Local Immune Response of Canarian Majorera Goats Primary Infected With Teladorsagia Circumcincta

Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GIN). Some of the most promising alternatives for their sustainability are based on the immune response of the host, such as the selection of genetically resistant breeds or the use of vaccines against these parasites. Given the limited information available on the immune response against GIN in goats, this study was carried out on the local immune response of goat kids of a Canary Islands indigenous breed (Majorera breed) experimentally infected with Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species. For this purpose, the relationship between different parasitological and immunological parameters at local level (both related to the humoral and cellular immune responses) have been analyzed at early (1 week post-infection –wpi-) and late (8 wpi) stages of infection. Primoinfection of goat kids with T. circumcincta L3 generates a complex immune response that could be dened as Th2 type, characterized by an increase of inltration in abomasal tissues of several effector cells as well as a progressive presence of specic antibodies against parasitic antigens in the gastric mucus. These cellular responses could be evidenced from 1 wpi onward, showing an increase in the gastric mucosa of antigen presenting cells and various lymphocyte subsets. The complexity of the responses developed is evidenced by the statistically signicant changes in numbers of all these subpopulations, as well as in the evolution of the relative cytokine gene expression. From a functional point of view, negative associations were observed between the number of most of these cells (CD4, IgA, IgG, and CD45R cells) and parameters that could be related to the fecundity of worms, a phenomenon that was especially evident when the number of IgG or CD45R cells or the specic IgA levels of the mucus were compared with parasitological parameters such as the lenght of female worms or faecal egg counts at 8wpi.


Introduction
Gastrointestinal nematodes parasites (GIN) are responsible for important economic losses in livestock farming worldwide, mainly in extensive production systems. Among other effects, they compromise animal welfare and can reduce productivity in terms of milk production and growth rates, carcass quality and reproductive disorders [1]. Even in subclinical infections, the presence of these nematodes results in an inadequate use of feeding resources and contributes to the development of other infectious diseases.
Among the various nematodes that affect the gastrointestinal tract of small ruminants, Teladorsagia circumcincta (T. circumcincta) outstands for its wide distribution and pathogenicity [2].
At present, control of these parasitic infections in ruminants relies heavily on treatments with anthelmintic drugs. However the emergence of parasite resistance to these products [3], as well as consumer concerns about the presence of chemical residues in meat and milk, have stimulated research towards new control strategies [4].
Through these alternatives, selection of resistant breeds [5], and improvement of the host immune system, by inducing protective responses [6], have been considered. Therefore, it would be of great interest to advance knowledge about the immunological mechanisms generated against ruminant GIN, information that would be very useful to optimize the development of vaccines against this group of parasites [7].
Though, there is a in increased interest in goat production as demonstrated by the continuous rise in this livestock population worldwide [8], most recent studies on the immune response to gastrointestinal nematodes have been conducted in cattle and sheep [9], and much less information is available for goats. In addition, information obtained in cattle or sheep can hardly be directly extrapolated to goats [10], due to GIN infections in goats are more severe than in other ruminants, there is a delay in the development of the immune response that makes it less effective [11,12] and nally, different goat breeds have shown genetic variability in resistance to GIN infections [13,14]. Therefore, it would be necessary to carry out speci c studies to elucidate the immune response generated in goats against this group of nematode parasites. Particular in goats, the GIN T. circumcincta constitutes one of the most relevant species due to: i) its high prevalence in many regions of the world [15,16]; ii) its pathogenic potential also in subclinical infections [17]; iii) and its particular tendency to develop resistance to anthelmintics associated with different factors such as the off-label use of these drugs in goats [18]. Therefore, the aim of this study is contribute to the knowledge of the local immunological responses in primary infections with T. circumcincta in goats, as a preliminary step in the recognition of the mechanisms involved in resistance against this nematode species.

