Physiological, immunological and genetic factors in the resistance and susceptibility to gastrointestinal nematodes of sheep in the peripartum period: A review

The peripartum relaxation of immunity happens when the circulating eosinophils and plasma antibodies decrease and remain low at end of pregnancy and during lactation in ewes. At local level, lower titers of antibodies (IgG1, IgM, IgA and IgE) as well as few cell counts (globule leucocytes, mast and goblet cells) in intestinal tissue (Beasley et al., 2010).

There is a transitory increase in the excretion of eggs in the faeces, especially during the last third of pregnancy and the first weeks of lactation (Hamer et al., 2019). This phenomenon has been widely studied in small ruminants, particularly in sheep, and is known as postpartum rise, lactation rise or peripartum rise (PPR) (Houdijk, 2008; Torres-Acosta & Hoste, 2008; Kidane et al., 2010; Goldberg et al., 2012a; Fthenakis et al., 2015). In ewes, the number of eggs per gram (EPG) excreted in faeces increases from four weeks before lambing, reaching a peak between the fourth and sixth week postpartum (Courtney et al., 1985; Goldberg et al., 2012a), subsequently decreasing towards the time of weaning (Vázquez-Hernández et al., 2006). The rise in the egg excretion rate in faeces is associated with the increase in the fertility of the parasitic females as a result of a group of immunological factors being depressed in the peripartum period. This event may be accompanied by clinical signs due to the effect of gastrointestinal parasitism (Ng´ang´a et al., 2004; Werne et al., 2013). Increased susceptibility to other infections may also occur during this period (Ng´ang´a et al., 2004; Karrow et al., 2014), especially in ewes with high susceptibility to GIN due to high variability within breed (Gonçalves et al., 2018).

In PPR, temporary alterations are characterised by physiological and metabolic changes associated to pregnancy and lactation (Ahmed et al., 2020). These changes also occur in cells and proteins of the immune system, that result in a low response of sheep against any infection, particularly those caused by GIN ( Gibbs & Barger, 1986; Barger, 1993; Kahn et al., 1999; Goldberg et al., 2012b; Jonsson et al., 2013; Pereira et al., 2020). The modulation of immune system is associated with disturbances in the endocrine system (Tembely et al., 1998; Beasley et al., 2010; Jonsson et al., 2013). Physiologically, it has been indicated that oestrogens stimulate the cellular and humoral immune responses by inducing direct effects on multiple cell types including immune and vascular cells (Trenti et al., 2018). In addition, in the peripartum period lactogenic hormones abound in the circulation and are antagonistic to oestrogens (Barger, 1993). Nutritional aspects (Jones et al., 2012), genetic influences (Kidane et al., 2010) and stress triggers also play important roles (Tembely et al., 1998). During the immediate prepartum period serum estrogens and estrone increase markedly. At partum withdrawal of progesterone the estrogens removes the block to lactation, and milk secretion results. Estrogen steroid decreased rapidly to low levels immediately postpartum and the release of growth hormone at parturition is required for normal lactation in ruminants (Convey, 1974) together with high levels of prolactin (Phillipps et al., 2020), as observed in Figure 1.

Fig. 1

Prolactin is responsible for initiating and maintaining lactation, with a stimulating effect by the suckling lambs (Fthenakis et al., 2015). It has a suppressive effect on the host’s immune system, by reducing the IgA levels necessary to prevent parasitic establishment at the intestinal level (Torres-Acosta & Rodríguez-Vivas, 1995; Kahn et al., 1999; Houdijk, 2008), favouring growth of the nematodes and the fertility of the female worms. The increased levels of prolactin at lambing coincides with low number of circulating eosinophils and by decreased total antibody and IgG1 titers (Fthenakis et al., 2015). Progesterone, the principal steroidal regulator of pregnancy, also has several immunosuppressive activities. However, in ovariectomized sheep, prolactin alone had a greater effect on the reduction of FEC (Fleming & Conrad, 1989). Although prolactin together with progesterone reduces the number of cells such as eosinophils, but in pregnancy the FEC are not high, suggesting that other mechanism and hormones are involved (Torres-Acosta & Rodríguez-Vivas, 1995; Kahn et al., 1999; Houdijk, 2008; Beasley et al., 2010; Fthenakis et al., 2015). The results show that Haemonchus contortus larvae have possible receptors to progesterone and respond to progesterone by inhibiting their moulting and, in consequence, their development. These results suggest that progesterone participates in larval arrest (Gutiérrez-Amézquita et al., 2017). In pregnant Angora goats, a high EPG value is associated with a high concentration of prolactin, compared to non-pregnant goats, which maintain low levels of prolactin and EPG, with a correlation of 0.83 between these characteristics (Rahman & Collins, 1992). In dairy goats, the level of resistance/resilience is negatively correlated with the amount of milk produced (Hoste & Chartier, 1993; Chartier et al., 1998).

