Parasitology

Factors that encourage interruption of development

Exposure to worsening climatic factors prior to infection Infection of immunologically resistant host Competition from resident adults?

why do pastural larval numbers decline in late autumn

Fewer eggs being released by animals, due not just to developing immunity, but also because old, dying parasites inside the host are not being replaced. Fewer eggs are developing successfully to [L3]. Some [L3] are dying, or being washed into soil (cold temperatures reduce their activity and hence their chances of returning). There are a few exceptions to this pattern. For example, Nematodirus filicollis, with its slow development to [L3] occurring in the egg, may overwinter unhatched, and [L3] may then appear in relatively large numbers in spring and early summer causing disease in lambs at this time: this occurs especially in areas with short summers and cold winters e.g. Southland, Canterbury. The blood-feeding nematode Haemonchus contortus, being highly prolific and responsive to climate, may build up to large numbers over a few weeks if conditions are suitable - sometimes at unusual times of the year. It is also highly pathogenic and a relatively poor stimulator of immunity in some sheep so that disease can occur in adult animals. This parasite is mostly a problem in the warmer parts of the North Island. It is thought that Haemonchus eggs and larvae struggle to survive the NZ winter and thus the parasite needs to overwinter inside animals.

factors that favour resumption

Improving external climate Stress on animal / decline in immune status Removal of competing resident adults Inhibited larvae are often found in a tissue niche, and are physically and metabolically inactive. As a result, they are often harder to remove with anthelmintics.

Praziquantel

("Droncit" and other trade names) is a very effective anthelmintic against all cestodes of domestic animals in New Zealand. First marketed in the late 1970s it has been off-patent for a several years and many generic products exist, usually in combination with anti-nematode anthelmintics. Its mode of action is to increase Ca2+ and Na+ influx leading to instant contraction of muscles. It also causes almost immediate vacuolation of the tegument. It is the only drug with high efficacy against Echinococcus and was the corner stone to the hydatid eradication campaign. Praziquantel was used in 6-weekly dosing of rural dogs and this programme was a key component of the eradication campaign to eradicate Echinococcus and Taenia ovis - successful for the former but unsuccessful for the latter. Praziquantel is also effective against some trematodes eg. Schistosoma spp but not Fasciola. At present almost total reliance is placed on praziquantel for control of cestodes in all animals with few other options being available. As mentioned earlier, several benzimidazoles have anti-cestode activity and are effective against Moniezia, and some against Taenia spp. Interestingly, there appears to be anthelmintic resistance in Moniezia to the benzimidazoles. In addition, morantel also has some cestocidal activity but it is limited - levamisole has no cestocidal activity.

peculiar features of acquired resistance to parasites

(a) Immunity can develop very rapidly in response to infection, (particularly where migration or tissue invasion occurs) but in some cases, notably with some helminths of the gut, especially strongylid nematodes, it may require several weeks or months exposure. For example, lambs and bovine calves may take 9-10 months or more to establish reasonable secure resistance to trichostrongylid nematodes with full adult immunity not established until 15-18 months of age. Recent research shows that this is dependent on host genetics with some animals able to make an effective response at an earlier age than others but even so it still requires several months. (b) Immunity to helminths and protozoa is not an absolute, sterile resistance. Immune animals commonly harbour small numbers of parasites while being able to prevent the establishment of numbers large enough to significantly affect the host. For example, adult ruminants may have small numbers of nematodes in their guts while ingesting thousands of infective larvae each day, though the nematodes that do develop may be smaller than normal, produce fewer eggs which are less viable and survive for less time than in a susceptible animal. Nevertheless, this small infection in adults may play an important role in the epidemiology of infection under certain conditions. (c) In some cases where parasites survive in hosts that are resistant to reinfection, this is termed concomitant immunity and it is often important to the survival of the parasite (e.g. persistence of hydatid cysts in liver and lungs, Taenia hydatigena (C. tenuicollis) larvae in the peritoneal cavity, Trichinella larvae in muscle, Toxocara larvae in tissues or schistosomes in blood vessels of resistant animals). This phenomenon is also important with some protozoa and will be discussed later this year. The persistence of such parasites in resistant hosts often depends on tricks parasites play. They can interfere with immune responses or avoid their consequences by such things as disguising themselves with host proteins, mimicking host antigens or varying their antigenic configuration. (d) Immunological resistance can be depressed by malnutrition, disease and some physiological states (e.g. lactation). For example, with sheep and cattle, good immunity to gastrointestinal nematodes is a relatively fragile state and poor nutrition such as the restricted feed supply often made available over winter can be sufficient to depress this immune response. Another example is footrot which can also suppress the immune response. In adult sheep the immunity wanes around lambing and during early lactation, especially in ewes with multiple lambs. Immunity may also wane in the absence of reinfection so that animals become susceptible again. It appears that protein supply is key to maintaining an effective immune response and partitioning into the late-term foetus takes priority. (e) Immune responses to nematodes may affect them in various ways, e.g.: depressing egg production reducing egg viability stunting growth inducing morphological changes, (e.g. loss of vulvar flap) inhibition of development of parasites in the animal where they remain as "inhibited larvae" within tissues of the host eg. Toxocara L2 larvae within somatic tissues of the host and Ostertagia ostertagi early L4 larvae inhibiting within the gastric glands. rejection of developing larvae expulsion of adults. There is a sequence that is generally followed as the immune response develops against GIT nematodes. Initially the egg production is reduced, at about this time larval establishment is declining and any resulting adults are stunted by comparison. When establishment has declined to low levels rejection of adults commences. Arrested development of larvae is also accepted to be at least partly induced by developing immune responses but this is not the only cause. This whole immune development phase can occur over several months. (f) In some infections, particularly protozoa such as Babesia and Theileria, resistance only lasts as long as low level infection persists: this is termed premunity - this will be discussed in relation to protozoa later this year. (g) There are some host-parasite systems where acquired resistance does not appear to operate very effectively. Cyathostomes in horses appear to be such a case as adult horses can be heavily infected. Why this should be is not understood. In the wild, where horses range over large territories, low levels of habitat contamination are likely to occur and this may also have reduced the necessity for the host to evolve effective immunological control mechanisms. Of course, it may simply be that cyathostomes are masters of disguise and evade the immune responses of the horse. Sheep (and probably goats) do not develop acquired immunity to the liver fluke Fasciola hepatica; why is not completely understood. It is generally acknowledged that goats do not develop a very effective immune response to gastrointestinal nematodes, especially by comparison with sheep. (h) Host immune responses, especially those involving hypersensitivity, are important in the pathogenesis of disease. In many (not all) cases it is the response of the host attempting to get rid of the parasite that is responsible for the pathology and clinical signs - not the effects of the parasite per se. Examples where this is evident include gastrointestinal nematode infections where the species are mucus browsers eg Ostertagia and Trichostrongylus. It can also be seen with schistosomosis and ascarid damage to livers. We will discuss similar examples with arthropods and protozoa. This is a key phenomenon in the development of clinical parasitism. (i) Another phenomenon termed resilience occurs in a small proportion of young sheep (and possibly other animals). In this case, the animals harbour large numbers of gastrointestinal nematodes and are apparently unable to control their numbers. However they grow well and do not suffer appreciable losses of production or, in most cases, develop clinical signs (Haemonchus infections are an exception and would produce signs because their effects depend largely on blood-sucking). In effect they are ignoring the presence of the parasites and not using the physiological effort to try and reject them. What makes animals resilient is not known but presumably arises from an inability to recognise and respond to the parasite antigens or to mount the effector responses that actually reject the parasites and induce the associated pathology. It has been shown that this trait can be selected for in sheep. Curiously, it seems that, in time, these animals do acquire a satisfactory level of resistance. The fact that resilient animals do not develop disease although heavily infected is an indication of the importance of host responses in the pathogenesis of parasitic gastroenteritis.

