THE EFFECT OF VITAMIN E SUPPLEMENTATION ON AN EXPERIMENTAL HAEMONCHUS CONTORTUS INFECTION IN DORSET LAMBS

Gastrointestinal parasitism in sheep, particularly lambs, results in substantial economic losses to producers worldwide. Nutritional status has been shown to play an important role in host immune response to parasitic infections. The objective of this study was to investigate the effect of vitamin E supplementation on an artificial Haemonchus contortus (H. contortus) infection in immature lambs. Twenty Dorset lambs were stratified into two treatment groups according to parasite susceptibility. Worm-free lambs, 28 to 32 weeks of age, were supplemented with vitamin E (d-α-tocopherol) for twelve weeks following the recommendations of the National Research Council for minimal daily requirement (VE5; 5.3 IU/kg body weight (BW)/day (d), n=10) or optimal immune function (VE10; 10 IU/kg BW/d, n = 10). Five weeks after initiation of vitamin E supplementation, lambs were infected with 10,000 H. contortus L3 larvae. Samples were taken weekly for determination of fecal egg counts (FEC), packed cell volume (PCV), and serum α-tocopherol. After six weeks of infection, the lambs were humanely slaughtered for the determination of tissue vitamin E content, worm burden and histologic evaluation of the abomasum. Increased dietary vitamin E supplementation had no effect on liver (P=0.08), muscle (P=0.62), or lymph node (P=0.38) α-tocopherol content. There was no effect of treatment on FEC or PCV, however there was a 49% reduction in total abomasal worm burden (P=0.002) in the VE10 group. These results indicate that elevated levels of vitamin E supplementation had a beneficial effect on the abomasal worm burden; however, there was no treatment effect on PCV or FEC.


Introduction
Gastrointestinal nematode parasites of small ruminants are responsible for staggering economic losses to producers on nearly every continent. The abomasal nematode causing the largest detriment to production is the barber pole worm, Haemonchus contortus, which poses particular risk to young animals due to their naïve immune system. The problem is compounded by the recent development of parasitic resistance to all classes of chemical dewormers used for the control of gastrointestinal parasite infections. Due to this evolution of parasite resistance to chemical anthelmintics, alternative approaches for control are needed . The control of parasitic infection in flocks and herds is heavily reliant on the integrated use of anthelmintics along with grazing management strategies.
Factors influencing the host-parasite relationship include age, genetics, and nutritional status. While past studies have demonstrated that supplementation with vitamin E, a free radical scavenging antioxidant, enhances immune responsiveness and performance in lambs , the effect of vitamin E supplementation on an immune response against parasitic nematodes is unknown. A recent increase in the recommended daily intake of vitamin E for sheep for optimum immune status based on studies showing the role of vitamin E in enhancing immune function in small ruminants (National Research Council, 2007), creates further need to investigate the effect of vitamin E supplementation on a lamb's response to parasitic infection. A dual approach that examines the potential of nutritional modulation to enhance immunity to parasitic infection is needed. This literature review will present a comprehensive background on parasitic infection in small ruminants and current research investigating sustainable parasite control methods.

Overview
The United States sheep industry is comprised of nearly 6.2 million animals and generates an estimated $392 million annually . Although it is difficult to determine precise figures for financial losses resulting from parasites, one study quantified sheep and goat parasitic production losses to exceed $270 million each year . Parasitic infection of sheep and goats is a particular problem in the Southeastern region of the country, as warmer climate conditions are conducive to parasitic growth and transmission. Seventy-five percent of 467 sheep producers surveyed within the southeastern region of the United States identified stomach and intestinal nematodes as a major concern and threat to production . The most recent survey of sheep producers conducted by the United States Department of Agriculture indicated that 74% of producers reported the predominant disease in their flocks to be due to stomach and/or intestinal worms .
While most economic losses are due to animal mortality, production losses are also significant contributors . Clinical signs of gastrointestinal nematode (GIN) infection contributing to production losses include decreased rate of gain, a reduction in feed efficiency, reduced meat, wool, and milk production, and a decreased reproductive efficiency . Control of GIN has been complicated by an increasing parasite resistance to traditionally used anthelmintics, resulting in drastic decreases in reported efficacy for all classes of available drugs . The dramatic emergence of resistance to multiple drug classes presents an urgent need for the reform of current small ruminant parasite control practices. Novel methods of GIN control are needed to prevent continued losses within the sheep industry. Understanding all aspects of the hostnematode interaction is necessary to ensure the future success and survival of pasturebased sheep production.

Gastrointestinal Nematodes of Small Ruminants
Small ruminants are affected by many species of nematodes, cestodes and coccidia that ultimately lead to production losses. Prophylaxis and treatment expenses are massive burdens in animal husbandry. The most common and pathogenic gastrointestinal parasites of small ruminants are discussed below.

Trichostrongylids
The predominant species of nematodes affecting sheep belong to the Superfamily Trichostrongyloidea and include Haemonchus contortus and Telodorsagia circumcincta, located in the abomasum, and Trichostrongylus colubriformis, Cooperia spp., Nematodirus spp. and Oesophagostumum spp., located in the small and large intestine respectively . Small ruminants on pasture are typically challenged with several nematode species at one time, contributing to the condition known as parasitic gastroenteritis (PGE) . Trichostrongylids cannot be differentiated from one another by microscopic examination of their eggs; only infective larvae and adult worms allow the determination of individual species .