During pregnancy, the placenta is the main provider of leptin. The levels increase at this stage and decrease gradually as the lambing date and lactation approaches (Ingvartsen & Boisclair, 2001; Mcfadin et al., 2002; Beasley et al., 2010). Ewes with high milk production and properly supplemented have a large amount of peri-renal fat, which in turn is related to a higher concentration of leptin in the blood and allows them to ensure fat tissue reserves and maintain good body condition (Rocha et al., 2011). On the other hand, leptin is an important regulator of the metabolic mechanisms of animals, and it has an important function in the immune system and in the activation of other hormones (Valderrábano et al., 2006). The function of leptin is linked to haematopoiesis and the induction of inflammation with activation of T lymphocytes, the production of Th1 cytokines and suppressing the production of Th2 cytokines (Ingvartsen & Boisclair, 2001). Beasley et al. (2010) found in infected Merino sheep approaching lambing that their leptin levels decreased and EPG increased. Ewes not supplemented or with reduced intake during pregnancy tend to lose body condition and energy reserves and presenting reduced leptin levels, which are related to loss of immune capacity against infections (Rocha et al., 2011).

Cortisol is a glucocorticoid that is released with any stressful event (Caroprese et al., 2010), as occurs just before lambing. Circulating cortisol stimulates the production of anti-inflammatory cytokines and inhibits the production of pro-inflammatory cytokines such as interferon gamma (IFN-γ), tumour necrosis factor (TNF-α) and interleukin-12 (IL-12); in addition, it inhibits the proliferation of T cells, modifies the action of Complement cells and alters the function of immunoglobulins (Aleri et al., 2016). All this leads to immunosuppression, increasing susceptibility to disease. At peripartum in ewes, the neutrophilia is commonly due to high cortisol levels at this time, which contributes to downregulation of surface adhesion molecules expression, in addition to an enhanced release of cells from the bone marrow (Ahmed et al., 2020). High cortisol levels in dairy cattle have been correlated with calcium and phosphorus deficiencies, causing hypocalcaemia problems in cows during the peripartum and provoking sensitivity to other infections (Kim et al., 2012).

Pepsinogen is a precursor to pepsin secreted by epithelial cells in the abomasum, the concentration of which generally increases at the time of expression of the immune response (Kidane et al., 2010), which coincides with the PPR from 4 weeks before lambing (Houdijk et al., 2000). An increase in pepsinogen concentrations is related to parasitism by Ostertagia spp. in abomasum (Simpson, 2000). As the parasite burden increases in the peripartum, the concentrations of pepsinogen and gastrin increase, the abomasum pH is altered, the abomasum permeability is increased and acid secretion is reduced (Houdijk et al., 2000; Davies et al., 2005; Angulo-Cubillán et al., 2007; Kidane et al., 2009), this being able to present diarrhoea (Miller & Horohov, 2006). In this sense, pepsinogen is considered as a marker of the integrity of the abomasum mucosa and a pathological indicator (Dominik, 2005; Kidane et al., 2009; Cei et al., 2016).

Protein levels in the diet are responsible for the concentrations of blood components during an infectious process (Louvandini et al., 2006). From two weeks before lambing and during lactation, ewes supplemented with high-protein diets maintain high concentrations of albumin and urea (Houdijk et al., 2000; Kidane et al., 2010), which coincides with a reduced excretion of eggs in the faeces; conversely, decreases in the level of albumin and urea favour an increase in EPG (Zárate Frutos et al., 2014). A low albumin concentration is also considered a pathological indicator of the presence of an infection (Dominik, 2005).