basic determinants of this seasonal pattern

(a) young animals are usually born in the spring. (b) from birth for the first 12-15 months of life ruminants are most susceptible to parasite infections. With experience of infection over this time immunity is slowly acquired. (c) conditions for larval development and, therefore, transmission of the parasites improve from spring into the autumn; in late autumn and winter, conditions for larval development deteriorate whilst conditions for larval survival are usually good.

Survival and movement of infective larvae in the environment

- [L3] are less susceptible to desiccation than earlier stages, but are not invulnerable. Moisture is also essential to provide water films on vegetation for movement away from the faeces. - there is an optimum temperature for survival of [L3] that is close to the minimum for development, i.e. approximately 10C. - above the optimum temperature for survival, [L3] are more active, but utilise food reserves more rapidly and therefore survive less well. - below the optimum for survival, cold is thought to produce physiological damage. At freezing temperatures, the formation of ice crystals within nematode tissue is very damaging (the same process that underlies frostbite). - movement increases as temperature increases; little or no movement occurs below 10C - [L3] move randomly when warm and wherever suitable moisture films occur on herbage or in soil, etc. A proportion of the population will move on to herbage and come into a position in which they may be ingested by a grazing animal. - the majority of larvae only move a few cm away from faeces and only ascend a short distance up the sward. Some larvae may eventually be washed into the soil by rainfall.

Factors predisposing to the development of resistance on a farm

1. "Excessive" or "unnecessary" drenching. This is commonly interpreted to be drench frequency and to some extent this is true but it is far from the whole story. Not every drench given has the same impact in terms of selecting for resistance. Drenching lactating ewes has been argued to select more than a drench to lambs in early winter - potential larval development and impact on the subsequent frequency of genes for resistance in the population is different - eggs deposited in winter are less likely to develop and the few that do are proportionally small in relation to the "autumn peak" (even though it will be declining over the winter) whereas those from ewes are laid down in spring at a time when there are fewer larvae on pasture than at any other time but the chances of developmental success for these eggs are higher. This is an extreme example but illustrates that it is a complex issue. It is not a simple issue of the number of drenches given. Drench frequency needs to be considered in respect to the refugia population that is available in that animal's environment 2. Underdosing. Again this is not a simple issue. Underdosing potentially facilitates survival of partially resistant worms. This is influenced by the relative dominance or recessiveness of the trait which will determine if heterozygotes will express any level of resistance. For many species to some anthelmintics this appears to be the case. For example, ivermectin resistance in H. contortus is considered to be a dominant trait, BZ resistance in the same species is a recessive/partially recessive trait. We don't know the situation for many anthelmintic/nematode species combinations but in general it is assumed that most will be recessive traits when the drench is given at the recommended dose rate. It is also influenced by the fact that not all nematodes are killed at the same dose rate. For every anthelmintic there are dose-limiting species which define the dose used (=label dose rate). Manufacturers determine the label dose rate as that required to kill the "hardest to kill" species they wish to make a claim for. However, there are usually other species that are easy to kill and these are thus routinely overdosed, often by 10+X that required to kill them. The importance of underdosing will probably vary for these different situations. 3. Accidental introduction of animals infected with resistant worms. This is an under-rated cause of resistance on farms and with the appearance of ivermectin-resistance in several species it is important farmers do not import their problem "on the back of a truck". 4. Inappropriate drenching. Included as a catch-all category and will become clearer when later lectures on control of parasites are considered. It includes practices that will increase the selection pressure for anthelmintic resistance. "Drench and Move": Drenching stock and moving them onto pasture that has few larvae present will mean that the majority of larvae on the "clean" pasture are only comprised of survivors of the drench. There may be few of them but the frequency of resistance genes in this population will be high. In time their numbers will increase. "Drench within the prepatent period": If stock are drenched repeatedly within the prepatent period then the only contamination of the pasture is from survivors of the drench. Consider the "selection pressure" that would be associated with repeated use of anthelmintics with a persistent activity such as moxidectin or slow release capsules.

How can dogs and cats become infected?