Parasite Distribution
Nematode populations thrive in warm, moist conditions, and are therefore widely distributed in tropical and subtropical regions of the world . In the United States, many studies have documented the higher incidence of production losses due to GIN occurring in the Southeast region of the country . The eastern South Central region of the United States (Alabama, Arkansas, Kentucky, Louisiana, Mississippi, and Tennessee) report the highest percentage (77.8) of sheep producers indicating their top health condition of concern to be stomach/intestinal worms .
In the temperate Northeast region of the United States, few studies have been conducted, and to our knowledge, none have been executed in recent years . These studies confirmed the species in the Northeast in order of predominance to be H. contortus, Nematodirus spp., Teladorsagia spp., and Trichostrongylus colubriformis . While all species can inhibit production, the abomasal nematode that poses the greatest threat to animal health and producer livelihood worldwide is Haemonchus contortus.

Haemonchus contortus
Haemonchus contortus is extremely pathogenic and the most economically devastating parasite of sheep and goats worldwide (Mortensen et al., 2003). Haemonchus contortus is commonly called the barber pole worm due to a distinct red and white striped appearance, caused by the helical winding of the white egg-filled uterus of the female adult worm wrapped around the digestive tract. The female adult worms are 18-30 mm long while the male adult worms are smaller and thinner than the females, at 10-16 mm long .

Signs and symptoms
Larval and adult stages of H. contortus feed on the blood of the host, inflicting severe anemia and death in heavily infected animals . Anemia is characterized by pale mucous membranes, particularly visible in the lower eyelid. Sudden deaths occur as a result of hemorrhagic anemia caused by severe blood loss from the gut.
Although animals with low levels of infection do not generally exhibit clinical signs, subclinical infections are also of great concern to producers due to weakness and significant decreases in weight, fertility, and milk yield within a herd or flock .
To feed on the host, H. contortus exposes an oral lancet used to slit capillaries of the abomasal mucosa open and feed on the released blood, simultaneously secreting an anticoagulant causing prolonged bleeding at the site . At peak levels of infection, H. contortus accounts for the loss of up to one fifth of the circulating erythrocyte volume per day from lambs, and one tenth of the circulating erythrocyte volume per day from adult sheep .
Disease is acute in animals with heavy worm burdens. H. contortus is extremely prolific; females can produce up to 10,000 eggs each day at a rate of one egg every ten seconds . Edema under the jaw, commonly called "bottle jaw", is another frequent sign of H. contortus infection caused by significant blood plasma and protein loss to the host . The effects of haemonchosis are most severe in very young or old animals as well as pregnant or lactating, undernourished, diseased or stressed animals due to a compromised immune system (Kaplan, 2006). Lambs are the most affected members of a flock, but mature sheep under physiological stress also may have fatal anemia .

Life cycle of Haemonchus contortus
H. contortus is dependent on a small ruminant host to develop and complete a 21 day life cycle. Adult female H. contortus worms begin laying eggs which exit the body in the manure of the host sixteen to twenty-three days post-infection . If environmental factors are favorable, eggs will hatch and molt from the first larval stage (L1) to the second stage larvae (L2). The cuticle of the L2 is retained as the larva develops into the infective L3 stage. While the time from initial egg hatching to reaching the infective L3 stage can be as little as five days, development may be delayed for up to months if environmental conditions are cooler . Infective L3 stage larvae leave the fecal pellet and climb up blades of moist grass on pasture to be consumed by grazing animals. After ingestion, the larvae shed their outer protective layer in the rumen and then invade the mucosa of the host abomasum, molting into the L4 stage larvae . After a final molt, the parasite becomes an L5 stage adult worm at which point a lancet develops, allowing the parasite to pierce vessels of the mucosa and begin feeding on blood   1996). This period of hypobiosis occurs in the abomasum of the host, with maturation of the larvae resuming when favorable conditions return in the spring.
Lactating sheep are particularly susceptible to H. contortus, and the re-emergence of the parasite from hypobiosis can account for a significant and rapid rise in parasitic infection levels around the time of lambing (Baker et al., 1999). The susceptibility during the periparturient period is attributed to the suppression of the immune system by increased levels of hormones and metabolic demands of pregnancy on the host, which is exacerbated by suboptimal feed intake in pregnant ewes . Leathwick et. al. (1995) found the establishment of larvae in the abomasum to increase 30% three weeks prior to lambing in pregnant ewes. While the precise mechanisms triggering hypobiosis are poorly understood, it is proposed to be influenced by both immune and environmental cues . It is clear that hypobiosis is an important adaptation that contributes to the immense success and survival of H. contortus.

Anthelmintics Used in the Control of Gastrointestinal Nematodes
Control of gastrointestinal nematodes is almost entirely based on the use of anthelmintics. There are currently three classes of anthelmintics used in sheep, including 1) benzimidazoles (including albendazole and fenbendazole), 2) cholinergic agonists (such as levamisole) and 3) avermectins (including ivermectin and moxidectin) . These drugs are FDA-approved for use in the treatment of gastrointestinal parasites in sheep.
Resistance of parasites to these traditionally used anthelmintics has become a serious problem in veterinary medicine, particularly in sheep husbandry .
The development of widespread resistance of gastrointestinal nematodes to chemical dewormers has left veterinarians and farmers with few options to effectively control parasites within a flock.