Globulins are altered during de PPR in sheep infected with GIN. Regardless of the protein content in the diet, globulin levels increase from four weeks before lambing (Houdijk et al., 2000; Zárate Frutos et al., 2014). The increase at final pregnancy stage is related to the presence of infectious or parasitic diseases (Zárate Frutos et al., 2014). Immunoglobulins constitute a natural defence mechanism in sheep, associated with inflammatory processes and the formation of antibodies. At the same time, increases in serum globulin levels are related to the quality of colostrum (Obidike et al., 2009).

Once the host is infected by the nematode larvae, the epithelial cells are stimulated to generate an immune response, together with complement fixation and mucus secretions constitute the innate response to resist the primary infection (Hendawy, 2018), characterised by the action of some cytokines (Klion & Nutman, 2004). In addition, the small proteins and various cell types are stationary, such as interferons (IFNs) and virus-infected cells, or mobile, such as circulating leukocytes, monocytes, dendritic cells and lymphocytes. Leukocyte-derived cytokines are known as interleukins (ILs), and those originating from monocyte-macrophages are called monokines, both of which are produced as a protection mechanism for the host animal (Finkelman et al., 1997; Lippi et al., 2013; Karrow et al., 2014). Despite the fact that the immunological mechanisms are not very clear, the innate and acquired response capacity are those that have the greatest influence in the grade of infection (Muñoz-Guzmán et al., 2006). The acquired response dependent on previous exposure of the host to a foreign agent (González-Garduño et al., 2019) and characterised by specificity and antigen memory is the more important (Karrow et al., 2014). The immune response focuses on humoral and cellular responses (Gauly et al., 2002; Sayers & Sweeney, 2005), as well as in inflammatory reactions (Angulo-Cubillán et al., 2007). These immunological bases are considered important because parasites, mainly those hosted in the intestinal lumen, are capable of producing immuno-modulatory substances that escape the host’s immune response (Moreau & Chauvin, 2010).

The innate response is the first line of defence, capable of recognising molecular patterns associated with the pathogen in a shorter time, and occurs mainly through the Complement system (Fujita et al., 2004). A group of plasma proteins interact with bound antibodies and surface receptors that promote the elimination of pathogens (Castellano et al., 2004). By activating the classical and alternate pathways, high amounts of the enzyme C3 convertase are generated, depositing a large number of C3b molecules in the pathogen, attaching some molecules (opsonins) to its surface (Castellano et al., 2004; Fujita et al., 2004; Muñoz-Guzmán et al., 2006); through this opsonisation processes the elimination of pathogens occurs. In addition to this, the generated C3a and C5a peptides facilitate the mobilisation of eosinophils and neutrophils, favouring the inflammatory reaction; Complement activation regulates the cytotoxicity of eosinophils against larvae in the early stages of infection (Muñoz-Guzmán et al., 2006).

In the peripartum period, the immunological differences between lactating and dry ewes are notable, so that the immunological relaxation in the peripartum include the low cellular and humoral response affected by hormonal and nutritional aspects modified by season changes (Beasley et al., 2012). The elements involved in the immune resistance or susceptibility of sheep consider the following concepts. T lymphocytes are responsible for directing the effector mechanisms once they are stimulated by antigens. When activated, CD4+ T cells differentiate into two groups: helper T cells or lymphocytes, Th1 and Th2 (Muñoz-Guzmán et al., 2006; Sykes, 2010). Th1 cells are responsible for increasing the expression of interleukins IL-2, IL-3, IL-13, IL-25, IFN-γ and TNF-α (Finkelman et al., 1997; Miller & Horohov, 2006; Hayward, 2013), as well as an increase in mRNA expression for IL-6 as an indicator of gene expression, while Th2 cells include IL-4, IL-5, IL-9, IL-10 and TGF-β, with effector mechanisms in the cellular immune response (Finkelman et al., 1997; Maza-Lopez et al., 2020). The expression of these components induces a local inflammatory reaction in which different types of cells, such as basophils, eosinophils, neutrophils and lymphocytes are involved, together with specific antibodies, the gastrointestinal mucosa and inflammatory mediators (Meeusen et al., 2005; Ingham et al., 2008; Karrow et al., 2014); in addition, dendritic cells (DCs) and natural killer cells (NK) appear (Angulo-Cubillán et al., 2007). These immunological mechanisms facilitate a reduction in the number, size and fertility of worms (Rowe et al., 2008). Intracellular parasites generally involve the Th1-type response, whereas GIN such as H. contortus involve the Th2-type response (Miller & Horohov, 2006; Muñoz-Guzmán et al., 2006; Moreau & Chauvin, 2010), although there are situations in which Th1 and Th2 responses are involved for certain parasites (Murphy et al., 2013). Results found by Bricarello et al. (2008) in Nelore cattle indicated that the immune response to Cooperia punctata was probably mediated by Th2 cytokines (IL-4 and IL-13) in resistant animals, and by Th1 cytokines (IL-2, IL-12p35, IFN-γ and MCP-1) in the susceptible group.