1. Ingestion of developed eggs containing the L3. 2. Migration of larvae (L3) across the placenta (dogs especially). 3. Ingestion of larvae in mother's milk (important for cats). 4. Ingestion of the tissues of a paratenic host such as a mouse. 5. Ingestion by mother of larvae voided by her young. NB only the first 4 are relevant to puppies, and 1, 3 and 4 for kittens. 5 relates primarily to nursing female dogs or cats. Hepatic-tracheal migration is required for 1 and 2, but may not be necessary for 3-5. This may be because these involve larvae that have already migrated at least once, but this is likely an oversimplification. Some studies have shown that larvae can migrate at least twice. Nevertheless, larvae that do not migrate may be spared some exposure to the immune system and this may favour them reaching maturity. Young puppies infected in utero can start releasing eggs when they are just over 3 weeks old and this can result in very heavily contaminated environments. Many pups will be much older before they are given any kind of anthelmintic, if they receive any at all. Imagine the potential for environmental contamination in puppy farms for example. Environmental contamination with Toxocara eggs is also important from the human health perspective. If humans ingest Toxocara eggs then the parasite attempts to migrate as it would in paratenic hosts, undergoing somatic migration. Whilst in most instances there may be no effects apparent, a minority of cases result in overt ill health. This, in human medicine, is termed visceral larval migrans. The worst effects occur when humans (typically children) ingest large numbers of eggs, or when larvae invade more sensitive tissues such as the CNS, sometimes the eye - see later lecture for more detail on VLM and its ocular form.

The behaviour of anthelmintics in the animal

1. Most modern anthelmintics are generally absorbed into the blood stream and secreted back into the gut before they affect parasites in the gut. Obviously they must be absorbed to affect parasites in other sites. Different sections of the gastrointestinal tract may vary as to how easily an anthelmintic is resecreted into that section. For example, the stomach/abomasum versus the small intestine. 2. The drugs are metabolised in the liver, commonly oxidised and conjugated before excretion. A variety of metabolites can be produced; these may have anthelmintic activity and in some cases the drug is only active after it has been metabolised (e.g. febantel). For some enterohepatic recycling can occur. For some anthelmintics urinary excretion is the main route and for others they are almost all excreted in faeces. 3. The rate of absorption, distribution, metabolism and excretion varies widely between drugs and depends on ionisation characteristics, lipid solubility and molecular stability. Peak activity can occur within minutes of administration or after several hours depending on the drug and formulation concerned. Formulation is very important in terms of achieving desired pharmacokinetic parameters. 4. Small variations in molecular structure can markedly affect activity, metabolism and toxicity. (See for example, avermectins and benzimidazoles) The rate of metabolism and excretion is important for three reasons. Firstly, the spectrum of activity and efficacy of a drug can be increased by its resistance to metabolism by the animal, as with later BZ derivatives. Secondly, it has recently been found that some animal species (e.g. goats, deer) metabolise drenches more rapidly than others (sheep, cattle) so decreasing drug efficacy - we still don't really know the most effective dose rates for deer, goats and camelids. Thirdly, persistence of drug can present potential residue problems necessitating the establishment of withholding periods, i.e. the time following use of a drug for which milk or meat may not be used for human consumption. It is important for these to be adhered to as, for many drugs, no detectable residues are tolerated and importers are liable to check shipments and reject them if residues are found. Only a small number of anthelmintics are licensed for use in lactating dairy cows.

Development (egg to [L3]) outside the host requires in Order Strongylida

1. Oxygen - usually in plentiful supply, but may be limiting at the centre of large, wet faecal pats. 2. Temperature - there is a minimum temperature of approximately 5 - 10C, below which no development occurs. - between approximately 10C and 30C development proceeds normally - the rate of development increases exponentially with increasing temperature. - at temperatures above about 30C, the rate of development continues to increase but the mortality of larvae is higher as the temperature exceeds what can be tolerated by the larva's physiological mechanisms. - thus there is an optimum temperature for development usually around 25-27C - optimum is taken to mean rapid development with a high survival rate. 3. Moisture Nematodes are essentially aquatic organisms. The larval stages, particularly the L1 and L2 are very susceptible to desiccation. Thus a lack of liquid water during larval development will lead to the inactivity and eventual death of most larvae. The initial water content of faeces varies between animals, with sheep tending to have drier pellets than is typical for cattle dung, and faecal moisture can vary depending on factors such as diet and even the effects of gastrointestinal parasites themselves (scouring). Once in the environment, rainfall and condensation (dew) will refresh faecal moisture, whilst evaporation will dry it out. In this respect, atmospheric humidity levels have no direct impact on larval development, but they do influence how quickly faeces will dry out.

Summary

1. Some gut parasites may physically damage the gut causing inflammatory reactions, haemorrhage etc. 2. Non-physical effects are much more important in the pathogenesis of disease and, in particular, the response of the host. 3. Competition for nutrients is of minimal significance in all but a few exceptions. 4. Effects on the structure and physiology of the host's gut and immunological responses are of major importance resulting in protein-losing gastroenteropathy, diarrhoea, anorexia and their consequences.

Spiroindoles - Derquantel

2-deoxyparaherquamide, now known as derquantel, is a synthetic derivative of paraherquamide. The parent compound paraherquamide was isolated from a penicilium fungus in 1981 but development as an anthelmintic was halted with the discovery of toxicity in mice and dogs. One company continued with exploratory research of semi-synthetic derivatives to develop derquantel which was first described in 2002 and is apparently much safer in dogs and mice. Mode of action: It is a cholinergic antagonist and blocks transmission of impulses between nerves and muscles i.e. in a different way to BZs and MLs. Its action against cholinergic receptors raises the question about difference from LEV and the AADs. Evidence indicates it is involved with a different set of cholinergic receptors to both of these and as an antagonist works somewhat differently. Described as a mid-spectrum anthelmintic it does not readily kill all trichostrongylid nematodes of sheep. It is not fully effective against Teladorsagia (Ostertagia) for example. Hence it was launched in mid-2010 in combination with abamectin ("Startect"). Interestingly, this combination is fully effective against ML-resistant Teladorsagia and work has now demonstrated it promotes true synergistic activity to achieve this surprisingly high efficacy against ML-resistant Teladorsagia. At present this molecule is only available as an anthelmintic for sheep with future plans to develop products for other animals unknown at this stage. The safety margin for sheep is indicated as 3 which is small and approximately the same as for LEV. Be aware this safety margin varies between animals and is very low for horses where the dose rate used in sheep is sufficient to kill horses. The combination, as marketed for sheep, has a low safety margin for cattle.

Loss of appetite (anorexia)

A common effect of parasitism at all levels of the gut is decreased food intake through loss of appetite (anorexia) probably resulting from neuro-hormonal changes in gut physiology caused by the infections. Since the factors controlling appetite are only partly understood, it is not surprising that the precise mechanisms are unknown. It can be very significant to production and the ability of the animal to compensate for other effects of parasitism (see later). It has been noted that the depression in appetite in chronic sub-clinical infections can range from 15-20%. Parasitised animals often become lethargic which may be related.

