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Helminthiasis in small ruminants production industry
Gastrointestinal helminths of domestic small ruminants.
Parasitism is a widely observed form of non-mutual symbiotic relationship between organisms. The parasite is an organism that depends on the host for completing its life cycle. On the other hand, the host does not depend on the parasite by any means and normally reacts to the parasite in order to clear it from its body. From an evolutionary point of view, when a parasite population is able to overcome the clearance reaction of a host, it becomes endemic and a path of co-evolution between the two organisms eventually begins [3]. However, in the context of small ruminant production systems, parasitism, as well as any other disease that alters the production performance, is considered a problem because it creates a significant economic loss. The mechanisms by which infectious diseases cause an economic loss are varied and mainly depend on the disturbances caused by parasites on the utilization of the nutrients fed to the host (FIG.1).
As to the helminths of the gastrointestinal tract of the domestic small ruminants, they experience their reproductive stage as adults within the intestinal lumen. The helminths of veterinary importance infesting the intestinal tract of domestic small ruminants are numerous and they are localised in different tracts of it. The helminths of the oesophagus and of the omasum are: Cotylophoron spp, Gonylonema pulchrum, and Paramphistomum spp. The helminths of the abomasum are: Haemonchus contortus, Marshagallia marshalli, Teledostertagia circumcinta, Ostartagia trifurcata, Parabonema spp., and Trichostrongulus axei. The helmints of the small intestine are: Avitellina centripunctata, Bunostomum trigonocephalum, Cooperia curticei, Cooperia surnabada, Gaigeria pachyscelis, Moniezia expansa, Nematodirus battus, Nematodirus filicollis, Nematodirus spathiger, Strongyloides papillosus, Trichostrongylus capricola and Trichostrongylus vitirinus. The helmints of the large intestine are: Chabertia ovina, Oesophagostomum columbianum, Oesophagostomum venulosum, Skjabinema ovis, Trichuris ovis and Trichuris skrjabini. However, most of the research in control strategies for containing the impact of these worms on small ruminant production is focused only on the most problematic nematodes, because of both their worldwide prevalence and of the extent of the economic loss they cause. These species are H.contortus, Teladostertagia circumcinta and Trichostrongylus spp, concerning the abomasum, and Trichostrongylus spp., concerning the small intestine [4]. H.contortus in particular is reported as being one of the most problematic because of its worldwide endemicity, the copious haemorrhage it causes and its marked resistance to anthelmintics [2, 5–7]. These features make it a good model organism for the study of gastrointestinal nematodiasis.
Economic impact of gastrointestinal nematodes on small ruminant industry
Gastrointestinal nematodes of small ruminants are raising growing concern across small ruminant production systems because of several reasons: the extent of the economic loss they cause is significant, their prevalence is extending outside tropical regions, and the growing number of parasites populations express resistance to anthelmintics.
The economic loss has been estimated on the order of millions of dollars per year in many countries. For example, the impact of gastrointestinal nematodes on the Australian sheep production system has been estimated to reach 1 billion dollar per year [8]; the cost of parasite control in New Zealand has been estimated as 29.3 million per year [9]; similar pictures appear in studies focused in Asia [10, 11]. The economic impact of gastrointesninal parasites is becoming relevant also in regions where its prevalence is not as high as in the tropical regions, such as Sweden, Netherlands, Denmark [12], France (Hoste et al., 2002) and UK [14] Due to the occurrence of free-living stages during their life cycle, which ensures transmission between hosts, these parasites are exposed to different environmental conditions. This feature resulted in a picture of the endemicity of these parasites which located them mostly within warm and humid environments, i.e. subtropical and tropical environments [5]. However, due to both their marked ability of adaptation [7], and to the current climate change, these parasites are recently adapting to temperate regions up to the neighbourhood of the polar circle. Indeed, the current picture of the prevalence of infectious diseases is very likely to change due to the current climate change [15, 16].
Until recently, small ruminant’s gastrointestinal parasites have been successfully controlled by the use of anthelmintics. However, many studies report an increasing resistance to these drugs among different populations of gastrointestinal parasites worldwide. This phenomenon further increases the economic loss due to gastrointestinal parasitism [14, 17–20]. Since most of the anthelmintics target single proteins, they are inevitably bound to lose efficacy because of the evolutionary potential of the parasites and their genetic variability. Resistance to anthelmintics, as well as the influence of the growing public concern for the use of drugs in food, production systems [21] have created a need for new strategies for controlling these parasites.