Anthelmintic Resistance
Resistance can be defined as the failure of the majority of a parasite population to be eliminated by an anthelmintic treatment, or the need for a greater concentration of anthelmintic to reach efficacy . The first resistance to commercial dewormers in the United States was recorded in 1957 to phenothiazine, followed shortly after by thiabendazole in 1964 . The problem has only grown in recent years, as many studies have documented the increasing inability of anthelmintics to control parasitic infection. To date, parasitic resistance to anthelmintics has been reported worldwide and exists to every class of available drugs .
The increasing resistance of gastrointestinal nematodes to chemical dewormers has become an epidemic, resulting in large revenue losses for sheep producers. While resistance to commercial dewormers had been documented in multiple parasites, the most dramatic resistance has been observed in H. contortus . Once considered to be native to the southern region of the United States due to warmer climates, H.
contortus has become one of the most significant obstacles to sheep producers in New England, particularly due to a growing resistance to routinely implemented commercial dewormers that has become evident in recent years. To date, resistance to every class of anthelmintics has been documented in the United States, with the first total failure to all available drugs documented in Arkansas in (Mortensen et al., 2003.

Factors Influencing the Development of Anthelmintic Resistance
The problem has only been exacerbated by previous recommendations by both parasitologists and veterinarians to rotate the anthelmintic used, to deworm all animals frequently, and to move animals to clean pastures just after deworming-practices which were once thought to control parasites, but have been recently shown to only exacerbate the problem . Repeatedly treating a parasite population with an anthelmintic puts selective pressure on individuals that have innate or acquired resistance to the drugs. Resistance is inherited by transmission of resistant alleles; even point mutations can lead to a decrease of susceptibility to a pharmaceutical compound, enabling the resistant proportion of the parasite population to survive treatment . Treatment becomes ineffective when the proportion of resistant genes increases and thereby dilutes the susceptible genes .
The ever decreasing efficacy of anthelmintics has resulted in reduced productivity and increased mortality in the flocks of sheep producers. It is clear that past methods of parasite control are not only ineffective in managing parasites but unsustainable as well, and many producers are looking to reduce or eliminate the use of chemical dewormers entirely.

Refugia
A key concept in sustainable parasite control is refugia, or the proportion of a parasite population that is not selected by drug treatment. Refugia is an essential factor in delaying the development of anthelmintic resistance . By treating only the animals that exhibit clinical signs of parasitic infection and require treatment and leaving the rest of the animals untreated, susceptible worm strains remain in the flock and have a diluting effect on the population of resistant worms . The goal of an integrated parasite control program is to keep refugia high by treating only affected animals and moving away from an overreliance on traditionally used chemical dewormers.

Best management practices for parasite control
Currently, the recommendation for best management practices (BMP) for sustainable parasite management incorporate a decreased reliance on commercial dewormers as well as the integration of alternatives to dewormer use in parasite control programs. An integrated parasite management program determines individual susceptibility to parasites. Deworming only those animals affected by parasites and controlling animal exposure to parasites through pasture rotation is currently recommended. Rotational grazing is a practice currently recommended to reduce infection. Moving a flock or herd to graze on different pastures periodically can substantially reduce pasture infectivity. Rotating more resistant mature animals with susceptible younger animals may also prove beneficial, although this strategy may be impractical for producers with limited acreage .

Selective Deworming
Research has concluded the major factor in the development of drug resistant parasites is the method of deworming all animals in a population simultaneously and frequently, a common practice implemented by most producers in the past. It is estimated that only 20-30% of the animals in a population harbor about 80% of the parasites (Kaplan, 2006). By deworming only the most susceptible animals rather than every animal as previously recommended, parasites can be more effectively controlled by lessening the evolutionary selection for resistant populations of worms. This method of selective deworming, or "smart drenching", can potentially slow the development of drug resistant parasites while reducing the number of treatments given, a clear financial benefit for producers. One of the most important aspects of smart drenching is a selective treatment approach based on the use of the FAMACHA© scoring system.

The FAMACHA© Method
A system to easily identify animals most affected within a population has been recently developed in South Africa at the Onderstepoort Veterinary Institute. This method was developed in consideration of small farms lacking resources, allowing producers to effectively and inexpensively assess animals without the use of lab equipment or diagnostics . In order to identify the most susceptible animals, producers can implement the FAMACHA © (Faffa Malan Chart) system to quickly assess the presence of anemia due to H. contortus infection based on mucous membrane pallor in goats and sheep, assessed by examination of the lower eyelid (Kaplan, 2004). Treatment can then be administered selectively to animals exhibiting anemia. The FAMACHA© method is a practical tool for implementing selective deworming, a management approach that operates on the premise that only animals showing clinical symptoms or reduced productivity should be treated with anthelmintics, thereby slowing down the development of anthelmintic resistance .
While other parameters can be used to identify parasitic infections in animals, such as body condition scoring, the presence of diarrhea, and fecal egg counts, the FAMACHA© system is a valuable tool producers can use to identify animals consistently infected and in need of treatment, as well as to reduce the number of animals treated and the cost of total treatment on a farm .