Materials And Methods
T. circumcincta strain used in this study was originally isolated from a sheep naturally infected with this nematode and, was kindly provided by Dr. Uriarte from the Centre for Agricultural Research and Technology of Aragón (CITA, Zaragoza, Spain).
Fifteen healthy goat kids (Majorera milk aptitude breed), three months old, were used in this study. These animals were reared under nematode-free conditions, until the beginning of experiment, when all goats were 6 months old. experiment started at 6 months of age. Afterward, all animals were randomly allocated in one of the following groups: group 1 (1 week infection) (n = 5), group 2 (8 weeks infection) (n = 5), group 3 (uninfected control, n = 5). Animals from groups 1 and 2 were orally inoculated with 8,000 T. circumcincta L3, and slaughtered at 1 (group 1) or 8 (group 2) weeks after infection (wpi) respectively. Group 3 animals were kept as uninfected controls and were also euthanized at 8 wpi.
In group 2 and 3, faecal samples were collected three times per week, from day 0 pi (post-infection) to the end of the study (8 wpi), to determine fecal egg counts.
At the end of the study, abomasa of all the animals were removed to perform parasitological (inmature and adult worms counts), immunological (speci c antibody levels in gastric mucus and antigenic recognition of proteins from T. circumcincta adult worms by immunoblot) as well as histological and immunohistochemical analysis. In addition, samples were also collected from gastric mucosa and abomasal lymph nodes to analize the relative expression of cytokines in both tissues.

Parasitological Analysis
Fecal egg counts were determined by the modi ed McMaster technique [19] and expressed as number of eggs per gram of faeces (EPG). At the end of the experiment, the abomasa of all the animals were washed with distilled water, and 200 mL samples were collected and preserved with formalin to determine the number of immature or adult worms (males and females). Thirty adult female worms from each animal were measured with a calibrated ocular scale, and the number of intrauterine eggs per female was microscopically determined. Finally, immature worm burden was established by digestion of mucosal scrapings with pepsin-HCl at 37 ºC. Digestion was stopped with formalin, aliquots of the digestión mix were examined to calculate the number of immature worms per gram of mucosa [20].

Analysis of Humoral Responses in Mucus
The antigen used to analyse the humoral responses was obtained from adult worms collected from the abomasum of monospeci c infected donor animals. The worms were homogenized on ice with a homogenizer (Ultra-Turrax T8, IKA® WERKE) in a solution of PBS (0.01M) containing EDTA (1mM) and phenylmethylsulfonyl uoride (PMSF) (10mM) (Sigma-Aldrich, USA). The homogenate was centrifuged at 5000 x g for 20 min at 4 o C and the supernatant, containing the soluble somatic antigen, was ltered and its protein concentration determined using a colorimetric method (Pierce BCA Protein Assay, ThermoFisher Scienti c, USA).
Mucus samples used to determine the levels of speci c local immunoglobulins, were obtained by super cial scraping of the abomasal mucosa and diluted in a buffer containing proteinase inhibitors (0.1M sodium phosphate, 0.05M sodium chloride, 3mM sodium acid, 1mM PMSF and 5mM EDTA; pH 7.1) at a rate of 2.5 mL buffer/gram of mucus. Samples were centrifuged at 18 000 x g for 30 minutes at 4 o C and supernatant used for the determination of speci c antibody levels [21].

ELISA test
Optimal test conditions were established based on the results obtained from two pools of positive and negative mucus samples. The nal concentrations of somatic antigen used for the determination of speci c IgG or IgA levels in mucus was 3.0 µg/mL or 5.0 µg/mL respectively. Samples were diluted (1:100 -IgG-or 1:25 -IgA-) in PBS, and conjugate (anti-goat IgG-peroxidase, -Sigma-Aldrich Inc., USA-or anti-goat IgA-peroxidase, -Acris GmbH, Germany-was diluted in PBS and used at a 1:1000 or 1:5000 dilution respectivelly. A citric acid-phosphate buffer containing 0.04 % (w/v) OPD and 0.1% (v/v) H 2 O 2 was used as substrate.

Immunoblot
Somatic antigens of T. circumcincta adult worm were fractionated by SDS-PAGE into a 12% (w/v) acrylamide gel under non-reducing conditions. The protein fractions were then electrotransferred onto a 0.22-µM-pore nitrocellulose lter membrane (Pure Nitrocellulose Blotting Membrane, Bio Trace™ NT, Life Sciences, USA), which was subsequently blocked with a 3% (w/v) solution of bovine serum albumin in PBS. After washing nitrocellulose strips with PBS-Tween 20 at 0.05% (w/v), they were subjected to an immuno-enzymatic reaction as previously described [22], in which gastric mucus samples were analized (diluted 1:1 in PBS). A 1/1000 dilution in PBS of the same conjugates used in ELISA test was employed. The immunorecognition was evidenced by incubation in a solution containing the chromogen 3-amino-9ethylcarbazole (AEC Staining Kit, Sigma-Aldrich, USA), following the manufacturer's instructions.