Benzimidazoles (BZs)

A group of related compounds. Farmers call this action family the "White Drenches" but this is not a term to be used in this course. This name relates to their insolubility and hence their opaque appearance when formulated. Some have colour additives and are not white. Some other anthelmintics can also be white when formulated. The original compound thiabendazole was released in 1961. Since then a number of chemical modifications have produced more effective compounds. Some of the early derivatives including thiabendazole are no longer marketed - these later formulations are colloquially termed "second" and "third" generation drugs but that has no sensible meaning and is effectively a marketer's ploy. Mode of Action: BZs bind to tubulin and interfere with the polymerisation and formation of microtubules in cells. Microtubules form a component of the intracellular skeleton and are important for a number of normal cellular functions including cell division. They form the mitotic spindles during mitosis. Later BZs have been modified so their elimination from the animal is slower, hence disruption of microtubules is more pronounced, hence efficacy is generally higher. They have also been found to be effective against the protozoan Giardia by the same mechanism. Currently marketed BZs include fenbendazole, oxfendazole, mebendazole, albendazole and oxibendazole. There are also pro-benzimidazoles which are metabolised by the body to BZs. These pro-BZs include febantel, thiophonate and netobimin but only the first of these is currently on the NZ market. BZs generally have a high safety margin as they don't bind to mammalian tubulin very well. They are generally effective against most GIT nematodes, some cestodes and some trematodes. In ruminants they bind to rumen particulate matter which slowly releases the compound in the rumen but mainly release it when they pass through the remainder of the GIT. Thus a single treatment in ruminants is the usual dose as the rumen acts as a "sink" for the chemical. Efficacy can be affected by dosing fed versus starved animals - rumen emptying is slower in starved than fed animals - and is affected by closure of the oesophogeal groove. In monogastric animals it is usually necessary to give repeat treatments to achieve effective blood levels for long enough for the drug to kill many nematodes. In dogs and cats BZs are incorporated into mixtures of anthelmintics to avoid the need to repeat dose the animal. BZs are given orally. Mechanism of Resistance: this involves loss of tubulin with high affinity receptor sites for BZs, thus microtubule polymerisation can proceed normally despite the presence of the BZ (you need to understand that microtubules are the cytoskeleton within cells and is in a constant state of flux being made at one end and broken down at the other - BZs affect the "making" of microtubules and "breaking down" continues normally so the integrity of the cells eventually collapses).

Other nematodes infecting newborn animals

A variety of other nematodes utilise transplacental and transmammary means of transmitting infection from the dam to her offspring. Some can do both, but for most, the transmammary route is typically more successful/important. Examples include: Ancylostomatoidea Ancylostoma caninum Dog Rhabditida Strongyloides westeri Horse Ancylostoma caninum is a voracious blood-feeding hookworm of dogs. It prefers warmer climates and is thus uncommon/rare in New Zealand. Because of transmammary infections (supplemented by percutaneous invasion by larvae across the skin) heavy infections can arise in very young puppies. Young milk-fed animals struggle to replace lost red blood cells (milk is low in iron) and can rapidly become anaemic - such (per)acute A. caninum infections can kill puppies relatively quickly.

Immunological mechanisms - acquired immunity

All parasites are antigenically complex and are able to stimulate the same immune mechanisms as can bacteria or viruses. In many cases this is very slow to develop (see later in this lecture). Some consider that immunity should imply total protection but for parasites this is rarely the case - there are often small burdens present in "immune" hosts. For this reason many scientists refer to "resistance" to parasites rather than "immunity" but this then gets some confused with the topic of anthelmintic resistance which is unrelated. The structural components of parasites are antigenic and these are referred to as somatic antigens. Parasites (particularly helminths and arthropods) also produce excretions and secretions that are antigenic: these are referred to as "ES" (excretion/secretion) or metabolic antigens. The distinction between "somatic" and "ES" antigens is important. The host normally reacts to both kinds of antigen from a particular parasite. The reaction may involve any or all of the following : humoral antibody production (IgM, IgG) secretory antibody production (IgA) cytophilic antibody production (IgE: immediate hypersensitivity) cell-mediated immunity (delayed hypersensitivity) + other cellular mechanisms. However, these immune responses do not necessarily provide actual protection against infection. In many parasite infections, protective immune responses involve "ES" antigens so that the development of effective immunity following natural infections depends on the presence of live parasites: responses to "somatic" antigens usually provide little or no protection though there are some exceptions (see later for vaccine development).

Effects on adult animals

Although in most host-parasite systems, the host acquires a variable and often substantial measure of immunological resistance to reinfection, parasites can still affect the productivity and health of adult animals. For example: 1. In some cases effective acquired resistance does not seem to develop (e.g. cyathostomes in horses). 2. Resistance can be depressed in some physiological or disease states, e.g. in ewes during lactation; in cattle with bovine viral diarrhoea; in all species with malnutrition. 3. If animals that are effectively resistant are exposed to challenge infections, significant effects on production and production efficiency can still occur. For instance, adult resistant ewes grazed on heavily infected pastures show decreased wool growth and efficiency of energy utilisation and losses of plasma proteins into the gut although FEW or NO PARASITES DEVELOP. They may also show signs of diarrhoea. The effect is directly related to the level of challenge and represents the cost of defence. There is increasing evidence to suggest that sheep selectively bred for their ability to develop resistance to nematodes at an early age are less productive than outbred animals when faced with similar levels of infection, perhaps for the same reason - they are more efficient at diverting resources to resisting infection.

pathogenesis of parasitic gastroenteritis

Although some parasites exert their main effects directly (e.g. by blood-sucking), most of the effects on the host are attributable to the host's responses to infection. Of major importance is the development of protein-losing gastroenteropathy as this seriously affects energy metabolism, nitrogen balance and protein synthesis. These effects may be aggravated by anorexia and diarrhoea. The latter is reflected in abnormalities of fluid balance and may be further complicated by hypoalbuminaemia decreasing blood colloid osmotic pressure. In some infections, blood-loss anaemias develop which not only decrease the oxygen carrying capacity of the blood, but also increase the losses of protein. Even in adult animals, expression of an immune response to significant challenge infection entails protein loss and energy cost that can have measurable effects on production. Despite the availability and widespread use of good anthelmintics, parasitism remains the most important production-limiting disease in farm animals. In extreme conditions of uncontrolled parasitism in sheep, mortalities of up to 30-40% may occur and decreased growth of survivors of up to 14 kg bodyweight over a 3-4 month period. The return on investment for the effective use of anthelmintics and other parasite control measures is immense. Without parasite control, whether by drenching or management, it would be impossible to raise animals under the intensive farming systems currently practised.

