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Immune response related to Pelibuey sheep naturally infected

with gastrointestinal nematodes in a tropical region of Mexico

Jocelyn Maza-Lopez, Martha Janeth Pacheco-Armenta, David E.

Reyes-Guerrero, Agustín Olmedo-Juárez, Roberto González-
Garduño, Sara Olazarán-Jenkins, Ma. Eugenia López-Arellano

PII: S2405-9390(20)30203-3
DOI: https://doi.org/10.1016/j.vprsr.2020.100422
Reference: VPRSR 100422

To appear in: Veterinary Parasitology: Regional Studies and Reports

Received date: 5 June 2019

Revised date: 5 May 2020
Accepted date: 31 May 2020

Please cite this article as: J. Maza-Lopez, M.J. Pacheco-Armenta, D.E. Reyes-Guerrero,
et al., Immune response related to Pelibuey sheep naturally infected with gastrointestinal
nematodes in a tropical region of Mexico, Veterinary Parasitology: Regional Studies and
Reports (2020), https://doi.org/10.1016/j.vprsr.2020.100422

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
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© 2020 Published by Elsevier.

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Immune response related to Pelibuey sheep naturally infected with gastrointestinal

nematodes in a tropical region of Mexico

Jocelyn Maza-Lopeza, Martha Janeth Pacheco-Armentaa, David E. Reyes-Guerrerob, Agustín

Olmedo-Juárezb, Roberto González-Garduñoc, Sara Olazarán-Jenkinsd, Ma. Eugenia López-


Arellanob

aFacultad de Ingenieros en Biotecnología. Universidad Politécnica del Estado de Morelos,

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Jiutepec, Morelos, México. jomal1993@gmail.com;janetharmm@gmail.com

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bCentro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Instituto Nacional

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de Investigaciones Forestales, Agrícolas y Pecuarias. Carr. Fed. Cuernavaca-Cuautla No. 8254,
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Jiutepec, Morelos, México, C.P. 62550. de.reyes.guerrero@hotmail.com;aolmedoj@gmail.com
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cUniversidad Autónoma de Chapingo, Unidad Regional Universitaria Sur-Sureste, Km 7.5, Carr.

Teapa-Vicente Guerrero, Tabasco, México. robgardu@hotmail.com

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dSitio Experimental Las Margaritas, Instituto Nacional de Investigaciones Forestales, Agrícolas y

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Pecuarias, Hueytamalco, Pue., México. saraolazaran@hotmail.com

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Corresponding author. López-Arellano ME, PhD. Carr. Fed. Cuernavaca-Cuautla 8534,

Progreso, Jiutepec, Mor., México, C.P.62550. Phone: +52.777.319.28.60 x146; email:

mlopez.arellano@gmail.com

Disclosure. The authors of this manuscript have no financial or personal relationship with other

people or organization that could inappropriately influence or bias the content of the paper

Abstract
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We analysed the immune response involved in sheep naturally infected with gastrointestinal (GI)

nematodes. Fifteen Pelibuey lambs were grazed in paddocks contaminated with GI nematodes for

13 weeks. To assess the infection, the number of eggs per gram (epg) and the percentage of

packed cell volume (pcv) were evaluated. Blood and abomasal tissue samples were collected at

week 8 post-infection to analyse the expression levels of IL-4, IL-5, IL-6, IL-8, IL-13, TGF- and

FC R1A genes. The nematode Haemonchus contortus was the main species identified. In

addition, two groups of lambs were classified based on the x  SE of epg and pcv values: G-1,

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with 151 28 and 29  0.33%, respectively, and G-2, with 475  59.5 and 26 0.38%,

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respectively. For G-1, upregulation of IL-4, IL-8, IL-13, TGF- and FC R1A genes from 2.42- to

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14.99-fold was observed in blood and abomasal tissue samples (p > 0.05), and IL-5, IL-8 and
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TGF-  genes had significant gene expression levels in blood (p < 0.05). For G-2, moderate gene
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expression levels, ranging from 1.22- to 3.45-fold, were observed in abomasal tissue (p > 0.05),

and the IL-5 gene presented significant gene expression in blood (p < 0.05). Strong positively
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correlated values (r) between pcv and IL-4, IL-8 and TGF- genes were observed in G-1. In

contrast, significant negative correlations between epg and IL-4, IL-5 and FC R1A genes indicate
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acute infection for G-2. Our results suggest that IL-4, IL-5, IL-13, TGF- and FC R1A genes are
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important modulators of GI nematode infections of Pelibuey lambs.