As cellular components of immunity, eosinophils with a cytotoxic effect are one indicator of a parasitic infection. These cells have importance in the immune protection of infected animals (Hohenhaus et al., 1998; Davies et al., 2005). In the presence of GIN infections, effector immune responses characterised by the production of IgE and peripheral and tissue eosinophils associated with the production and activation of interleukins IL-4 and IL-5 are induced (Finkelman et al., 1997; Klion & Nutman, 2004). Once in circulation, eosinophils release the content of their toxic granules or metabolites onto the nematode cuticle, increasing cellular cytotoxicity, with release of proteins and mediators of inflammation (Klion & Nutman, 2004), favouring a lesion of the cuticle and the adherence of more eosinophils. In this way, regulation of the growth of GIN occurs together with the expulsion of eggs in the faeces of the host. Ewes in peripartum show a reduction in eosinophil count (Beasley et al., 2010; Pereira et al., 2020).

With GIN infection, the pro-inflammatory cytokines IL-4, IL-5, IL-9, IL-10 and IL-13 are involved in different mechanisms of the humoral response. One of these is through the IgA, IgG, IgM and IgE antibodies, which, being antigen receptors, prevent the invasion of pathogens through endocytosis of the antigen (O’Sullivan & Donald, 1973; Beasley et al., 2010); another is due to delayed maturation, reduced fertility and induction of parasite death. They are also involved in intestinal contractility, allowing the expulsion of worms (Miller & Horohov, 2006; Sayers et al., 2007; Houdijk, 2008; Hayward, 2013; Murphy et al., 2013; Wilkie et al., 2015). The surrounding IgA levels in the mucosa have been associated with reductions in the fertility and length of H. contortus, as well as a reduction in the parasite burden at the intestinal level (Amarante et al., 2005; Davies et al., 2005; Karrow et al., 2014; Hernández et al., 2016). Some studies have associated plasma IgA with FEC as an important immune response (Bowdridge et al., 2015; González-Garduño et al., 2018). High IgE levels are also involved in the expulsion of worms and in the regulation and activation of mast cells, eosinophils and basophils (Alba-Hurtado & Muñoz-Guzmán, 2013; Murphy et al., 2013; Karrow et al., 2014). The expulsion of worms due to the effect of IgE occurs through the release of vasomotor amines: compounds that stimulate the contraction of smooth muscle and increase vascular permeability, allowing fluid to escape into the intestinal lumen, resulting in displacement and expulsion of most of the nematodes implanted in the intestinal mucosa of the animal. IgG concentrations are associated with a reduction in excreted eggs in faeces (Murphy et al., 2013).

Resistance against GIN is polygenic in nature (Sayers & Sweeney, 2005; Zvinorova et al., 2016) and is quantitative; that is, it is influenced by a large set of genes or loci with small effects (Hayward, 2013; Karrow et al., 2014). The identification of genes related to resistance to GIN involves procedures based on molecular genetics, through the use of molecular markers, and the application of different strategies such as mapping of quantitative traits locus (QTL) regions by linkage disequilibrium, identification of candidate genes, the use of maps of high-density single nucleotide polymorphism (SNP) and complete genome (genome-wide) association studies (Keane et al., 2006; Bishop & Morris, 2007; Wilkie et al., 2015; Zvinorova et al., 2016), as well as microarray analysis, are very useful in determining post-infection gene expression. All these procedures offer advantages and opportunities to investigate resistance genetics and parasite–host interactions (Hayward, 2013).

The ability to express resistance between and within breeds is genetically regulated (Stear & Murray, 1994), which is why selection can be made either directly through identifying the genes or alleles involved by means of molecular genetic techniques or indirectly through phenotypic indicators such as worm counts, FEC (Beh & Maddox, 1996; Davies et al., 2005; Good et al., 2006), packed cell volume (PCV), antibody levels, specifically IgA and IgE (Karrow et al., 2014), eosinophil count, pepsinogen concentration (Beh & Maddox, 1996) or other variables related to the immune response. One of the main variables by which the parasite burden is determined has been the EPG count, and this is one of the main phenotypic selection criteria. It must be evaluated by measurements over time, establishing the dynamics of the curve in the peripartum period and the correlation with other productive characteristics (Goldberg et al., 2012b).