Mast Cells

An increased number of mucosal mast cells are a common sequel to gastrointestinal nematodiasis. Such mast cells can release a variety of mediators including leukotrienes, prostaglandins, platelet activating factor, 5-hydroxytryptamine and various proteases. Some of these are considered as being important in the rejection of gastrointestinal nematodes. Some of these mediators are still being defined. Release of these mediators will follow cross-linking of bound IgE and IgG on the surface. Such cross-linking will be specific to certain antigens i.e. ES product from 1 specific nematode, but the mediators are likely to be effective against any nematodes. Thus it seems likely that the actual rejection of nematodes in the gut may depend on the release of these highly active biochemicals by binding of specific antibody to cells such as mast cells and then the cross-linking of these by specific antigens. Remember that release may be specific but these biochemicals act nonspecifically. So their release may be prompted by Trichostrongylus in the small intestine but they will also kill other trichostrongylid nematodes in that area such as Cooperia or Nematodirus. A subset of mucosal mast cells are the Globule Leukocytes. Their specific role is still unknown but they are located between the epithelial cells and they are very numerous in animals that are responding to the presence to parasites. Their presence is a useful indicator that parasites are present. Normal mast cells are located in the lamina propria beneath the basement membrane which separates the epithelial cells and the lamina propria. It is common for pathologists examining tissues from an animal to report large numbers of Globule Leukocytes and relate this to chronic parasitism. Curiously, even after considerable research over many years we can not yet clearly define the specific mechanisms that a host will use to reject a parasite burden.

Avoid underdosing

Animals should receive at least the recommended dose rate of drug: it is better to overdose rather than underdose though care must be taken with drugs which have narrow safety margins: it is critically important to read the instructions to make sure the correct volume of drench is given for the weight of the animal. - animals should preferably be weighed and dosed accordingly. - where only a sample of a mob is weighed, they should be dosed according to the maximum weight, not the average. - where scales are not available, it is important to ensure that estimates of body weights are as realistic and accurate as possible: weighbands can be used for some species. - both before and during drenching, checks should be made to see that drench guns are delivering the correct volume. - drenches should not be mixed (to produce home-made combinations) unless designed for the purpose, or minerals etc. added which may interfere with drench effectiveness Underdosing is also likely to be important where resistance genes are present in a heterozygote and where the gene is recessive. i.e. a standard dose of anthelmintic is effective but a reduced dose may convert the gene into a dominant character and allow these heterozygote worms to survive

Current Situation in New Zealand

Anthelmintic resistance is widespread in NZ in sheep, goats, cattle and deer. First reported in 1980, the prevalence has increased exponentially since then. Most reports in sheep initially involved BZs, particularly to Nematodirus spathiger, but since then all trichostrongyloids and strongyloids have been involved. In the last few years there have been reports of levamisole resistance and more recently multiple resistance to the combination of levamisole and BZs. The first case of ML resistance was in sheep in the 1990s but they are now also common in sheep, cattle, goats and deer. The latest prevalence figures will be given in the lecture. ML resistance has been seen in Teladorsagia (Ostertagia) circumcincta in goats and sheep for several years. However, a 2004/5 survey showed the prevalence was about 30+% of sheep farms so is likely to be higher now. Cooperia curticei is now also being reported as being ML resistant from a number of sheep properties. So far the only formal reports of Trichostrongylus colubriformis resistant to MLs have been made from goats where a recent survey indicated they were very common. Some anecdotal reports are now starting to be made of recovering these same ML-resistant isolates from sheep but none yet formally confirmed. There is one confirmed report of Haemonchus contortus showing resistance to ivermectin but there are obviously likely to be more - such cases are very common in Australia and South Africa. So far the other trichostrongylids have not been involved in ML resistance. In cattle there are an increasing number of reports involving ivermectin resistance in Cooperia oncophora and BZ resistance in this and other trichostrongylids. To date BZ resistance in O. ostertagi is reasonably common but reports of resistance to MLs in this species are still minimal - there have only been 2-3 cases confirmed - such resistance is hard to detect and there are likely to be more and this will invariably increase and become particularly problematic for nematode control in young cattle. Fortunately levamisole continues to be effective against C. oncophora and this has provided relief for the last several years. Unfortunately levamisole has never been particularly effective against O. ostertagi and has always struggled to get better than a 95% kill rate so it doesn't rate as a particularly good fall-back position for this species. Collectively the situation for cattle is looking severe as we expect ML resistance in O. ostertagi to become more common. There was a national survey for anthelmintic resistance in sheep or cattle which was conducted in 2004/5 (=10 years ago) and overall the results indicated about 60-70% of sheep farms had anthelmintic resistance to BZs and LEV and about 30% to MLs. For cattle >90% of farms had evidence of ML resistance in Cooperia. In the 10 years since we know there are more cases being seen but no formal surveys to record the increasing prevalence. In horses, anthelmintic resistance is also believed to be very common in cyathostomes to BZs although no formal surveys have been published. To date there is no overt evidence of ML resistance in cyathostomes but it has been reported overseas. There is also evidence of ML resistance in Parascaris equorum and Oxyuris equi.