Keywords: Sheep, nematodes, natural infection, immunology, phenotype

1. Introduction

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Sheep production in tropical and temperate regions is frequently affected by parasitic nematodes,

requiring recurrent anthelmintic treatments. However, nematodes have developed genetic

mechanisms to survive these drugs (Besier et al., 2016; Leathwick and Luo, 2017). The

integration of other control strategies is therefore important to achieve good animal health and, as

a consequence, adequate production. Different alternatives for parasitic control are currently

under study; one is the selection of sheep resistant to gastrointestinal (GI) nematodes (Li et al.,

2007; Chiejina et al., 2010; Wilkie et al., 2017). Identifying animals resistant to GI nematodes

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could be an approach to avoid the high number of unnecessary treatments, which could also

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reduce resistance (Leathwick and Luo, 2017; Zvinorova et al., 2016). The acute infection period

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of GI nematodes is defined via the inflammatory process when larvae invade the host tissues, and

numerous genes are activated to regulate this biological event (Li et al., 2007; Greer and Hamie,
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2016; Hendawy, 2018).
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The blood-feeding nematode Haemonchus contortus is one of most economically important

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pathogens within the strongylid nematodes that affect domestic ruminants. The infection of GI

nematodes involves TH1 and TH2 cells related to interleukin-4 (IL-4), IL-5, IL-10 and IL-13 and
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immune modulators, i.e., immunoglobulin E (IgE) and eosinophil cells (Allen and Sutherland,
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2014; McCrae et al., 2015). Previously, a group of Pelibuey sheep has displayed immune

tolerance against H. contortus infection, since a weak nematode infection was observed, with

500 eggs per gram (epg) and high relative expression of immune genes against GI nematodes

(Gonzalez et al., 2008; Estrada-Reyes et al., 2017). The Pelibuey sheep is an indigenous breed

with widespread distribution in tropical regions of Mexico. It is recognised for its adaptation to

tropical climates and it has been selected for meat production (Galina et al., 1996). Considering

the importance of this breed for Mexican farmers, the objective of the present study was to

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evaluate the parasitological (epg and pcv values) and relative expression profiles of the immune

response in Pelibuey lambs naturally infected with GI nematodes under tropical conditions.

2. Material and Methods

2.1 Area of study and meteorological data

This study was performed at the ovine experimental station Las Margaritas in Hueytamalco,

Puebla, Mexico. The station is situated at 1921´N 9720´W, 500 m above sea level, in a

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subtropical climate Af (c), with a mean annual rainfall of 3,000 mm and an average annual

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temperature of 21C. The estimated prevalence of benzimidazole- and ivermectin- resistant GI

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nematodes was around 80% (Campos et al., 1990; Gonzalez et al., 2008; Torres-Acosta et al.,

2012).
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2.2 Animal population

The 15 Pelibuey ewes used in this study (before pregnancy) have previously been identified as
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resistant using phenotypic traits, eggs per gram (epg) and packed cell volume (pcv) (Gonzalez et
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al., 2008; Estrada-Reyes et al., 2015). Resistant ewes were selected following the phenotypic

parameters citied by Gonzalez et al. (2008) and Estrada-Reyes et al. (2015): pcv 30%, epg < 500
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and clinical signs such as non-diarrhoea, emaciation and pale mucous membranes.

Ewes and their offspring were kept in pens with concrete floors after lambs were born to avoid

potential infections. Fifteen 4-month-old lambs, post-weaning, were housed and treated with

7.5 mg/kg of levamisole to eliminate any potential GI nematode infection before grazing. Lambs

were grazed on paddocks with Cyanodon plectostachyus grass and supplemented with 100 g per

day per animal of 16% crude protein for 13 weeks; water was provided ad libitum. Three lambs

without GI nematode infection were used as controls for molecular studies and kept in a sheep

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shed on a concrete floor. Control lambs were fed with sugar cane, 20% sorghum, 1% urea and

4% soy.

2.3 Phenotype collection

The studied lambs grazed in contaminated paddocks with GI nematodes for 13 weeks. Number of

epg and percentage of pcv were determined every 7 days. Samples were collected from June 6 to

August 28 post-infection, 2017. The number of epg was estimated using the McMaster

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parasitological technique (Coles et al., 2006), and the percentage of pcv was determined using the

micro-haematocrit technique (Gonzalez et al., 2008).

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2.4 Sample collection for gene expression analysis
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Small tissue samples (1 cm) were collected from the abomasal fundic region using surgical

procedures according to the Mexican Ethical Norms for Animal Health (NOM-051-ZOO-1995,
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NOM-062-ZOO-1995, http://www.gob.mx/senasica) at 8 weeks post-infection. Tissue samples

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were washed several times with phosphate-buffered saline solution (PBS, 1X, pH 7.4) and stored

at –80°C with RNAlater solution (Sigma, St Louis, MO, USA) until use. In addition, blood
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samples were collected from the jugular vein of each lamb 8 weeks post-infection in Paxgene
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Blood RNA tubes (Qiagen, Hilden, Germany) and kept at –80C until use.