Pathophysiology of haemonchosis
In order to understand what are the known biological determinants of abomasal nematodiais and what is their influence on the alteration of the productive performance of the parasitized animal, this chapter aims at briefly describing the organs and the cellular populations mainly involved during the interaction between the abomasal parasites and their host. An inventory of the known biological mediators playing a major role in the regulation of this interaction will also emerge.
Morphofunctional description of Haemonchus contortus
H.contortus is the pathogenic agent of Haemonchosis. It belongs to the phylum Nematoda, which includes worms featuring the following characteristics. Nematoda are commonly known as round worms because their body has a cylindrical shape thinning at the extremities. The body is covered with a transparent cuticle, secreted by the hypoderm, which can also form various structures depending on the species. The cuticle of H.contortus features two cervical papillae, which fulfil both sensory and mechanic functions. The hypoderm deepens within the muscular tissue below to enclose the two excretory grooves along the sides and both the dorsal and ventral nerves inside the respective cords. The innermost membrane is composed of muscular cells, which form the celomatic cavity filled with fluid. The celomatic cavity contains the filamentous organs of the digestive and reproductive systems. The digestive system is formed by the oral cavity, the oesophagus, the intestine and the anus. The oral cavity of H.contortus features a lancet, which enhances the haemorrhage from the blood vessels of the host. The reproductive system of the female is formed by the ovary, the uterus and the vulva. The reproductive system of the male is formed by one testicle, and a ductus deferens which ends into the cloaca. The vulva of H.contortus presents a vulvar flap, which facilitates fecundation together with the spicules and the asymmetric bursa of the male’s reproductive system (Fig.2, Fig 3, [6]. A large inventory of excretory/secretory products (ESP) is also involved in the interaction between the parasite and the host. Most of them have been characterized as proteases [22], whereas others have been hypothesized to play a crucial role in the regulation of the host response but have not been identified yet.
The interactions between the host and the parasite
Life cycle of H.contortus
This paragraph depicts the life cycle of H.contortus, which explains how the parasite and the host come into contact for their interaction to occur. The life cycle of H.contortus is similar to the direct life cycle of Trichostrongylidae and includes stages outside the host’s digestive lumen and inside of it. The stages outside the host’s digestive tract are: the egg, and the free-living larval stages. The egg is deposed by the adult females reproducing in the host’s abomasum. After having been excreted together with the faeces, it develops to an L1 larva. L1 feeds on the bacteria encountered within the faeces and develops to L2. During the L1 and L2 stages, the individual stores energy which allows the development to the infesting L3 larva, as well as its survival, because the protective enclosing the L3 does not allow it to feed anymore. The timing of the whole development process and the percentage of egg successfully developing to L3 depends on the environmental conditions it occurs in [23–26]. The L3 larva migration patterns are driven both by passive and active transport; however, negatively geotropic patterns are only explained by active migration [27]. This increases the likelihood to be ingested by a potential host. After having been ingested by a suitable host, the L3 turns to L4: the L3 cuticle is shred and the intestine is developed, allowing for the beginning of the histotrophy within the abomasal mucosa. At this point, the cycle can either continue to the development of the adult stage or undergo a phase of hypobiosis. Hypobiosis is a state of arrested development which keeps the L4 to an early stage of development. The factors governing hypobiosis include both the environmental conditions experienced by the free-living stages and the environment occurring inside the host’s digestive tract [28–30]. Once the L4 develops to an adult, it establishes in the abomasum and feeds by disrupting the blood vessels of the abomasal mucosa by its lancet. When the adults are sexually mature, they mate for producing eggs and the cycle begins again.
Immune response associated with resistance to H.contortus
The immune response of the host is due to many cellular populations derived both from myeloid stem cells and lymphoid stem cells. These cells differentiate into several subpopulations, fulfilling different roles when activated by the contact with antigens of non-self organism. The immune response is classified into two main types: the innate immune response, which refers to the reaction of the organism to any antigen recognised as non-self, and the acquired immune response, which refers to the reaction of the organism aimed at previously encountered antigens. The former mostly involves myeloid-derived cells, whereas the latter is mostly due to lymphoid-derived cells. The immune response is regulated by a vast inventory of mediators, produced by a variety of cellular populations, which drive the type of immune cells recruited against the antigen, the healing of damaged tissues and build the “memory” of the acquired immune response. In general, the role of the effector cells is to cause damage to the neighbouring cells and the role of the mediators molecules is to maximise the localization of the activity of the effector cells to any organism expressing non-self antigens. Furthermore, the mediators molecules (cytokines) also regulate the mobility and the activation of some sub-populations of effector cells and stimulate the tissue repair pathways. Indeed, especially in the case of parasites which have co-evolved together with their host, not all immune response mechanisms have an effect on controlling the parasite population [31]. Some of the effective mechanisms which have been associated to resistance to Haemonchus contortus will be discussed in the following section.