Ivermectin vs Moxidectin vs Abamectin

As members of the ML group they share the same mode of action. However there are pharmacological and pharmacokinetic differences between them. Moxidectin is extremely lipophilic and is immediately sequestered into the body's fat depots from where it re-emerges over a period of time. When given orally it operates as a two compartment model - one is the body's lipid depots and the other is the remainder of the body, especially the bloodstream. As the level in the blood is depleted it is reinforced by chemical leaving the lipid depots. This results in a long "tail" of moxidectin in the bloodstream (and hence mucosa) of the animal. The doselimiting species for which the label dose is set are killed but for those that are easy to kill the declining blood levels continue to kill for a considerable period of time. In sheep, for Teladorsagia (=Ostertagia), Haemonchus and Dictyocaulus efficacy is maintained for 2+ weeks and this is reflected in a claim for persistent activity for moxidectin against these species on the label. One injectable formulation of moxidectin in sheep is specifically formulated to leave a long-persistence depot around the oily (=lipid) content of the injection at the injection site as well as then distribute around the body - this gives up to 100 days control against "easy-to-kill" species. These is considerable debate about the effect this declining tail has on selection for anthelmintic resistance as partly resistant parasites are exposed to declining levels of anthelmintic and can establish in the animal much earlier than fully susceptible parasites. This will give them a "headstart" in reparasitising the animal and partly resistant nematodes will then breed with each other with some of these progeny being even more resistant. An interesting situation has developed with these products. Isolates of T. circumcincta and H. contortus have been noted where there is obvious ivermectin resistance but moxidectin continues to kill them. This reflects a different feature of moxidectin. When the original dose rate is set it is designed to kill the "hardest-to-kill" species which usually means that other easy-to-kill" species are getting exposed to more drug than needed. This is the case for moxidectin - at the recommended dose rate moxidectin is more potent against some species = it requires less in terms of mg/kg dose to kill these two species than does ivermectin, hence resistance can be expressed to ivermectin but not yet to moxidectin as even though the LD95 for moxidectin has increased it is still below the recommended dose rate whereas for ivermectin the LD95 is above the recommended dose rate and resistance is expressed. It does invariably mean that even though the worms in the animal at that time may be killed by moxidectin, the persistent activity for moxidectin against these two species is substantially reduced or even absent. Note that the recommended dose is set to kill the hardest to kill species and there doesn't appear to be a fundamental difference in activity between moxidectin and ivermectin against these dose-limiting species. Abamectin is generally more potent than ivermectin against these easy-to-kill species but slightly less so than moxidectin. Abamectin behaves like ivermectin in terms of not having a persistent effect.

Significance of protein-losing gastroenteropathy (PLGE).

As noted earlier, this result from increased mucosal cell turnover rates, increased mucus, leakage of plasma proteins (equivalent to 20-125g protein/day), increased IgA secretion and, with parasites like Haemonchus, from blood-loss (as much as 10% of circulating blood volume/day). What happens to this "lost" protein depends on the level at which the loss occurs. (Of course, most naturally acquired infections of lambs, for example, comprise many species and involve the abomasum, upper small intestine and large intestine). Estimates suggest 70-85% of the host protein lost into the stomach, or small intestine can be digested and reabsorbed in the small intestine (most protein digestion and absorption occurs in the distal small intestine which is further down than most nematodes live). The total loss of protein into the gut which isn't recovered in the lower ileum amounts to several grams of nitrogen per day. In experimental, subclinical, single species infections 4-6 gm N/day were recorded as leaving the terminal ileum. Any of this protein entering the large intestine or lost into the lumen at that level cannot be reabsorbed; it can be: • broken down by bacteria to ammonia which is absorbed and excreted as urea by the host • lost in the faeces as bacterial protein or passed out unchanged. Decreased serum albumin levels initially cause the liver to increase production to compensate but, as parasitism continues, the animal switches a high proportion of its protein synthesis to the gut mucosa to cope with increased cell turnover. It has been estimated that the alimentary tract accounts for about 5% of total body protein but contributes 25-45% of body protein synthesis. An increased requirement for protein synthesis in the gut can markedly influence the body's protein synthesis diverting it away from muscle etc. It can shift this equilibrium markedly to the right. Availability of amino acids for metabolism of/for peripheral tissues can be reduced by as much as 30%. The consequences of these changes are very important to the host. 1. The digestion of "lost" protein and increased synthesis of protein n in the gut wall require large amounts of metabolic energy. This is thought to be the major contribution to decreased efficiency of ME utilisation which is a very serious effect of parasitism (see experimental data attached). Note that parasitised animals require much larger intakes of nutrients than parasite-free animals for equivalent amounts of growth and production. 2. The diversion of protein synthesis to the gut and attempt to compensate for losses in faeces and urine leaves fewer amino acids available for producing structural proteins - muscle, bone, wool etc. (Skeletal growth may also be affected by impaired phosphorus absorption in some infections). Depending on protein supply this may require loss of muscle and other tissue protein to provide for the GIT requirements. 3. Decreased plasma albumin decreases colloid osmotic pressure of the blood which may result in the formation of transudates and oedema (see diarrhoea). Increased globulin synthesis in response to continual antigenic stimulation also diverts amino acids away from structural proteins.

SLOW-RELEASE DEVICES for cattle

At present there are none available. One which released ivermectin but used a different type of capsule than the one used in sheep was available but withdrawn in 2001 due to marketing difficulties. It was driven by an osmotic pump and delivered ivermectin for 135 days. It proved too expensive to use. There are other slow release devices overseas for oxfendazole and morantel but these have not been released in New Zealand, mainly because of cost.

Significance of anaemia

Broadly speaking, anaemias associated with parasites fall into two categories - those caused by blood loss and those that are not. Blood loss anaemias result from blood-sucking parasites (e.g. Haemonchus, Ancylostoma) or haemorrhage from ulcers (e.g. hyostrongylosis). These are regenerative anaemias because the bone marrow becomes hyperactive. To start with, normocytic normochromic anaemia is seen but continuing blood loss is likely to lead to iron deficiency and a microcytic, hypochromic anaemia. Loss of blood involves loss of plasma and cell proteins additional to other losses from PLGE. Some of these proteins may be digested at some energy cost. A shortage of amino acids for protein synthesis may impair bone marrow activity. Chronic infections with parasites that do not suck blood or cause haemorrhage (e.g. Ostertagia, Trichostrongylus) are sometimes associated with anaemia caused by decreased bone marrow activity because of decreased amino acid availability. These anaemias are non-regenerative. Blood loss anaemias are potentially serious and important. Anaemia is the principal cause of death in haemonchosis and the clinical signs shown by non-fatal cases. The non-regenerative anaemias are generally only moderate in degree and are not life-threatening.