Alternatives to commercial dewormers
The rapid and dramatic spread of parasitic resistance to anthelmintics as well as concern about their possible detrimental effects on the environment has encouraged researchers to initiate the development of alternative parasite control methods. Since the main method of controlling H. contortus has become relatively ineffective, other methods need to be investigated.
Although initial field trials have shown potential, to date no vaccine has been developed to successfully prevent haemonchosis .
The discovery of a fungus capable of trapping nematodes has been shown to be highly quebracho extract from tree bark has been shown to reduce fecal egg count and female fecundity of the barber pole worm in goats . Terrill and co-workers (2009) reported that sericea lepsideza, a CT-containing hay resulted in reduction of both fecal egg count and adult worm numbers in the abomasum of small ruminants. A significant number of other studies have shown supplementation with copper oxide wire particles (COWP) to demonstrate anthelmintic activity in experimentally infected sheep Nyman, 2000).
Several other novel techniques of control, including breeding genetically resistant sheep, implementing alternative management practices such as intensive rotational grazing, and nutritional modification are also currently being studied as alternative GIN control. Current research has made some progress in the discovery of groundbreaking, non-chemical solutions to the sustainable management of H. contortus and other economically important gastrointestinal nematodes worldwide.

Ruminant Immunity
In a 2002 study, Binns et al. reported approximately 10-35% of lambs die within the first 6 months of life, a mortality rate nearly double that of mature sheep. These losses can be attributed to an underdeveloped, naïve immune system of young ruminants.
In the first 3-4 months of life, ruminant neonates are almost entirely dependent upon maternal antibodies passively transferred through colostrum. The majority of maternal immune cells are T cells, which provide early cell-mediated immune system . While maternally derived antibodies provide protection, they concurrently inhibit the lamb from mounting an antibody response of its own . Due to this inhibition, lambs are not vaccinated against infectious diseases until they are at least 3-4 months old . Lambs are particularly vulnerable to infection when maternal antibodies are no longer present and have not yet begun to effectively mount their own immune response.

Th1/Th2 response
The immune system is comprised of widespread individual effector cells, tissues and organs with complex interactions that allow a rapid response to any foreign pathogens entering the body . Cytokines, low weight signaling proteins involved in immune cell communication, are produced by the activation of CD4+ T helper cells and allow for immune attacks against intracellular or extracellular pathogens, respectively . T helper lymphocytes are differentiated into two subsets, T helper type 1 (Th1) and type 2 (Th2), based on the cytokines they secrete .
Th1 type cells are responsible for the activation of cellular immunity, and a Th1 type response is typically seen in response to viral infection, intracellular pathogens and the activation of delayed-type hypersensitivity reactions. A Th1 type response typically responds to viruses and vaccinations, and is characterized by the cytokines IL-2, tumor necrosis factor, and interferon-γ which mobilize macrophages, T cells, and natural killer cells to promote proliferation and inflammation .
A Th2 response is characterized by the increased production of immunoglobulin secretion by B cells, particularly IgG and IgE and the cytokine IL-4, as well as increased eosinophils and mast cell recruitment (Shallig, 2000). A Th2 type response is associated with allergic reactions  and is also activated upon parasitic infection (LaCroux et al., 2006;. It is hypothesized that there is a balance between the expression of both the Th1 and Th2 responses, although challenges with different pathogens may alter the type and ratio of cytokines expressed, such as the increase of Th2 type cytokines during a parasitic infection (Kidd, 2003).

Host Response to Parasitic Infection
Inflammation during parasitic infection is important in larval and adult stage worm expulsion from the mucosa   . Effector cells degranulate, releasing vasoactive molecules, which physically expel worms from the abomasal mucosa .
Eosinophils are small granulocytes produced in bone marrow and are closely associated with parasitic infection, activated by Th2 cytokines IL-3 and IL-5 (Balic et al., 2000). Mediators released by eosinophils include major basic protein, peroxidases, and neurotoxins, and have been shown to have lethal effects against gastrointestinal nematodes in vitro (Kilon and Nutman, 2004). Mediators act as potent vasodilators, inducing smooth muscle contraction and subsequent expulsion of parasites with the abomasum .  found that sheep with low egg counts had higher numbers of circulating eosinophils, which have been also been associated with larval migration inhibition (Douch et al., 1984).
Infection with H. contortus is associated with the production of mucosal mast cells and globule leukocytes within the GI tract of the sheep (Miller, 1996). Sheep that are more genetically resistant to parasites have more globule leukocytes than more susceptible breeds . Mucosal mast cells, recruited from bone marrow by Th2 cytokines such as IL-4, release histamine, ultimately becoming globule leukocytes after degranulation (Miller, 1996). Many studies have documented the role of mast cell degranulation in worm expulsion (Douch et al., 1986, but simply quantifying mast cells numbers upon necropsy is not an accurate reflection of activation status and can therefore be misleading (Balic et al., 2000). Quantification of globule leukocytes has been shown to provide a more accurate measure of mast cell activity .
Resistance to parasitic infection is defined by an animal's ability to prevent the establishment of parasites mediated by immune mechanisms. Animals with a greater tolerance to infection may still harbor a parasitic infection but may not exhibit clinical signs, and are said to be resilient. While resilient animals remain productive during a parasitic infection, they also still maintain the number of worms on pasture infecting a flock .