Genetic selection is dependent on the heritability (h²), which in the case of FEC is considered moderately inheritable and highly repeatable (Bishop & Morris, 2007; Saddiqi et al., 2010), similar to other productive characteristics. Values from 0.15 have been observed (Vanimisetti et al., 2004), but other authors indicate values of 0.25 to 0.30 (Sréter et al., 1994; Kahn et al., 1999) and even up to 0.63 (Miller & Horohov, 2006; Alba-Hurtado & Muñoz-Guzmán, 2013). Selective breeding of the maternal line for nematode resistance has potential epidemiological benefits by reducing pasture infectivity (Vineer et al., 2019).

The genetic basis of resistance is closely related to the immunological component, most of the loci involved with immunological processes are located in the Main Histocompatibility Complex (MHC), a highly polymorphic region that consists of a group of closely linked genes involved in the presentation of antigens in the host’s immune system. Association of the FEC with the MHC I and MHC II regions adjacent to chromosome 20 of sheep has been found (Stear & Murray, 1994; Karrow et al., 2014) with the amount of FEC (Keane et al., 2006; Karrow et al., 2014; Zvinorova et al., 2016). The DRB1 locus of MHC II has been associated with resistance to GIN, particularly with increases in IgA and IgE levels (Dominik, 2005; Hassan et al., 2011; Hayward, 2013; Karrow et al., 2014). Also, with IFN-γ gene located on chromosome 3, a strong association between one allele of this gene has been detected with the reduction in FEC and an increase in the levels of specific antibodies (IgA) against Ostertagia circumcincta in lambs (Karrow et al., 2014). However, some authors have cited that expression of the IFN-γ gene does not directly influence resistance to GIN (Dervishi et al., 2011; Alba-Hurtado & Muñoz-Guzmán, 2013; Karrow et al., 2014). Likewise, with the IgE gene (Díaz et al., 2005; Pettit et al., 2005; Keane et al., 2006; Sayers et al., 2007; Karrow et al., 2014) a strong Th2 cell response has been detected during infections, with overexpression of IL-13, IL-5 and TNF-α (Alba-Hurtado & Muñoz-Guzmán, 2013). The OMHC1-188 and OLADRB2-282 alleles of the MHC influence the differentiation between genotypes in the antigen-presenting mechanisms (Alba-Hurtado & Muñoz-Guzmán, 2013). Of the TLR variants, the TLR4 gene is reported to be involved with the immune response to parasitic infections, as are other nearby genes, TNFSF8 and TNFSF15, which encode cytokines belonging to TNF-α (Lin et al., 2016).

After evaluating the ALOX15, CD109, CD163, CPA3, EMR3, IL-13, KIT and MAP3K5 genes, it was found that ALOX15 and IL-13 play important roles in resistance to GIN (Wilkie et al., 2015) and were significantly increased in resistant animals and expression was negatively correlated with FEC. The expression of different genes at the intestinal level has been evaluated in two divergent lines by means of microarray analysis, with the highest expression in susceptible animals of HLA-A, MSH6, GPX2, IF135, UBD, SERPING1 and TFF3, the expression being generally associated with the stress caused by parasitic infection, such as in the case of GPX25; in contrast, resistant animals showed higher expression of the RAC2, ITGB2, DAP3 and TRADD genes associated with neutrophilia and post-infection cellular processes and of importance in innate immunity (Keane et al., 2006). As indicators of the genetic influence on resistance, the presence of QTL regions for EPG on chromosomes 1, 2, 3, 6, 14, 19 and 20 has been revealed for different nematodes, including H. contortus, and on chromosome 1 for haematocrit (Bishop & Morris, 2007). Other authors have reported different QTL regions in sheep for H. contortus, such is the case of chromosomes 1, 3, 6, 8, 14, 20 and 22 (Miller & Horohov, 2006; Zvinorova et al., 2016).

The physiological, immunological and genetic effects that influence the resistance or susceptibility of ewes to GIN infections in the peripartum period are determined by the interaction of various factors: