lectin (MBL), surfactant proteins (SP-A and SP-D) and conglutinin and purified with PEG8000 (Sambrook et al., 2001). (CGN) are the best characterized (Hansen and Holmskov, 1998; Veldhuizen et al., 2011). The binding of collectins to microorganisms 2.1.4. Identification and analysis may facilitate microbial clearance through aggregation, complement Plasmids of the selected clones were submitted for capillary se- activation, opsonization and phagocytosis activation. In addition, the quencing (Macrogen, Inc.), and a similarity analysis of the inserted DNA collectins can modulate inflammatory response and the adaptive im- and the deduced protein sequence was conducted using BLASTN, mune system (Van De Wetering et al., 2004a,b). BLASTp and Transeq. Open readings frame (ORF) predictions were Conglutinin is a protein in the collectin family that is involved in the obtained using http://www.expasy.org/tools/. Sequence alignment innate immune defense, showing antiviral and antibacterial activity was measured using Clustal W and Bioedit software. Phylogenetic (Drickamer, 1992). This protein interacts with the complement system diagrams were constructed by calculating the proportion difference (p- to bind carbohydrates on the degradation product, iC3b (Laursen et al., distance) of the aligned amino acid sites from full-length peptide se- 1994), and stimulates the oxidative burst in phagocytes (Dec and quences (including the goat bovine - CGN Capra hircus) using the Wernicki, 2012). In this study, we characterize the conglutinin gene in maximum likelihood method and MEGA5 software. goats and evaluate differences in the gene expression profiles of con- glutinin, surfactant protein A, galectin 14 and 11, interleukin 4 and 2.2. Expression profiles of the lectins and cytokines interferon gamma in goats monitored over a four-month period who were characterized as resistant or susceptible based on their average 2.2.1. Experimental design EPG count values. Animals were selected from a herd of 172 goats, F2 generation from ½ Anglo-nubiana × ½ Saanen, raised at Embrapa Experimental Unit 2. Materials and methods Goats and Sheep (Unidade Experimental da Embrapa Caprinos e Ovinos), Sobral, Ceará in Northeast Brazil (latitude 3°42′59.01″ and 2.1. Goat conglutinin characterization longitude 40°23′21.57″). Animals of both genders aged between four and five months were placed in the experimental area of an irrigated 2.1.1. Animals tissue pasture with Tanzania grass (Panicum maximum Jacq. Cv. Tanzania) Samples of the abomasum, large intestine, lung, liver and spleen naturally contaminated by nematode larvae. were collected from one Anglo-Nubian and Saanen crossbred goat, At the beginning of the experiment, all animals were dewormed male, approximately 12 months old. The tissues were aliquoted and with four different chemical groups: Closantel (10 mg/kg BW), stored in RNA stabilizer solution (RNAlater®, Applied Biosystems, Ivermectin (0.2 mg/kg), Levamisole (7.5 mg/kg) and Moxidectin Foster City, CA, USA) and maintained at −80 °C until RNA extraction. (0.2 mg/kg). The animals were monitored using eggs per gram of feces (EPG) counts (Ueno and Gonçalves, 1998) until the count was zero. 2.1.2. RT-PCR After that, the animals were transferred to the Tanzania grass pasture For the RNA extraction, 50–100 mg fragments of tissue frozen with naturally contaminated with nematode larvae (Haemonchus sp., Tri- liquid nitrogen were ground with a mortar and pestle and homogenized chostrongylus sp. and Oesophagostomum sp.). The individual fecal sam- in Trizol reagent (Invitrogen, USA). RNA concentration was estimated ples were examined weekly, and the number of nematode EPG counts by fluorimetry (Qubit, Invitrogen, USA), and the quality was assessed were determined for each animal as previously described by Gordon by agarose gel electrophoresis. After DNase treatment, cDNA was syn- and Whitlock (1939) and modified by Ueno and Gonçalves (1998). thetized using Superscript III RT (Invitrogen, USA) with Oligo(dT)12–18 When the mean EPG reached 800, blood and fecal samples were col- primers and RNase inhibitors according to the manufacturers re- lected over three days, and the animals were dewormed again with the commendations. same anthelmintics. Animals were subjected to a second challenge PCR was conducted with a final volume of 20 μL using PCR buffer when the fecal EPG count was zero, to discriminate different levels of (1×), 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer resistance to the parasitic infection (Coutinho et al., 2015).