Innate immune response
The innate immune response is mainly activated by one class of antigens referred to as pathogen-associated molecular patterns (PAMPs), which include molecules featuring high steric redundancy across various pathogens [32]. The very first defence line inhibiting the establishment of larvae is the mucous secreted by the surface mucous cells of the abomasum. The mucus reduces larval motility by mechanical impedance and by factors such as leukotriens [33, 34], secreted by mast cells and globule leukocytes. Mast cells also release histamine, which both increases peristalsis for mechanically clearing the parasites and initiates the inflammation process. Gastric secretions and motility result in reduced establishment and reduced fecundity of the worms [35].
The innate immune response is basically the inflammation which any tissue builds when damaged, which results in increased vascular permeability and increased blood circulation in the neighbourhood of the damage. In consequence, the concentration of circulating molecules, among which the complement has an effect on the larvae of H.contortus, increases in the surrounding of the larvae. The complement is a complex of many polypeptides which bind together by a cascade of covalent bounds. This cascade can, in turn, be triggered by one of the complement’s polypeptides binding either to the antibodies coating the invading organism’s surface (classical activation pathway) or directly to the carbohydrate structures of invading organisms (alternate activation pathway). The activation of the complement results in direct cell membrane damage and release of citokines. H.contortus causes the activation of the complement via the alternate way, which results in the generation of chemokines such as C3a and C5a which attracts eosinophils to the sorroundings of the larvae [36–38]. Eosinophils, together with neutrophils, are also attracted by the ESP products of H.contortus.
The effector cells of the innate immune response are scattered across the connective tissues of the body and those mainly involved in the innate response to nematodes are: eosinophils, basophils, mast cells and antinflammatory macrophages. The mucosal mast cells release several cytokines, among which IL-13, IL-4 and IL-5 result in the following consequences: increasing intestinal motility, acting as a mechanical defense for the expulsion of the parasite. Mucosal mast cells are also involved in the regulation of the IEC cyokines release and when they are activated to infiltrating mast cells, they recruit and act together with eosinophils and neutrophils by releasing the content of their cytosolic granules [39]. The cytosolic granules contain several compounds. Reactive oxigene species, which inflicts direct oxidative damage to the parasites cuticle. Histamine, which increases intestinal motility and vascular permeability. Proteases, such as MCP-1 reducing fecundity of the adults and enhancing the activation of other effector cells. Chemokines, which recruit circulating basophils and eosinophils [40].
Other cell types involved in the innate immune response are the γδ T cells, by secretion of IL-5 and IL-13 [41]. In addition to this contribution to the innate immunity, the cytokines of the IEC, mast cells, DC and natural killer (NK) also contribute to steering the develompent of the acquired immune response towards a type 1 hypersensitivity response, by means of activating the T helper 2 lymphocytes (Th2) and B cells [42].
Acquired immune response
The acquired immune response is mediated by receptors, coded by MCH genes, which bind to specific antigens of the invading host and allow the lymphoid cells to focus their activity on the pathogen. These receptors can be both expressed on the surface of the lymphoid cells (cell mediated immune response), and released from their surface (antibody-mediated immune response) [43]. An effective specific immune response is activated against helminths when the MHC class II receptor of an antigen presenting cell, DC2 being specially effective, contacts a CD4+ T cell receptor. IL-4 secreted by DC2 cells, together with IL-1 secreted by macrophages, also contribute to the activation of Th2 cells. The activated Th2 cells secrete a number of interleukines which contribute to building up an antibody-mediated humoral immune response by inducing the B cells to shift into antibody producing producing plasma cells in the lymph nodes [31, 32]. The antibodies produced by the plasma cells are of different types and functions. As far as the response to helminths is concerned, immunoglobulins G (IgG) are mostly associated to reduced worm burden [44–46]; the circulating isoform of IgA has been associated to reduced worm growth and fecundity, whereas its mucosal and fecal form has been associated to reduced worm burden and ESP [45, 47]; IgE allow the localized degranulation of basophils and eosinophils to the worm surface by its affinity to th Fc receptor of these cells and have been associated with reduced worm burden but possibly also with immune- mediated tissue damage [48, 49]. DC cells also produce IL-10 and TGF-β, which induce T cells to differentiate into regulatory T cells (Treg), involved in the regulation of the immune response and the inflammation in the gastrointestinal tract [50].