2.5 RNA extraction and cDNA synthesis

Total RNA was isolated from blood and frozen abomasal tissue samples using QIAzol Lysis

Reagent and RNeasy Plus Mini Kit, respectively (Qiagen, Hilden, Germany), following the

manufacturers’ protocols. The RNA concentration and purity were determined at 260 nm and

OD260: OD280 ratio (NanoPhotometer NP80, IMPLEN, USA). Integrity was confirmed by 1%

agarose gel electrophoresis via staining with ethidium bromide (20 g/mL). Total RNA

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(250 ng/L) was used to synthesise first-strand cDNA using an RT2 First Strand Kit (Qiagen,

Hilden, Germany), according to the manufacturer’s guidelines. The Polymerase Chain Reaction

(PCR) methodology was carried out using the RT2 SYBR® Green qPCR MasterMix 2X (Qiagen,

Hilden, Germany), following the manufacturer’s instructions.

2.6 PCR array

Nucleotide sequences of seven genes (IL-4, IL-5, IL-6, IL-8, IL-13, TGF- and FC R1A genes)

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related to the immune response against H. contortus were selected for primer design, using the

Custom RT2 Profiler PCR Array Data Analysis Cat. Num. CAPB11185R (Qiagen, Hilden,

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Germany). Sequences were obtained from the National Center of Biotechnology
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(https://www.ncbi.nlm.nih.gov). The gapdh and  -actin housekeeping genes were selected as
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reference (Table 1). All PCR assays were performed in a Rotor-Gene (Corbet 6000, Qiagen). The
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conditions for cycling were 10 min at 95°C for 45 cycles of initial amplification, followed by 15 s

at 95°C for denaturation and 30 s at 60°C for annealing. Also, melting ramps from 65 to 95°C
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were implemented per assay. The reaction efficiency of each target gene was evaluated at 0.80–
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1.0, considering positive PCR controls (PPCs) and CT (cycle where the amplification plot crosses
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the threshold; Estrada-Reyes et al., 2017).

2.7 mRNA quantification analyses

For relative quantification, the comparative C T method was used (Pfaffl, 2001). The CT value for

each cytokine and receptor gene were subtracted from the average C T value of gapdh and β-actin

genes to normalise the expression; this value is called ΔC T . The ΔΔC T average value was

estimated from the ΔC T value in the infected and non-infected groups to determine the expression

level of each gene. The fold change was analysed by ΔΔC T ; the CT value of each replicate (n = 3)

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of each gene and each group was analysed by Student´s t-Test (p = 0.05). All data were analysed

using the Qiagen platform-web GeneGlobe (https://www.qiagen.com).

2.8 Statistical analysis

The epg and pcv data were normalised using UNIVARIATE analysis (SAS, version 9.0, 2004,

Institute Inc. Cary, N.C.) and the Kolmogorov-Smirnov statistic test, respectively. Non-normally

distributed data were log (epg + 1) transformed prior to analysis to reduce the variance and bring

the model closer to a normal distribution; the pcv percentages were transformed using Arcsin√X.

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Statistical analysis was carried out with transformed data for all response phenotypic traits (i.e.,

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epg and pcv values) by the multivariate linear mixed-effect regression model using PROC

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MIXED in SAS (Version 9, 2004; SAS Institute Inc., Cary, NC). Through a model of measures
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repeated over time, different covariance structures were tested for each study variable. The
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unstructured (UN) was selected and adjusted to each model by presenting lower AIC and BIC

values. The statistical model was calculated as follows:

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Yijkl =μ + θi + ζ j(i) +γk + (θγ)ik + εijk,

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where Yijkl = response variable (epg, pcv), μ = overall average, θi = fixed effect of lamb group (i
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= G1, G2), ζ j(i) = random error associated with j-th lamb nested in i-the group, γk = effect of the k-

the period, (θγ)ik interaction between group and period, ε ijkl = random error associated with

repeated measures of the animal ~ IIDN (0, σ 2 ). The means obtained from the parasitological

parameters per animal were compared using least square means (differences were considered

statistically significant if p  0.05.