parasites in ruminants

Calves and lambs are born parasite-free. As they start to graze they ingest small numbers of larvae; these larvae may have overwintered on pasture from the previous autumn and in the late spring and early summer (particularly in sheep) may be supplemented by newly developed larvae derived from eggs deposited on pasture by adult animals. This early infection is generally of little significance to the young animal's health and growth, but further eggs will be dropped onto the pasture. Larvae can develop relatively quickly from these eggs because temperatures are rising, however, some of these larvae may die from desiccation because it is often dry in the latter half of the summer. Conditions for larvae improve dramatically when the wet weather returns in autumn, when it is still warm enough for rapid larval development and populations of larvae on pasture can rise very rapidly at this time of year. These larvae are largely derived from eggs dropped onto pasture by the young animals themselves. The prospects for larval survival are also improving in late summer to autumn - as conditions become cooler. The autumn rise in larval numbers occurs at a time when nutritional requirements of young animals are still rising while the amount and quality of feed available is usually declining. It is because these two factors - high larval numbers and declining plane of nutrition - occur together that most of the disease and poor growth caused by trichostrongyloid nematodes occurs between late summer and mid-winter in the first year of life.

Physical damage

Damage to the mucosa that destroys its continuity will lead to an inflammatory response which may be aggravated by bacteria or other noxious materials being able to reach the lamina propria. Ulceration of the mucosa may follow. The inflammation involves vasodilation and loss of proteins into tissues and the gut lumen. If blood vessels are damaged, haemorrhage will result. Such damage occurs with "plug-feeding" strongylids that browse on the mucosa. The extent of the damage is thus related, in part, to the size of the buccal capsule. For example, Chabertia ovina, with its large buccal capsule is relatively more pathogenic than Oesophagostomum venulosum which has a much smaller buccal capsule. In a slightly different way Triodontophorus (horse colon) commonly feed in tight "nests" of several nematodes and combined together they cause ulceration. Blood-sucking parasites such as hookworms and Haemonchus physically attack the mucosa in order to obtain blood. Attachment by cestodes and acanthocephalans also causes physical trauma which may lead to abscessation or ulceration. Large parasites such as ascarids may physically partially obstruct the gut lumen; on rare occasions the gut may be completely obstructed or ruptured causing death. They can also rarely enter the bile duct and totally obstruct that.

Significance of diarrhoea

Diarrhoea results from the osmotic effect of electrolytes lost into the gut resulting in water retention in the gut and very probably the release of inflammatory mediators by mast cells (and possibly others) in the lamina propria which stimulate smooth muscle such that passage of material through the intestinal tract is reduced in terms of time. Abnormal loss of water in faeces causes the animal to conserve water by reducing urinary flow and body secretions and to increase intake by drinking more. In ruminants, water intake is related to dry matter intake so that anorectous animals may not drink as much as they need to compensate for losses, worsening the situation. When loss of water exceeds supply, the animal becomes clinically dehydrated. The first signs are: • loss of skin elasticity - because to maintain circulating fluid volume, water moves from the extravascular extracellular compartment into the circulation. • dry mucous membranes - to reduce losses, secretion is reduced. If loss continues, water passes from the circulation to maintain intracellular water levels. This leads to haemoconcentration. Remember that haemoconcentration may obscure anaemia. If hypoalbuminaemia is present, decreased blood colloid osmotic pressure leads to oedema and transudate formation and further haemoconcentration. Thus a severely dehydrated hypoproteinaemic animal shows: loss of skin elasticity, dry mucous membranes, subcutaneous oedema, transudates in body cavities and haemoconcentration. These signs are seen in the terminal stages of severe parasitic gastroenteritis.

Anti-Cestode Drugs

Drugs effective against Echinococcus (=hydatids) and safe to use have been difficult to produce. Some cestodes such as Moniezia spp. are much more easily killed than taeniids; even oral copper sulphate is highly effective and cupric acetoarsenite, which is more effective and safer, was once commonly used against Moniezia in lambs. Again this indicates a difference in metabolism between Moniezia and the taeniids. One of the earliest drugs used against cestodes was arecoline, originally obtained from the betel nut (Areca catecha) but now made synthetically. It is a purgative, was not very effective at removing scolices, mainly resulted in loss of the strobila and is no longer used. It was the purgative used during the period of dog testing in association with the eradication of Echinococcus from New Zealand.

Vaccines

Failure of somatic antigens to induce a protective immune response has been a major obstacle to the production of vaccines against parasites. Until recently the only successful ones make use of attenuated live parasites (e.g. X-irradiated larvae) or collected "ES" antigens from in vitro culture of parasites. The development of molecular biology techniques enabling the identification and production of protective antigenic molecules presents new possibilities and they are being applied to a wide variety of parasites. Some progress is being made towards the production of effective vaccines with a few parasites - one for use in sheep against the cestode Taenia ovis infection and another for the cestode Echinococcus have been developed (although neither has yet been marketed). It has been shown that it is possible to develop a vaccine for Haemonchus based on antigens from the intestinal cells of the parasite - there are a small number of such antigens that have been investigated by different research groups. These are proteins the host does not normally come into contact with and so are termed a "novel antigen" or "hidden antigen". One is from an antigen from the intestinal pilae. With this vaccine it is thought that ingested antibody bind to these pilae and interfere with intestinal function to the extent that the nematode dies. It is thought that such antigens may be the key to producing other vaccines for gastrointestinal nematodes. Experimentally, this antigen has been shown to act as a vaccine and produce quite high levels of protection in sheep. A commercial vaccine for H. contortus using this antigen is being developed and is likely to be available within 2-3 years but its usefulness in New Zealand is debatable. The main problem has been to find a system to express the antigen with the appropriate tertiary structure to retain its antigenic properties. Several other novel antigens to protect against H. contortus but targeting different hidden antigens are being developed and some are apparently showing some effect against other trichostrongylid nematodes as well. None of these are as developed as the intestinal pilae vaccine mentioned above. The only "novel antigen" vaccine so far marketed was for ticks and was marketed in Australia but is no longer produced as it was not a commercial success. Thus, despite the above, molecular biology has not provided the rapid solutions that some expected. For example, despite the involvement of numerous research teams around the world and the expenditure of hundreds of millions of dollars, attempts to produce an effective vaccine for malaria have so far failed. The same is generally true for gastrointestinal nematodes of ruminants other than Haemonchus. Research teams in Australasia have been attempting to develop vaccines for Trichostrongylus for many years and despite promises none have appeared as yet. The reasons for such failures are not fully understood. Vaccines against mucosal browsers such as Trichostrongylus, which lie in contact with the mucus layer on the mucosa, are proving more difficult to develop vaccines against, especially those with novel antigens. With Haemonchus the parasite sucks blood and thus will ingest a large number of circulating antibodies. However, mucosal browsers will not ingest this circulating antibody, only those such as IgA that are found within the gastrointestinal tract.