Pathogenesis
The anatomy of the abomasum
The abomasum of small ruminants is the organ of the digestive system most similar, both for its morpholgy and for its physiology, to the stomach of monogastric species. Its main function concerning digestive process is the proteolysis. It is a luminal organ composed of four main layers: the tunica serosa, the tunica muscularis, the tunica submucosa and the tunica mucosa. Parasitism concerns mainly the tunica mucosa, the innermost of them, where the histotrophic phase of the L4 and the haematophagic phase of the adults occur. The tunica mucosa is composed of three further layers: the lamina muscularis, the lamina propria and the lamina epithelialis. The lamina muscularis is composed of smooth muscle tissue. The lamina propria is composed of connective tissue which contains the blood vessels which the adults feed from. The lamina epithelialis is composed of heterogeneous populations of cells, which determine the functional subsetting of the tunica mucosa in two different regions: the fundic region and the pyloric region. These regions are identified according to the cellular populations found inside the gastric glands, the latter formed by the introversions of the lamina epithelialis deep within the lamina propria. In fact, the cells of the lamina epithelialis found outside these glands, which form the luminal surface of the abomasum, are mostly surface mucous cells and do not differ much across the three regions. These cells produce the mucous covering the luminal surface of the abomasum, which is mostly composed of mucin (MUC5AC) and forms the so-called mucosal barrier, protecting the mucosa itself from the gastric juice.
The pyloric is region located close to the pylorus and is characterised by the presence of the pyloric glands. These type of glands further fulfill a regulatory function by producing both endocrine and paracrine mediators. The characteristic cells of the pyloric glands are the G cells (gastrin produing), the D cells (somatostatin producing) and the enterochromaffine cells (atrial natriuretic peptide producing).
Table of contents :
1. General introduction
2. Literature review
2.1 Helminthiasis in the context of small ruminants industry
2.1.1 Gastrointestinal helminths of domestic small ruminants.
2.1.2 Economic impact of gastrointestinal nematodes on small ruminants industry figures
2.2 Phathophysilogy of haemonchosis
2.2.1 Morphofunctionl despription of H.contortus
2.2.2 The interactions between the host and the parasite
2.2.2.1 Life cycle of H.contortus
2.2.2.2 Immune response associated with resistance to H.contortus
2.2.2.2.1 Innate immune response
2.2.2.2.2 Acquired immmune response
2.2.2.3 Pathogenesys
2.2.2.3.1 The anatomy of the abomasum
2.2.2.3.2 The physiology of the abomasum and its regulation
2.2.2.3.3 Symptoms of haemonchosis figures
2.3 Control strategies
2.1 Focus on genetic selection
2.4 Quantitative genetics applied to livestock improvement
2.4.1 Genetic variation
2.4.2 Definition of inbreeding and relatedness
2.4.2.1 Computing the genetic relationship matrix from pedigree information only
2.4.2.2 Computing the genetic relationship matrix from marker information only
2.4.2.3 Computing the genetic relationship matrix from both pedigree and marker information.
2.4.3 Estimation of the additive genetic variance
2.4.4 Estimation of the breeding values
2.4.5 Estimation of the QTLs’ allelic substitution effects
2.4.6 Prediction of the response to selection
3. Experimental studies
3.1 Article1 – Genetic parameters for growth and faecal worm egg count following Haemonchus contortus experimental infestations using pedigree and molecular information (published)
3.1.2 Abstract
3.1.3 Introduction
3.1.4 Materials and Methods
3.1.4.1 Experimental design
3.1.4.2 Genotypes
3.1.4.3 Phenotypes
3.1.4.4 Statistical analysis
3.1.4.5 Significance tests
3.1.5 Results and discussion
3.1.5.1 Phenotypic variation
3.1.5.2 Genetic variation
3.1.5.3 Standard errors
3.1.4 Conclusions
3.2 Article 2 QTLs for faecal egg counts and packed cell volume detected during two subsequent experimental infestations with Haemonchus contortus on creole goats. (manuscript in preparation)
3.2.1 Abstract
3.2.2 Introduction
3.2.3 Materials and methods
3.2.3.1 Phenotypic informaation
3.2.3.2 Genetic information
3.2.3.3 Heritabilities
3.2.3.4 QTL detection
3.2.4 Results and discussion
3.2.4.1 Descriptive statistics
3.2.4.2 Heritabilities
3.2.4.3 QTL detection
3.2.5 Conclusions
Figures and tables
4. Discussion and perspectives
4.1 Experimental design: weak and strong points
4.2 The biology underlying the observed phenotypic variation
4.3 Genetic parameters
4.4 Impact of molecular information
4.5 Practical implications
4.6 Modelling
5. Conclusions
6. References