Histology and Immunohistochemistry
Tissue samples from abomasal mucosa were cut (4 µm thick) and stained with Giemsa and hematoxylineosin stain in order to determine the number of eosinophils, globule leukocytes and mast cells. Cell counts were carried out at a 500x magni cation in 40 randomly selected elds of 0.044 mm 2 , at the upper and lower third of the mucosa. Eosinophils and globule leukocytes were counted in the hematoxylin-eosine stained sections and mast cells in the Giemsa sections. The results were expressed as numer of cells/mm 2 [23].
All samples were incubated for 90 min at 25 o C. Positive reactions were demostrated by incubation with biotinylated rabbit anti-mouse immunoglobulins (Agilent Technologies, USA) diluted 1:20 in RPMI medium (Sigma-Aldrich Inc., USA) and a solution of avidin-biotin peroxidase (ABC) complex at a 1:100 dilution in PBS. Finally, slides were reacted with 0.035% (w/v) 3-3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) containing 0.01 % (v/v) hydrogen peroxide. Counterstaining was performed using Harris' hematoxylin stain, and immunoreactive cells were counted in 40 elds located in upper and lower third of the mucosa [21].

Determination of Relative Cytokines Gene Expression by Real Time PCR (RT-PCR)
Abomasal lymph node (approximately 0.5 g) and abomasal mucosa samples (0.5x1x1 cm) were preserved in Trizol (TRI-Reagent, Sigma-Aldrich, USA) at -80°C prior to RNA isolation. Total RNA extraction was performed using a double phenolic extraction technique [24]. To eliminate possible contamination with genomic DNA, the samples were treated with DNase (RQ1 RNase-Free DNase Promega, USA). RNA samples were quanti ed using a spectrophotometer (NanoDrop-1000 Spectrophotometer, ThermoFisher Scienti c, USA) at 260 nm, and their purity was estimated by determining the OD ratio 260/280 nm, while RNA integrity was determined by means of the RNA6000 Pico Kit (Agilent Technologies, USA).
cDNA synthesis was performed on a total volume of 20 µL, using 1 µg of total RNA and a reverse transcriptase kit following the manufacturer's instructions (iScriptTM cDNA Synthesis Kit, BioRad Lab., USA). The resulting cDNA was diluted in 80 µl of DNA-free water (dilution 1:5) before ampli cation.
RT-PCR was performed using the GoTaq® qPCR Master Mix kit containing BRYT Green™ dye as uorophore (Promega, USA). The reaction mixture was prepared according to the manufacturer's instructions, however, for most assays a higher concentration of magnesium chloride (MgCl 2 ) was used ( The sequence of the primers used for the evaluated cytokines gene expression (IL-2, IL-4, IL-10, INF-γ and IL-17) and β-actine (housekeeping gene), the size and melting temperature (Tm) of the ampli ed products, as well as the concentration of MgCl 2 used in each reaction are detailed in Table 1. A relative quanti cation of gene expression was performed following the method ΔΔCt [25]. The data were normalized using β-actine gene as housekeeping gene.

Statistical analysis
Data were analyzed statistically using IBM SPSS Statistics software for Windows, version 22.0 (IBM Corp. USA). The differences between the experimental groups in single day parameters were determined using the nonparametric Mann-Whitney U test. The Spearman correlation test was used to analyse the the association between different parameters assessed in the study. Probabilities of p < 0.05 were considered as signi cant.

Parasitological Analysis
Infected animals slaughtered at 1 wpi (group 1) showed counts of 1,450 ± 228.1 larvae (mean ± SEM) in the gastric contents, with a mean length of 3.14 ± 0.22 mm. After digestion of the gastric mucosa, 77 ± 33.4 larvae/gram of mucosa were detected.
In group 2, slaughtered at 8 wpi, the mean value (± SEM) of adult worms was 2,039 ± 480.75, of which 63% were female worms which showed a mean length (± SEM) of 8.32 mm ± 0.63 mm, as well as a mean number of intrauterine eggs (± SEM) of 9.03 ± 2.1. Number of larvae in the mucosa was 6.6 ± 1.6 larvae/gram. Correlation analysis by Spearman's test (r) of all these data, showed a noticeable trend of negative association between the number of worms in the gastric content with their length (r = -0.7) and the number of intrauterine eggs (r = -0.5), although a positive association between length and number of intrauterine eggs showed a statistical signi cance (r = 0.9; p < 0.05).
The prepatent period of experimental infection in group 2 was 25 days post-infection (dpi). However, one of the animals showed fecal egg counts later on, in day 35 post-infection. The maximum mean value was reached at 47 dpi (620 epg) showing a mean FEC of 370 epg ± 80.5, at the end of the experiment (Fig. 1).

Analysis of Humoral Responses in Mucus
Speci c IgA anti-T. circumcinta At a local level, the presence of speci c IgA in the gastric mucus was much more evident in infected animals in which the worms reached the adult stage (group 2), when compared to group slaughtered at one wpi (group 1) and the uninfected control group (Fig. 2). Mucus samples from group 1 and 3, developed a similar and poor antigenic recognition against somatic proteins of adult worms by immunoblot. In contrast, in animals slaughtered at 8 wpi (group 2), IgA anti-T. circumcincta developed a strong reaction against antigenic fractions with molecular weights of approximately 22, 39, 50, 84 and > 120 kDa (Fig. 3). In this group 2, the levels of speci c antibodies of the IgA isotype present in the gastric mucus were negatively associated with the length of the female worms and the fecal counts of eggs at the end of the study (r = -0.9; p < 0.01).
Speci c IgG anti-T. circumcinta As described for IgA response to somatic antigens, the highest speci c IgG levels were detected in samples obtained from group 2 (8 wpi), while animals from group 1 and uninfected controls showed a poor speci c response (Fig. 2). These levels of mucus-speci c IgGs in group 2 were negatively associated with worm length and fecal egg counts at slaughter (r = -0.6), but were highly variable within the group, so no statistical signi cance could be proven. Despite relatively high levels if speci c IgG isotype recorded in group 2, recognition by immunoblot could only be clearly evidencied with protein fractions with a molecular weight of approximately 39 kDa.

Histological and Immunohistochemical Analysis
Infected groups 1 and 2 showed a mean number of eosinophils in ltrated in the gastric mucosa clearly higher than the uninfected control group, but those differences did not reach statistical signi cance. The counts of globule leukocytes and mast cells were also higher in the gastric mucosal from animals of the infected groups (p = 0.06) (Fig. 4). It was also remarkable the negative correlation observed between the number of globule leukocytes and the length of immature worms in the animals from group 1 (1 wpi) (r = -0.894; p < 0.05). However, no statistical association could be established between the number of eosinophils and mast cells and the parasitological parameters studied.
The immunohistochemical analysis showed that the monoclonal antibodies used displayed immunereactivity with different cell subpopulations of the abomasal mucosa of goat kids. In both infected groups (groups 1 and 2), all the subpopulations analized were increased with respect to the control group, with statistical signi cance in the number of WC1, CD4 + , MHCII + and IgG + cells in group 1, and γδ + , WC1 + and IgG + cells in group 2 (Fig. 5).

The analysis of the correlations between these subpopulations and different parasitological parameters
showed that there was a relation between the presence of high number of worms in the abomasums and the in ltration of various cellular subsets in the gastric mucosa. Thus, signi cant positive associations could be observed (p < 0.05) between the number of IgA + cells and the number of immature worms at 1 wpi, as well as between CD4 + , γδ + and IgG + cells and female worms at 8 wpi. In addition lymphocyte populations CD4 + , γδ + , IgG + , IgA + and CD45R + were negatively associated with the length of the worms (both at 1 or 8 weeks p. i.) as well as the fecal egg counts (at 8 weeks p. i.), although these correlations only showed statistical signi cance in the group 2 when the relationship between FEC and IgG + (r =-0.9; p < 0.05) or CDR45R + (r= -0.9; p < 0.01) lymphocytes were determined.

Relative Cytokines Gene Expression
All experimental groups showed detectable levels of gene transcription of IL-2, IL-4, IL-10, IL-17 and INFγ, in both lymph nodes and gastric mucosa, and although some trend in the relative gene expression between infected and control groups was observed, these differences did not reach statistical signi cance. In particular, it was remarkable the up-regulation of IL-4-and IL-17-gene transcription (4-fold and 6-fold respectively, in comparison with controls) which was detected in gastric lymph nodes one wpi (group 1), whereas this encreased gene expression was not detected once the adult worms had developed (group 2). In this latter group, only a slight increase in INF-γ-gene transcription (2-fold in relation with control group) was detected in the gastric mucosa.

Discussion
Despite the homogeneity of the animals included in the different experimental groups in terms of age, sex, breed, origin or feeding, the different parasitological parameters analyzed in the present study showed a high variability, as it has been observed in other experimental infections with T. circumcincta in canarian goats [22,26,27]. Variability within a group was re ected by high SEM values in some of the parameters assessed, such as larvae and adult worm counts at the end of the experiment, indicating that immunoprotection against the parasite may differ among individuals, as it has been reported in some ovine breeds infected with T. circumcincta [28][29][30]. This nding has also been described in other goat breeds infected by different GIN species [31], which would indicate that important individual factors are involved on the natural defense mechanisms developed against GIN in goats.
Similarly, the prepatency period in our study was approximately 25 dpi, which seems to indicate a slight delay in larval development compared to sheep, which has also been observed in other trials in goats using other isolates of T. circumcincta [10], a phenomenon which could be linked to the use of sheepadapted strains in both cases. The prepatent period reached 35 days in one of the experimentally infected animals, furthering the idea of the presence in this animal population of a different degree of natural resistance that could be manifested by a delay in the endogenous cycle of the parasite.
Among the mechanism associated with resistance to T. circumcincta infections, at least in sheep, is the reduction in FEC, mainly linked to decreased female fecundity rather than number of parasites [32]. In our study, the fecundity was assessed by the intrauterine egg counts of the female worms, which showed a logical negative correlation with the length of these worms, a feature found in other studies for this parasitic species [29], and the faecal egg counts observed at the end of the experimet. All of those parasitological parameters were negatively associated with the speci c humoral immune response of the two analyzed isotypes (IgG and IgA), pointing to the possible role of the humoral response in the fecundity regulation of T. circumcicnta in goats. Negative correlations reaching statistical signi cance when analyzing the association between speci c IgA levels and length of females.
This predominant role of IgA in resistance to infection against T. circumcicnta has been also observed in sheep infected with this parasite [33][34][35][36]. In sheep have also shown that some of those protective responses are associated with an increased antigenic recognition of parasite proteins by IgA immuoglogulins [37]. The prominent IgA level in the gastric mucus in the current trial could be related with the higher number of antigenic fractions recognized, in relation with IgG, shown by means immunoblot analyses. Accordingly, no association between IgG in mucus and parameters related to resistance to parasite development could be evidenced. The scarce local humoral response detected at the earliest stages of the infection, which does not seem to play a relevant protective role, supports the lack of previous sensitization of the animals.
The humoral response observed in the gastric mucus would match with a Th2 immune response, which is considered a common feature in GIN infections. In group 2, during the 8 weeks of the study, a development of this type of response characterised by an increase of antibodies of different isotypes (including IgA, IgG) at local level (as can be seen when comparing these parameters between infected animals and non-infected controls). This type of immune response is usually accompanied by increased mucosal eosinophil in ltration, as well as mast cell and globule leukocyte hyperplasia [38], also observed in this experimental group 2, where the increase in tissue eosinophil counts was close to statistical signi cance, although no relationship could be established of this tisular eosinophilia as a defensive mechanism against the parasite, in contrast to reports in T. circumcincta infected sheep [39].
The increase of effector cells, such as eosinophils and mast cells in the gastric mucosa, has been related as mechanisms of resistance against GIN [40]. Both cell populations increased in our study, especially at 8 weeks p.i. The increase in the number of these effector cells in the gastric mucosa one wpi was also observed and could be associated with innate defensive mechanisms [41]. Mast cells and eosinophils were also rapidly increased in the abomasal mucosa from goats primoinfected with H. controtus [42,43]. However, these authors could not determined an increase in the number of globule leukocytes in the early stages as it was observed in our study. This increase shown an interesting negative association with the length of the immature worms, whose potential role as a natural resistance mechanism in this goat breed should be evaluated [44]. Eosinophil in ltration, although enhaced throughout the study -a phenomenon associated with a certain degree of resistance to H. contortus infection in some sheep breeds [23]-, could not be associated with any effect on the parasitological parameters investigated.
The present results also showed a rapid recruitment of lymphocyte subpopulations in the abomasal mucosa of goats primarily infected with T. circumcincta, similar to those found in goats infected with other gastric nematodes, such as H. contortus [45]. This increase, already observed in animals one week after experimental infection, was associated with the number of worms and with cell types involved in Th2 responses against GIN in sheep, such as antigen presenting cells (MCHII + ) and CD4 + lymphocytes [46,47]. This celular ltration, from a functional point of view, highlighs a negative association between several populations such as CD4 + , γδ + , IgA + , IgG + and CD45R + , and fecundity re ected by a reduction in female worm length and FEC, as observed when the local humoral immune response was analyzed. The presence of a signi cant CD8 + lymphocyte in ltrate observed here was also previously detected in goats and sheep infected with H. contortus from 7-10 days post-infection, but similary to what occurs in our study, no relationship could be established with the parasitological data [23,45].
The in ltration of γδ + lymphocytes and their WC1 + suppopulation in the gastric mucosa from the rst week of infection was also evident here. These cells constitute an important percentage of lymphocytes in goats, especially in young animals [48], and are considered to have important defensive functions, playing a relevant role in the interrelation between innate and adaptive responses against bacteria, viruses, and protozoan parasites, but there is not much information available on its functionality against nematode parasites in ruminants [49]. As it has been observed in goats primarily infected by H. contortus [45], this cell population also displayed an increase in the gastric mucosa from the rst wpi., showing a negative association with the length of immature worms (group 1) and female worms (group 2). This nding has also been observed in canary sheep resistant to H. contortus, a phenomenon associated, as in current trial, to the local humoral response developed by speci c IgA antibodies [23] as it was previously discussed. However, this observation contrasts with results derived from sheep infected with T. colubriformis, where no signi cant defense mechanisms attributed to γδ + cells could be identi ed [50], which could indicate differential biological functions of this cell type on the basis of parasite species, tisular location and/or host particularities.
Although cytokine expression in lymph nodes and gastric mucosa in infected and control animals was not conclusive, a rapid onset in IL-4 expression was observed -as previously described in sheep [51]-, a cytokine that could stimulate B-cell proliferation and in ltration of CD4, MCH-II and IgG cells in the gastric mucosa, as observed in our study. Regarding the observed increase in IL-17 expression, although it is a cytokine for which not much information is yet available on its role against ruminant GIN, it appears to have an important regulatory function in the defensive responses necessary to maintain immune tolerance [52].
When the experimental infection progressed, although a humoral response could be detected and some effector cell subpopulations were incresed, the gene expression of mediators of the immune response was even less evident than during the rst week of infection. Thus, only a slight increase in the relative expression of INF-γ in the gastric mucosa of group 2 compared to control group was observed. This nding, which has also been resported in the course of H. contortus or O. ostertagi infection in goats and calves, respectively, could be contradictory if we take into account the inhibitory effect of INF-γ on Th2 responses [53]. Such observation has been explained as the beginning of the shift towards an antagonistic Th1 response, which could occur in susceptible animals to GIN after a period of development of Th2 responses, as a result of a mechanism of evasion of the immune response [54][55][56], or even the expression of a mixed Th1/Th2 response that could be effective against GINs [57] In conclusion, the primoinfection of goat kids with T. circumcincta L3 generates a complex immune response that could be de ned as Th2 type, characterized by an increase of in ltration in abomasal tissues of several effector cells (eosinophils, mast cells and globule leukocytes) as well as a progressive presence of speci c antibodies against parasitic antigens in the gastric mucus. These celular responses could be evidenced from 1 wpi onward, showing an increase in the gastric mucosa of antigen presenting cells (MCHII+) and various lymphocyte subsets (CD4, CD8, γδ, CD45R, IgA, and IgG). The complexity of the responses developed is evidenced by the statistically signi cant changes in numbers of all these subpopulations, as well as in the evolution of the relative cytokine gene expression observed in the abomasum and abomasal lymph nodes at 1 week and 8 weeks p.i.
From a functional point of view, negative associations were observed between the number of most of these cells (CD4, IgA, IgG, and CD45R cells) and parameters that could be related to the fecundity (such as the length of female worms or FEC at the end of the experiment), a phenomenon that was especially evident when the number of IgG or CD45R cells or the speci c IgA levels of the mucus were compared with parasitological parameters such as the lenght of female worms or FEC at 8 wpi.
Although more studies (including a higher number of experimental animals and serial infections) would be necessary, all this information should be taken into account to evaluate alternative control strategies against T. circumcincta based on the host immune response of the host, such as selection programs for resistant goat breeds or vaccination protocols against this nematode species. Figure 1 Eggs per gram of faeces (EPG) expressed as mean EPG ± SEM in goats orally infected with 8,000 T. circumcincta L3 (group 2).

Figure 2
Total IgG and IgA anti-T. circumcincta in mucus from groups 1, 2 (orally infected with 8,000 T. circumcincta L3 and slaughtered at 1 or 8 weeks p.i., respectively) and uninfected control group. Results are mean optical density (OD) at 492 nm ± SEM. Levels of effector cells (eosinophils, mast cells and globule leucocyte counts) in the gastric mucosa in goats from groups 1, 2 (orally infected with 8,000 T. circumcincta L3 and slaughtered at 1 or 8 weeks p.i., respectively) and uninfected control group. Results are mean number cells/mm2 ± SEM.