3. Results

3.1 Phenotypic parameters

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The epg and pcv means and the standard errors (x  S.E.) from Pelibuey lambs infected with GI

nematodes are presented in Figure 1 and 2. The dominant GI nematode species identified in all

samples was H. contortus. Due to differences in parasite infection between infected lambs, two

groups were recognised as Group 1 (G-1, n = 8) and Group 2 (G-2, n = 7), based on thex  S.E.

of epg and pcv values. Following these criteria, G-1 showed 151 28 epg and 29  0.33% pcv,

and G-2 presented 475  59.5 epg and 26  0.38% pcv. Group 1 showed an increase > 500 epg

only at week 6 post-infection, whereas G-2 increased its epg rate at weeks 4, 6, 7, 8 and 12 post-

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infection (p  0.05). Differences in pcv levels were observed between the groups. The lowest pcv

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values presented by G-1 and G-2 were 25  1.0% and 20  1.0% (p  0.05), respectively. From

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weeks 8 to 12 post-infection, pcv levels were similar between G-1 and G-2. Increased pcv levels
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were observed for G-1 during the last weeks of H. contortus infection until they reached pcv
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levels similar to those of G-2.

3.2 Blood and abomasal samples and relative quantification

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The relative gene expressions and p-values for each group are shown in Table 2. In abomasal
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tissue from G-1, six genes evaluated in the present study were upregulated, and the highest gene
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expression levels were observed for TGF- , FC R1A and IL-5 genes (14.99, 13.66 and 9.95-fold,

respectively). For G-2, IL-6 and TGF- genes were upregulated (3.45, 2.05-fold) and IL-5 gene

was downregulated (0.18-fold). In peripheral blood from both groups, IL-8 gene showed the

maximum gene expression level, and upregulated expression levels were observed for FC R1A,

TGF- , IL-13 and IL-4 genes. Also, IL-5 gene was downregulated in both groups.

3.3 Correlations between phenotype and immune genes

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In group 1, a positive correlation coefficient was observed (r = 0.7, p < 0.05) between the pcv

value and IL-4, IL-8 and TGF-  immune genes in blood samples (Table 3). In addition, we

observed a strong positive correlation (r = 0.9; p  0.001) between IL-4 and FC R1A genes for

abomasal and blood samples and for IL-5 with IL-4, IL-13 and FC R1A genes in abomasal tissue

from G-1. However, no correlation between epg as the main phenotypic value of GI nematode

infection and immune gene expression from G1 was identified. The G-2 showed significant

differences, with negative r-values between epg and IL-4, IL-5 and FC R1A gene expression (r =

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–0.7, p = 0.05) in blood samples. Also, significant differences with negative r-values were

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observed between epg and FC R1A gene expression (r = –0.7, p = 0.05) in abomasal tissue. In

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addition, strong positive correlations were observed between IL-4 and IL-5, IL-13 and FC R1A
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genes and between IL-5 and IL-8, IL-13 (r = 0.9, p = 0.001) (Table 4).
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4. Discussion
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Parasitism caused by GI nematodes is the major constraint to efficient sheep production in

tropical and temperate regions. Current parasitic controls rely upon the frequent use of
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anthelmintics, inducing the selection of GI nematodes resistant to anthelmintic drugs. These

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problems affect the economy and deworming programmes to control parasites (Roeber et al.,

2013). However, the use of alternative controls against nematodes requires the study of resistant

phenotypic traits and the biological mechanisms of the immune response (Gonzalez et al., 2008;

Estrada-Reyes et al., 2015).

In the present study, selected Pelibuey sheep were classified into two groups, G-1 and G-2, based

on epg and pcv. For each group, the relative expression of immune genes related to the nematode

infection was analysed, showing important variations between groups. Results showed lower epg

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values for G-1, with increased levels of pcv, probably caused by reduction in H. contortus

infection. Contrary results were observed for G-2, in which animals presented higher levels of

parasitic infection and lower pcv values. Although the climate conditions (25°C annual

temperature and 80% humidity) throughout the experimental period were ideal for high nematode

prevalence (Gonzalez et al., 2008), no anthelmintic treatment was required. Several studies have

reported similar results when animals were observed over long grazing periods, and the

information generated from these studies has helped to reduce anthelmintic treatments in tropical

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and temperate regions (Gonzalez et al., 2008; Chiejina et al., 2010; Estrada-Reyes et al., 2015;

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Wilkie et al., 2017).

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Analysis of immune gene expression in G-1 suggests that resistance to GI nematodes was
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associated with IL-4, IL-5, IL-13, TGF- and FC R1A genes. These results are similar to
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previous immunological studies with H. contortus- and Tladorsagia circumcincta-infected sheep,

in which resistant phenotypic traits and increased cytokine activity were observed (Gossner et al.,
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2013; Wilkie et al., 2016; Estrada-Reyes et al., 2017, 2018). The inflammatory mechanisms

presented during GI nematode infections involve activation of anti-inflammatory cytokines such

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as TGF- and cytokines IL-4 and IL-13, which regulate IgE switching (Allen and Sutherland,
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2014). Previous studies performed with sheep infected with H. contortus suggested that

upregulation of the IL-4 gene was associated with an increase in the amount of IgE produced by

the host to attack the parasite (Kooyman et al., 1997; Gomez-Muñoz et al., 1999). A similar

immune response was observed in the present study, where H. contortus was the dominant

nematode species, and the immune response in G-1 was attributed to FC R1A, TGF- , IL-4, IL-5

and IL-13 genes. These cytokines were upregulated, and significantly correlated responses

between gene expression levels and parasitological parameters were observed. Promotion of IgE

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class switching has been related to IL-4/IL-13 due to helminth allergens to attract inflammatory

and regulatory cytokines i.e., TGF- (Wynn, 2003).

Correlation coefficient analysis from Pelibuey lambs naturally infected with H. contortus

contributed to a better understanding of the differences observed between groups, G-1 and G-2,

based on the relative expression level of immune genes and the parameters pcv or epg. The

strong positive correlation between pcv and immune genes of G-1 suggested immune regulation

of haemonchosis, since reduction of epg was observed throughout the experimental period, which

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was relatively long and without any anthelmintic treatment. In previous studies, Li et al. (2007)

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and Thomas et al. (2012) mentioned the importance of selected TH2 cells, i.e., IL-4 and IL-5, to

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regulate GI nematode infection in ruminants, and a similar cytokine response was observed for
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Pelibuey lambs of G-1. In contrast, infected lambs from G-2 displayed a negative correlation
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between epg and immune genes, mainly with those immune cytokine genes related to nematode

control. Lambs from G-2 showed difficulties to reduce the level of H. contortus infection,
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displaying a strong positive correlation between inflammatory genes, i.e., chemokine IL-8 and

TGF- , to attract other cytokine genes whose function is associated with nematode pathogenesis.
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Small ruminant nematode infections are a great concern for producers; thus, it is recommended to

include animals that may regulate infection in the flocks. Otherwise, acute and chronic infections

could continue affecting animal health and production. Under these circumstances, the

development of immune resistance is a potential tool to effectively regulate nematodosis, thereby

improving husbandry in regions with high prevalence of infection and anthelmintic resistance

issues.

5. Conclusions

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Natural infection of Pelibuey lambs with H. contortus involved different levels of inflammatory

and regulatory TH2 cells in lambs with different phenotypic traits, pcv and epg. Persistence of the

nematode infection was associated with susceptible lambs from G-2 due to active inflammatory

cells and the negative correlation between epg and immune genes related to nematode control.

Contrary observations were made in lambs from G-1, where the role of IL-4 and FC R1A

appeared to be important to regulate haemonchosis in lambs natural infected with GI nematodes.

Pelibuey sheep are used for their adaptation to tropical regions, but the animals should be

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protected against prevalent pathogens such as GI nematodes, improving control strategies. The

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selection of naturally resistant hosts requires time, but results in the conservation of synthetic

anthelmintic drugs.
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Competing interests
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The authors declare that they no competing interests.

Authors’ contributions
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López-Arellano ME conceived and designed the present study and drafted the manuscript.
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Reyes-Guerrero DE contributed to design and to performance molecular techniques

Maza-Lopez J and Pacheco-Armenta M contributed to carry out experimental molecular studies

corresponding to blood and abomasal tissue.

Olmedo-Juárez A and González-Garduño R carried out statistical analysis.

Olazarán-Jenkins S carried out the selection of resistance Pelibuey-breed through phenotype data.

Acknowledgments

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The authors would like to thank Dr Pedro Mendoza-de-Gives for his kind comments to this

manuscript. We also want to express our gratitude to Gabriel Ramírez-Vargas for his technical

assistance.

Financial support

This study was partially supported by Instituto Nacional de Investigaciones Forestales, Agrícolas

y Pecuarias (National Institute for Forestry, Agriculture and Livestock Research) Grant Number

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11555219663.

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Conflict of interest. None

Ethical standards
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The experimental sheep were strictly maintained and treated under the Norma Oficial Mexicana
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(Official Rule Number) NOM-051-Z00-1995 (www.senasica.gob.mx) as well the Ley Federal of

Sanidad Animal (Federal Law for Animal Health,

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www.diputados.gob.mx/LeyesBiblio/ref/Ifsa.htm). These guides specify that all procedures

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performed in studies involving animals must follow the Federal Law and Official Rule in

accordance with the ethical standards of INIFAP.

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References

Allen, J.E., Sutherland, T.E., 2014. Host protective roles of type 2 immunity: Parasite killing and

tissue repair, flip sides of the same coin. Sem. Immunol. 26, 320-340.