Factors Affecting Host Response to Parasitic Infection
Age is the most important factor in an animal's ability to effectively combat parasitic infection. The immune system matures with age; hence young lambs harbor the most severe H. contortus infections and account for the largest financial losses among producers as a result of reduced weight gain and death in heavily infected hosts . The effects of haemonchosis are also more pronounced in pregnant or lactating, undernourished, diseased or stressed animals due to a compromised or underdeveloped immune system (Kaplan, 2006).
Genetic factors also play a role in an animal's resistance to GIN. Several tropical and subtropical breeds of sheep are documented to have more natural resistance to parasitic infection. Resistant breeds include St. Croix (Zajac et al., 1990), Florida Native , and Gulf Coast Native . Resistance in these breeds is defined as reduced establishment of larvae and subsequent development into adult stages . The nutritional status of an animal is also a factor in the ability to respond to GIN infection.

Parasite and Nutrition Interaction
Nutritional status is a key component of the immune response of the host and the host's subsequent ability to mount an effective defense against gastrointestinal nematodes (Knox et. al., 2003). It is well documented that animals on diets deficient in protein or key vitamins have a decreased capacity to withstand parasitic pathogenesis, and concomitantly gastrointestinal nematodes significantly inhibit the host's ability to efficiently utilize nutrients (Knox et al., 2003). Lambs fed a diet high in protein harbor less severe parasitic infections due to an increased immune response .
Studies have shown that supplementation with protein (Kahn et al., 2003;, energy , and trace elements  improves host resistance and resilience to parasitic infection. Few studies have examined the role of vitamins in parasitic infection.

Structure and Source
Vitamin E is the general name for eight individual tocopherol and tocotrienol isomers naturally occurring in green leafy vegetables, nuts, wheat germ, and seeds . Based on the position and number of methyl groups attached to the aromatic ring structure, both isomers have the four forms α, β, γ, and δ (Stocker, 2004). The most biologically active form of vitamin E found in nature is αtocopherol .

Vitamin E and Immunity
Vitamin E is a fat-soluble vitamin best known for its role as an antioxidant (Combs, 1990). In vitro studies have documented the role of vitamin E in enhancing membrane stability and cell communication , reduction of oxidative stress (Lee and Wan, 2002), and the proliferation of T cells through the upregulation of the cytokine interleukin 2 (IL-2) .
Past studies provide evidence that the Th1/Th2 balance of an animal may be shifted to the Th1 type response during vitamin E supplementation. In studies of diseased and aging humans and mice, vitamin E has been shown to up-regulate Th1 associated cytokines and decrease the Th2 associated cytokines. The same studies provided evidence that oral supplementation with vitamin E increased the production of IL-2 and decreased IL-4 production, a key Th2 cytokine . Vitamin E has been shown to inhibit inflammatory cytokine responses but its effect on the Th1 or Th2 balance is undetermined in the young, immune system of lambs .

Vitamin E and Ruminant Immunity
Research investigating the role of vitamin E in regulating the immune system of ruminants has yielded conflicting results. Studies in livestock have linked supplementation with vitamin E to increased antibody secretion and improved immune responses in ruminants . Supplementation of vitamin E during the last month of gestation resulted in a 5-20% decrease in lamb mortality . When ewes were supplemented with vitamin E, their lambs were shown to be born with greater IgG levels (Gentry et al., 1992), and calves supplemented with vitamin E also had higher levels of IgG in addition to higher IgM as compared to control animals . Despite these documented effects of vitamin E on immunoglobulin synthesis, other research on supplementation with vitamin E has yielded no effect  Recent recommendations of the National Research Council (NRC) have significantly increased previous guidelines for daily vitamin E requirements in sheep 20 times to 10 IU/kilogram of body weight daily (NRC, 2007) from 0.5 IU/kilogram of body weight daily (NRC, 1985). Recommendations were based on studies linking enhanced immune function in sheep supplemented with vitamin E ) and a study indicating previous recommendations of vitamin E did not prevent myopathy and hepatic lipidosis in lambs .

Vitamin E and Parasitic Infection
Although it is apparent that vitamin E may enhance the immune response of the host through greater immunoglobulin production as well as the production of a stronger immune response, it may actually impede the ability of the host to mount an effective immune response to GIN infection by suppressing the Th2 type response needed to combat haemonchosis. Studies in mice confirm that oral supplementation with vitamin E has a significant effect on the Th1/Th2 balance . Vitamin E supplementation enhances a Th1 type response, causing an increase in IL-2, while decreasing Th2 type cytokines, interleukin-4 and interleukin-5-those that would normally be induced during a parasitic infection . A Th2 type response required to successfully combat parasitic infection is characterized by an increase in immunoglobulins IgG and IgE, as well as the abomasal infiltration of effector cells such as globular leukocytes, mast cells, and eosinophils (Miller, 1996). Vitamin E supplementation has also been shown to decrease mast cell proliferation (Kempna et al., 2004), and important gut inflammatory response required by the host to successfully expel worms from the host in a parasitic infection (Miller, 1996). This reduction of the inflammatory response induced by vitamin E supplementation may interfere with the ability of the host to combat a parasitic infection. Data collected from this study will lay the groundwork for future studies of the mechanisms involved in vitamin E and parasitic infection

Haemonchus contortus Infection in Dorset Lambs
This thesis is written in manuscript format and includes a condensed paper written in the style and formatting of the journal Veterinary Parasitology. This manuscript is currently unpublished but is prepared for future submission to the journal of Veterinary Parasitology. Additional details on the methods used throughout the study are included in the appendices. Samples were taken weekly for determination of tissue α-tocopherol content, worm burden and histologic evaluation of the abomasum for inflammation and infiltration of eosinophils, globule leukocytes and mast cells. There was a 49% reduction in total abomasal worm burden (P = 0.002) in the VE10 group. VE10 lambs did not differ from VE5 lambs in serum or tissue α-tocopherol, FEC, PCV or histologic parameters. Further studies are necessary to elucidate the mechanism of action of vitamin E on gastrointestinal nematode infection of lambs.

Introduction
Gastrointestinal nematode parasites in sheep account for staggering economic losses to producers worldwide. The problem has been compounded by the development of parasite resistance to all classes of chemical dewormers used to control gastrointestinal parasites . The bloodfeeding nematode Haemonchus contortus, commonly called the barber pole worm, is of particular concern due to its highly pathogenic nature. Larval and adult stages of H.
contortus feed on the blood of the host, resulting in severe anemia and death in heavily infected animals , Mortensen et al., 2003. The effects of haemonchosis are most pronounced in young, pregnant or lactating, undernourished, diseased or stressed animals (Kaplan, 2006).
An effective immune response mounted during parasitic infection is characterized by a T helper type 2, or Th2-type reaction . Upon parasitic infection, antibodies, lymphocytes, eosinophils, globule leucocytes and mast cells act upon larval stages of H. contortus embedded within the mucosa and adult worms inhabiting the lumen of the gastrointestinal tract . Effector cells, including mast cells, degranulate and release vasoactive molecules, acting to physically expel worms from the abomasal mucosa .
Nutritional status has well documented effects on the host's ability to combat heavy parasitic infestation, and concomitantly gastrointestinal nematodes significantly inhibit the host's ability to efficiently utilize nutrients (Knox et al., 2003). Animals on diets deficient in protein or trace nutrients have a decreased capacity to withstand parasitic pathogenesis, while lambs fed a diet high in protein harbor less severe parasitic infections due to an increased immune response . Studies have shown that supplementation with protein (Kahn et al., 2003;, energy , and trace elements  improve host resistance and resilience to parasitic infection. Vitamin E is a fat-soluble vitamin best known for its role as an antioxidant (Combs, 1990). In vitro studies have documented the role of vitamin E in enhancing membrane stability and cell communication , reduction of oxidative stress (Lee and Wan, 2002), and the proliferation of T cells through the up regulation of the cytokine interleukin 2 (IL-2)  .

Past studies in ruminants supplemented with vitamin E have resulted in improved
immune responses and performance in sheep and lambs, as well as decreases in lamb mortality in supplemented pregnant ewes Turner, 1996, Kott et al., 1998).
Numerous studies in ruminants provide evidence that vitamin E may enhance ruminant immune response through increased antibody production (Tengerdy et al., 1983), and higher levels of immunoglobulins G and M . No studies have examined the effect vitamin E supplementation may have on parasitic infections in lambs.

Studies in mice and humans have demonstrated the ability of vitamin E to promote
a Th1 type response through increased production of interleukin 2 and interferon-γ, Th1 associated cytokines, during a challenge with influenza . Other studies suggest that vitamin E may simultaneously dampen a Th2 response, as evidenced by a reduction in Th2 cytokines interleukins 4 and 5 in mice challenged with asthma (Okamoto et al., 2006), decrease IgE levels in mice challenged with an allergen (Bando et al., 2003), and the inhibit mast cell proliferation (Kempna et al., 2004).
Vitamin E supplementation has also been shown to decrease mast cell proliferation (Kempna et al., 2004), an important gut inflammatory response required by the host to successfully expel worms from the host in a parasitic infection (Miller, 1996). A study by  evaluating antioxidant status in goat kids and lambs found that goat kids had consistently higher plasma antioxidant levels than lambs as well as significantly higher total worm burdens than lambs. The higher antioxidant status in this study was correlated with decreased resilience to infection .
Supplementation of vitamin E and selenium to mice challenged with T. gondii was resulted in higher numbers of tissue cysts and increased severity of meningoencephalitis (McCarthy et al., 2003). These findings suggest vitamin E may inhibit an animals' ability to effectively respond to parasitic infection.
The most recent recommendations of the National Research Council (NRC) significantly increased daily vitamin E requirements in sheep from 0.5 IU/kg body weight (BW) (NRC, 1985) to 10 IU/kg BW (NRC, 2007) for optimal immune function. The current NRC recommendation (2007) was based on studies indicating that the previous dietary recommendation for vitamin E intake did not prevent myopathy and hepatic lipidosis in lambs  in addition to studies showing enhanced immune function in sheep supplemented with vitamin E

Study Subjects
Twenty Dorset lambs were housed at the University of Rhode Island's Peckham Farm. Lambs were weaned between 2 and 3 months of age. During the study, lambs were group fed 0.94 kg of grain/lamb/day as well as 1.36 kg hay/lamb/day for the duration of the study. This study was conducted with the approval of the Institutional Animal Care and Use Committee of the University of Rhode Island.