Besier, R.B., Kahn, L.P., Sargison, N.D., Van Wyk, J.A., 2016. The pathophysiology, ecology

and epidemiology of Haemonchus contortus infection in small ruminants. Adv. Parasitol. 93, 95-

143.

13
 Journal Pre-proof

Campos, R.R., Herrera, R.D., Quiroz, R.H., Olazarán, S., 1990. Resistencia de Haemonchus

contortus a bencimidazoles en ovinos de México. Téc. Pecu. Méx. 28, 30-34.

Chiejina, S.N., Behnke, J.M., Musongong, G.A., Ngongeh, L.A. 2010. Resistance and resilience

of West African Dwarf goats of the Nigerian savannah zone exposed to experimental escalating

primary and challenge infections with Haemonchus contortus. Vet. Parasitol. 171, 81-90.

Coles, G.C., Jackson, F., Pomroy, W.E., Prichard R.K., von Samson-Himmelstjerna, G.,

of
Silvestre, A., Taylor, M.A., Vercruysse, J., 2006. The detection of anthelmintic resistance in

ro
nematodes of veterinary importance. Vet. Parasitol. 136, 167–185.

-p
Estrada-Reyes, Z.M., López-Reyes, A.G., Lagunas-Martínez, A., Ramírez-Vargas, G., Olazarán-
re
Jenkins, S., Hernández-Romano, J., Mendoza-de-Gives, P., López-Arellano, M.E., 2015. Relative

expression analysis of IL-5 and IL-6 genes in the tropical sheep breed Pelibuey infected with
lP

Haemonchus contortus. Parasite Immunol. 37, 446-452.

na

Estrada-Reyes, Z., López-Arellano, M.E., Torres-Acosta, F., López-Reyes, A., Lagunas-Martínez,

A., Mendoza-de-Gives, P., González-Garduño, R., Olazarán-Jenkins, S., Reyes-Guerrero, D.,

ur

Ramírez-Vargas, G., 2017. Cytokine and antioxidant gene profiles from peripheral blood
Jo

mononuclear cells of Pelibuey lambs after Haemonchus contortus. Parasite Immunol. 39,

e12427.

Estrada-Reyes, Z., Tsukahara, Y., Goetsch, A.L., Gipson, T.A., Sahlu, T., Puchala, R., Wang, Z.,

Hart, S.P., Mateescu, R.G., 2018. Effect of Ovar-DRA and Ovar-DRB1 genotype in small

ruminants with hemoncosis. Parasite Immunol. 40, e12534.

Galina, M.A., Morales, R., Silva, E., López, B., 1996. Reproductive performance of Pelibuey and

Blackbelly sheep under tropical management systems in Mexico. Small Rumin Res. 22, 31-37.

14
 Journal Pre-proof

Gomez-Muñoz, M.T., Cuquerella, M., Gómez-Iglesias, L.A., Méndez, S., Fernández-Pérez, F.J.,

de la Fuente, C., Alunda, J.M., 1999. Serum antibody response of Castellana sheep to

Haemonchus contortus infection and challenge: relationship to abomasal worm burdens. Vet.

Parasitol. 81, 281-293.

Greer, A.W., Hamie J.C. Relative maturity and the development of immunity to gastrointestinal

nematodes in sheep: an overlooked paradigm? 2016. Parasite Immunol. 38, 263-272.

of
Gonzalez, J.L., López-Arellano, M.E., Olazaran-Jenkins, S., Liébano-Hernandez, E., de Gives,

ro
P.M., Vázquez-Prats, V., Vega-Murillo, V., Calderon, R., 2008. Phenotype characterization of

-p
Pelibuey native lambs resistant to Haemonchus contortus. Ann. N.Y. Acad. Sci. 1149, 177-179.

Gossner, A., Wilkie, H., Joshi, A., Hopkins, J., 2013. Exploring the abomasal lymph node
re
transcriptome for genes associated with resistance to the sheep nematode Teladorsargia
lP

circumcincta. Vet. Res. 44, 1-13.

na

Hendawy, S.H.M., 2018. Immunity to gastrointestinal nematodes in ruminants: effector cell

mechanisms and cytokines. J Parasit Dis. https://doi.org/10.1007/s12639-018-1023-x .

ur

Kooyman, F.N., Van Kooten, P.J., Huntley, J.F., MacKellar, A., Cornelissen, A.W., Schallig,
Jo

H.D., 1997. Production of a monoclonal antibody specific for ovine immunoglobulin E and its

application to monitor serum IgE responses to Haemonchus contortus infection. Parasitology.

114, 395-406.

Leathwick, D.M., Luo, D., 2017. Managing anthelmintic resistance-variability in the dose of drug

reaching the target worms influences selection for resistance? Vet. Parasitol. 243, 29-35.

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 Journal Pre-proof

Li, R.W., Sonstegard, T.S., Van Tassel, C.P., Gasbarre, L.C., 2007. Local inflammation as a

possible mechanism of resistance to gastrointestinal nematodes in Angus heifers. Vet. Parasitol.

145, 100-1007.

McCrae, K.M., Stear M.J., Good, B., Keane, O.M., 2015. The host immune response to

gastrointestinal nematode infection in sheep. Parasite Immunol. 37, 605-613.

Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR.

of
Nucleic Acids Res. 29, e45.

ro
Roeber, F., Jex, A.R., Gasser, R.B., 2013. Impact of gastrointestinal parasitic nematodes of

-p
sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance –

an Australian perspective. Parasite. Vector. 6, 1-13.

re
lP

Thomas, D.G., Rückerl, D., Maskrey, B.H., Whitfield, P.D., Blaxter, M.L., Allen, J.E., 2012. The

biology of nematode- and IL4-dependent murine macrophage polarization in vivo as defined by

na

RNA-Seq and targeted lipidomics. Blood 120, e93-e104.

ur

Torres-Acosta, J.F, Mendoza-de-Gives, P, Aguilar-Caballero, A.J, Cuéllar-Ordáz, J.A, 2012.

Jo

Anthelmintic resistance in sheep farms: update of the situation in the American continent. Vet.

Parasitol. 189, 89-96.

Wilkie, H., Gossner, A., Bishop, S., Hopkins, J., 2016. Variations in T cell transcription factor

sequence and expression associated with resistance to the sheep nematode Teladorsagia

circumcincta. PLoS One. 11, e0152396.

Wilkie, H., Riggio, V., Matika, O., Nicol, L., Watt, K.A. Sinclair, R., Sparks, A.M., Nussey,

D.H., Pemberton, J.M., Houston, R.D., Hopkins, J., 2017. A candidate gene approach to study

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 Journal Pre-proof

nematode resistance traits in naturally infected sheep. Vet. Parasitol. 243, 71-74.

Wynn, T.A., 2003. IL-13 effector functions. Annu. Rev. Immunol. 21, 425-456.

Zvinorova, P.I., Halimani, T.E., Muchadeyi, F.C., Matika, O., Riggio, V., Dzama, K., 2016.

Breeding for resistance to gastrointestinal nematodes – the potential in low-input/output small

ruminant production systems. Vet. Parasitol. 225, 19-28.

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Figure and table legends

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Figure 1. Faecal egg counts in Pelibuey lambs during natural H. contortus infection in a tropical

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grazing paddock. a, b, Letters in the same column are different significantly (Tukey, p < 0.05).
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Figure 2. Packed cell volume in Pelibuey lambs during natural H. contortus infection in a tropical
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grazing paddock. a, b, c, Letters in the same column are different significantly (Tukey, p < 0.05).
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Table 1. Gene information and annealing temperatures (Tm, ° C) for the primers used in the

custom PCR array design (CAPB11185R).

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Table 2. Relative expression of immune-related genes in Pelibuey lambs during natural H.

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contortus infection in a tropical grazing paddock.

Table 3. Correlation coefficients (r) between phenotype parameters and gene expression levels

for G-1 Pelibuey lambs naturally infected with H. contortus.

Table 4. Correlation coefficients (r) between phenotype parameters and gene expression levels

for G-2 Pelibuey lambs naturally infected with H. contortus.

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Table 1. Gene information and annealing temperatures (Tm, °C) for the primers used in the

custom PCR array design (CAPB11185R).

GenBank Access Tm
Genes
Code (°C)

IL-4 NM_173921.2 79.7

IL-5 NM_001009783.1 80.6

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IL-6 NM_001009392.1 81.6

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IL-8 NM_173925.2 80

IL-13

TGF-
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NM_174089.1 79.3
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NM_001166068 81.2

FCR1A
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NM_001100310.1 81.75

gapdh NM_001190390.1 84.4

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-actin NCB1-U39357 87.4

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Tm = melting curve temperature; IL = Interleukin; TGF=

Transforming Growth Factor, FC R1A= Immunoglobulin E receptor.
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Table 2. Relative expression of immune-related genes in Pelibuey lambs during natural H. contortus
infection in a tropical grazing paddock

Group 1 Group 2

Gene ( 28 pcv;  450 epg) ( 28 pcv;  450 epg)

Abomasum Blood Abomasum Blood

IL-4 8.71 2.42 1.79 4.17

IL-5 9.95 0.15*** 1.96 0.27**

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IL-6 5.29 0.60 3.45 0.91

IL-8 1.12 7.57* 0.18 12.4

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IL-13 7.7 2.52 1.22 2.72
pcv =FCpacked
R1A cell volume;13.66
epg = eggs per gram;  R1A = IgE receptor;
IL = Interleukin; FC1.66 TGF=

0.01; * = p ≤ 0.05.
4.97

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Transforming growth factor beta 1; red letter = upregulated expression level; black letter =
TGF-expression level;
unchanged blue letter = downregulated
14.99 4.28*** expression level;
6.01

2.05 *** = p ≤ 0.001;3.63

** = p ≤
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Table 3. Correlation coefficients (r) between phenotype parameters and gene expression levels

for G-1 Pelibuey lambs naturally infected with H. contortus

Blood samples Abomasal tissue samples

IL- IL- IL- FCR ep pc IL- FCR

epg pcv IL-4 IL-8 IL-4 IL-5 IL-8 IL-13
5 6 13 1A g v 6 1A

0.2 0.2
pcv
9 9

- -
0.78 0.3
IL-4 0.2 0.4
** 3

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2 7

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0.4 0.95*
IL-5 0.0 0.60 0.60 0.5
2 **
8 2

IL-6
-
0.2
6
0.46 0.44
0.7
1* -p -
0.1
7
-
0.2
7
-0.14 -0.04
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- -
0.0 0.70 0.76* 0.4 0.4 0.3
IL-8 0.4 0.72* 0.70* 0.1
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9 * * 0 9 6
8 2

- -
0.0 0.5 0.5 0.2 0.96* 0.91* 0.84*
IL-13 0.61 0.31 0.04 0.5 0.2
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3 4 1 7 ** ** **
2 6

- -
FCR 0.91* 0.3 0.4 0.1 0.3 0.93* 0.95* 0.0 0.84* 0.94*
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0.3 0.63 0.75* 0.6

1A ** 9 5 4 4 ** ** 4 ** **
3 5
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- -
TGF- 0.75 0.6 0.6 0.88* 0.3 0.79* 0.2 0.78* 0.88* 0.3 0.70* 0.85*
0.0 0.85 0.5 0.54
 * 7* 2 ** 2 * 5 * ** 6 * **
5 7

G-1 = group 1; epg = eggs per gram; pcv = packed cell volume; IL= Interleukin; *** = p ≤ 0.001; ** = p ≤
0.01; * = p ≤ 0.05

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Table 4. Correlation coefficients (r) between phenotype parameters and gene expression levels

for G-2 Pelibuey lambs naturally infected with H. contortus

Blood samples Abomasal tissue samples

IL- IL- IL- FCR pc IL- IL- FCR

epg pcv IL-4 IL-5 epg IL-4 IL-5 IL-8
6 8 13 1A v 6 13 1A

0.0
pcv 0.01
4

-
0.2 0.2 0.2
IL-4 0.77
0 1 2

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*

- -

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0.2 0.92* 0.2 0.90
IL-5 0.74 0.0
3 ** 5 **
* 5

IL-6
-
0.69
0.4
3
0.84*
*
0.64
-p 0.5
2
0.6
6
0.65 0.43
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-
- 0.1 0.2 0.3 0.88 0.93* 0.4
IL-8 0.27 0.47 0.0
0.57 9 5 3 ** ** 2
9
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- -
- 0.92* 0.7 0.1 0.3 0.77 0.94* 0.3 0.89
IL-13 0.0 0.82* 0.3
0.57 ** 0 5 1 * ** 0 **
1 5
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- - -
FCR 0.4 0.93* 0.94* 0.8 0.4 0.7 0.4 - 0.3
0.77 0.7 0.09 0.0 0.13
1A 3 ** ** 2* 6 5 0 0.07 4
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* 6* 3

- - - - - -
TGF- - 0.7 0.1 - -
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0.1 0.20 0.49 0.1 0.31 0.5 0.3 -0.44 0.3 0.2 0.31
 0.52 5* 0 0.49 0.57
3 3 2 2 9 2

G-2 = group 2; epg = eggs per gram; pcv = packed cell volume; IL= Interleukin; *** = p ≤ 0.001; ** = p ≤
0.01; * = p ≤ 0.05

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Highlights:

1. The immune response of infected Pelibuey against H. contortus was evaluated

2. Differences in parasitological parameters and gene expression levels were observed in

lambs

3. Immune genes of blood and tissue samples from infected lambs were upregulated

4. Lambs with low infection level had positive correlation between pcv and some cytokine

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gene expression

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5. Lambs with severe infection level had negative correlation between epg and immune

genes -p
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