Experimental Design
Twenty Dorset lambs, 2-3 months of age were dewormed and housed in an environment free of parasites. Each lamb was administered a primary artificial infection of 10,000 Haemonchus contortus stage L3 infective larvae to stratify lambs according to parasite susceptibility. Three weeks post-infection, the lambs were dewormed with 0.2 mg/kg ivermectin and 1.5 mg/kg praziquantel per kg of body weight (EquiMax, Pzifer Animal Health, Eaton, PA) and levamisole hydrochloride (8 mg/kg) (Pfizer Animal Health, Eaton, PA). Lambs were stratified, using fecal egg shedding rates and sequentially assigned to two treatment groups. The male: female ratio was balanced between treatment groups.
Lambs were supplemented with vitamin E (d-α-tocopherol) for 11 weeks via a commercially available oral supplement (Emcelle, Stuart Products, Bedford, TX). The VE5 group of lambs was supplemented with the minimal amount of vitamin E recommended by the National Research Council (5.3 IU/kg body weight/day, n=10), while the VE10 treatment lamb group was supplemented with the National Research Council recommendations for optimal immune function (10 IU/kg body weight/day, n=10). The vitamin E was mixed into the grain fed to the lambs during the morning feeding and was dosed according to the body weight of the heaviest animal in each group.
At week 5 of supplementation, lambs were artificially infected with 10,000 H.
contortus L3 larvae. Body weight, feed intake, fecal egg count (FEC), packed cell volume (PCV), and serum -tocopherol levels were determined weekly. Lambs were humanely slaughtered 6 weeks post infection. Histologic samples of the abomasum were examined for inflammation and infiltration of globule leukocytes, mast cells, and eosinophils. Sections of muscle, lymph node, and liver were collected and analyzed for -tocopherol content. Abomasal contents were collected at necropsy for parasite recovery, quantification and identification of sex and stage of development.

Blood and tissue collection
Blood samples were collected via jugular venipuncture into sterile SST and EDTA vacutainer tubes (Becton Dickinson Biosciences, San José, CA) for α-tocopherol analysis and PCV determination, respectively. Serum tubes were centrifuged (946 x g, 10 min, 25° C) and serum was aliquotted and stored at -80° C until analysis for αtocopherol content. PCV was determined by the micro-hematocrit centrifuge method.
Liver, lymph node, and muscle tissue samples were collected at necropsy for αtocopherol analysis, snap frozen in liquid nitrogen, and stored in -80° C until analysis.

Feed sampling
Grain and core samples of hay fed to the lambs were sampled weekly throughout the study. Feed samples were kept at -20° C until analysis. Composite samples of hay and grain were analyzed for α-tocopherol content (Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI) as well as nutritional analysis (Dairy One Inc., Ithaca, NY).

Histology
At slaughter, sections of the cardiac, fundic, and pyloric regions of the abomasum were fixed in 10% formalin buffered saline (

Parasitology
Rectal fecal samples were collected for fecal egg count determination according to the modified McMaster technique (Whitlock, 1948). At necropsy, the abomasum was opened along its greater curvature and contents were collected. Content volume was brought up to two liters and a 10% aliquot was taken and fixed with equal volume of 10% FBS. After washing, the abomasum was incubated overnight in saline at 37º C to obtain embedded larvae. Contents were collected and fixed with equal volume 10% FBS. All worms from the 10% aliquot were counted from each sample, with total burdens estimated by extrapolation of aliquot counts. For each animal, the first 150 worms were sent to the Virginia/Maryland Regional College of Veterinary Medicine and were identified according to species, sex, and developmental stage.

Statistical Analysis
Serum α-tocopherol, FEC and PCV data were analyzed using mixed procedure in SAS with repeated measures (SAS Inc., Cary, NC). Each multivariable model included terms treatment, week, and treatment x week. Histological and parasite recovery data were compared using a Student's t test. Significance was defined as P ≤ 0.05 and results are reported as mean + SE.

Feed Analyses
Nutrient analysis of the hay and grain fed to the lambs is presented in Table 1.

α-tocopherol analysis
Serum α-tocopherol concentrations of all groups across time are shown in Figure 1.
Lambs in both VE5 and VE10 treatment groups had increased serum vitamin E levels after week 0; however, there was not a significant difference in serum alpha-tocopherol concentrations between groups. There were no differences in muscle (P = 0.62) or lymph node (P = 0.38) tissue α -tocopherol content between VE5 and VE10, however, the difference between VE5 and VE10 approached significance in the liver (P = 0.08) ( Figure 2).

Blood packed cell volume
The PCV of VE5 and VE10 did not differ (P = 0.39). The PCV decreased over time across treatment (P < 0.0001). There was a 21% decrease in blood PCV across treatment groups over the infection period ( Figure 3).

Fecal egg count
Mean FEC increased over the infection period in both treatment groups. There was a trend towards a reduced FEC in VE10 lambs versus VE5 lambs (P = 0.10, Figure 4).
There was no treatment x week effect (P = 0.44).

Parasite Recovery
Lambs were slaughtered 6 weeks post infection. The sex ratios of adult worms recovered did not differ between groups (P = 0.4). VE10 lambs had a lower abomasal worm burden than VE5 lambs (P = 0.002, Figure 5). There was a 49% reduction in total abomasal worm burden in the VE10 group as compared to VE5 lambs ( Figure 5).

Abomasal Inflammation
Abomasal tissue samples were assessed for overall levels of abomasal inflammation by a pathologist and based on a subjective scale, ranging from 0 to 4 ( Table 2).
Inflammation scores of all study lambs ranged from 2 to 2.4 ( Table 2). There were no significant differences between treatment groups in mucosal abomasal inflammation (P = 0.389).

Eosinophil and Globule Leukocyte Numbers in Abomasal Mucosa
There was a wide variation in the individual response within the VE10 lambs for all cell types evaluated. Consequently, the difference in the means for eosinophils and globule leukocytes, though greater in VE10 versus VE5, did not rise to the level of significance ( Figure 6). Mucosal mast cells were unable to be quantified.

Discussion
The objective of this study was to determine the effect of vitamin E supplementation at 5.3 or 10 IU d-α-tocopherol/kg BW/d on the progression of an experimental Haemonchus contortus infection in lambs. Abomasal worm burdens were significantly lower in VE10 lambs. This finding is also reflected in the trend observed in the fecal egg counts, as the VE10 lambs had consistently lower counts overall than VE5 lambs. The PCV slowly decreased in both treatment groups throughout the duration of the study, although there were no significant differences between the two groups. There was no difference in serum and tissue α-tocopherol concentration between VE5 and VE10 however, the difference between VE5 and VE10 in the liver approached significance.
There was no treatment effect of α-tocopherol on abomasal inflammation, but it is clear that there was some effect of vitamin E on eosinophil and globule leukocyte infiltration, as the most reactive animals were found in the VE10 group ( Figure 1, Figure 6, Table 2).

Fecal egg counts have been shown to be an accurate indicator of parasite burdens in
sheep . Trichostrongyle type eggs were observed in fecal samples starting at 3 weeks post exposure to oral drenching with 10,000 infective L3 Haemonchus contortus larvae, consistent with the prepatent time period of this abomasal nematode . The large variation in FEC can be accounted for by the fact that parasitic infections are often over dispersed with a minority of the animals in a population shedding the majority of the eggs . As expected, the pattern of increase in FEC over time corresponded with a gradual decrease in PCV over the duration of the 6 week infection. No lambs experienced PCV falling below the acceptable range of 25-45 % for sheep .
Lambs used for this study were stratified by parasite susceptibility and then sequentially assigned to treatment groups. The Dorset sheep breed is considered to be susceptible to parasitic infection, but within-breed variation in susceptibility or resistance is well documented . Lambs experienced both a primary and secondary infection therefore the lambs may have developed some level of immunity to the parasites, resulting in less severe clinical signs than may have been seen in comparison to a single naturally acquired pasture infection .
Repeated challenge of the immune system with infective parasitic larvae leads to significant acquired immunity . It is possible that differences between groups may therefore be less dramatic as a result of both the lack of substantial establishment of adult worms and acquired resistance in the study lambs due to multiple experimental infections.
Although serum -tocopherol concentrations below 2.0 g/ml have historically been considered deficient (McMurray, 1982;Maas et al., 1984), the considerably lower values of 0.60 to 1.50 g/ml are currently considered adequate for lambs (DCPAH, Lansing, MI). The average liver -tocopherol concentrations were 72.4 and 85.4 g/g dry weight in VE5 and VE10, respectively, well above the 20-40 ug/g dry weight considered adequate (Diagnostic Center for Population and Animal Health, Lansing, MI, . There were no significant differences between the groups in serum or tissue α-tocopherol concentrations, although liver αtocopherol content within VE10 approached significance (P=0.08).
The lack of significant difference in serum α-tocopherol levels in the current study could be partially explained by the finding that after blood becomes saturated with the vitamin, it is deposited into the tissues, causing a decline in serum tocopherol levels . It is possible that the two dosages of vitamin E supplementation recommended by the NRC used in this study (        10. Remove the needle from the skin, and place a finger over the area to prevent bleeding.
11. EDTA tubes should be immediately placed in ice. SST should sit upright for 30 minutes at room temperature.
5. With a transfer pipette, pipette plasma into cryogenic vials. Cover vials with yellow caps, and label cap with the last 2 digits of the animal identification number.
6. Place samples in appropriate freezer box, and store in a -80° C freezer.
6. The sample is fixed with 200 ml 10% phosphate buffered formalin (PBF) and stored until nematode quantification and identification.
Abomasum Soak: A soak is performed to collect any larvae that may be embedded within the tissue of the abomasum.
1. Once the wash is completed, the abomasum is spread out on in a pan, covered with saline, and covered with aluminum foil.
3. The contents of the soak are collected and the abomasum is gently washed with water and collected.
4. Contents are allowed to settle for at least 2 hours.
5. In order to collect the settled contents a serological pipet is used to remove as much liquid without disturbing the bottom as possible.
6. The remaining sample is collected into 500 ml jars, 200 ml in each, and fixed with 200 ml 10 % PBF each.

Standard Operating Procedure for Measuring the Vitamin E Content of Serum by High Performance Liquid Chromatography
Standard operation procedure modified from a protocol from the Diagnostic Center for Population and Animal Health at Michigan State University.