Check drench efficacy

Farmers are recommended to check their drench is working at least once a year. This could be just checking egg counts after drenching within the prepatent period or a more formal Faeceal Egg Count Reduction Test - the latter is more reliable but the former will give some crude indications. In itself this testing won't delay the emergence of resistance but will make the farmer aware of resistance emerging and allow him to make changes. It generally has also been found to promote more rational use of anthelmintics.

Levamisole (this group is also referred to as the Imidothiazoles)

First released in the early 1960s as tetramisole it was found that most of the anthelmintic activity is in the laevo-isomer (little in the dextro-isomer) and this is now the only available formulation marketed and is called levamisole (LEV). It is a broad-spectrum against most GIT and respiratory nematodes but not cestodes or trematodes. It can exist as different salts. For oral dosing it is usually as the HCl salt but for injection it is normally as a phosphate salt. It is also formulated as a pour-on for cattle either alone or in combination with abamectin. At present in the NZ market only the HCl salt is available. Mode of Action: They act as cholinergic agonists of nematodes paralysis. These affectdifferent types of cholinergic receptors to those activated by the AADs or antagonized by derquantel - see below. Nematodes have a range of different cholinergic receptors and LEV will bind with just a limited range of these but in most nematodes this is sufficient to kill the nematode. Mechanism of Resistance: this is associated with a decrease in susceptible ACh receptors with their replacement by insusceptible ACh receptors but the actual mechanism is poorly understood. The safety margin is small by modern anthelmintic standards and care should always be used to avoid gross overdosing - LEV will bind to mammalian cholinergic receptors. For example, it is not used in horses because the margin between therapeutic and toxic doses is too close. In sheep this ratio is quoted between 3-7 fold.

Other egg-infective nematodes

For several other nematode taxa the infective stage develops inside the egg, which will not hatch until ingested, e.g.: Oxyurida Oxyuris equi DH: horse, Stage in infective egg: L2 (possibly L3) in egg Enoplida Trichuris vulpis dog L1 in egg The pinworm of horses Oxyuris equi has an unusual lifestyle. The large (up to 10cm) females with long tapering tails, accumulate eggs within their uteri rather than shedding them gradually. When eventually ready to lay her eggs, the female protrudes her anterior end (including her vulva) out of the anus of the horse and smears the eggs on the perineal skin in a large sticky mass. The eggs can persist on the skin for the short time it takes for them to become infective (in as little as a 3-5 days). Eventually the egg mass dries and many eggs fall off into the environment. The presence of the egg mass can be associated with anal pruritus (itchiness) in infected horses and this facilitates spread of infection since horses may rub themselves on e.g. fenceposts leaving behind a few eggs which then get licked up by another horse. Infection can also be transmitted by grooming equipment. In marked contrast to T. canis, the canine whipworm Trichuris vulpis is seldom found in young puppies. The PPP is 10 weeks hence the much lower chance of finding eggs in the faeces of very young puppies. Whipworm infections are seen sporadically in dogs of diverse age groups which suggests two things: The parasite does not provoke a good immune response (despite what some textbooks state) favouring the development of chronic infections, and: when faeces is left contaminating the environment dogs can re-infect themselves and infections build over time.

Immunological mechanisms - acquired immunity

Helminth parasites are antigenically complex and are able to stimulate immune mechanisms. In many cases effective immunity is variable or is very slow to develop and total protection (sterile immunity) is rarely achieved - there are often small burdens present in "immune" hosts. For this reason many scientists refer to "resistance" to parasites rather than "immunity" (NB not to be confused with the topic of anthelmintic resistance which is unrelated).

Effects on the structure and function of the gut

Helminths cause changes in the histology and physiology of the gut. This has been most thoroughly investigated with nematode infections of ruminants but similar changes occur in other animals. (a) Gastric stomach - revise normal histology of gland structure. When nematode larvae enter the gland crypts, the mucosa responds by increasing its mitotic and cell turnover rates, especially in and around parasitised glands. The glands become deeper than normal in 1-2mm diameter focal areas and appear as nodules. This is termed focal hyperplasia (also called nodular hyperplasia). In the parasitised gland and those immediately around it, there is a decrease in acid secreting parietal cells and enzyme secreting cells, especially Chief cells producing pepsinogen. These are replaced by low cuboidal, poorly differentiated, cells. This is termed focal metaplasia. Remember that cells in the glands are replaced by cells moving down from the neck region and which normally differentiate as they mature. These poorly differentiated cells can still produce some pepsinogen (but no acid). This limited production combined with the increased number of such cells can maintain pepsinogen production. There is also an accumulation of inflammatory cells around these developing nodules, especially neutrophils, lymphocytes, eosinophils and plasma cells. Another common response is an increase in mucus production. When the larvae emerge from the glands, these changes often become more widespread, probably associated with the migration of adults onto the surface and thus more widespread release of ES material. With heavy infections the changes may involve most of the mucosa. The hyperplasia and metaplasia are now "generalised". Grossly, the mucosa may appear thickened and wrinkled and, especially in cattle, appearing like "Morocco leather". The changes, as described, are always seen in Ostertagia and Trichostrongylus infections of ruminants. Similar lesions occur in pigs infected with Hyostrongylus. With chronic Hyostrongylus infections and to a lesser extent with chronic Trichostrongylus axei infection these changes may also induce disruption to the epithelial layer and result in formation of ulcers. This is not a result of physical damage by the nematodes but is a result of host response. In pure Haemonchus infections in sheep the same focal changes occur but it is generally accepted that only the hyperplasia becomes generalised, the gland crypts recover their normal cell populations and hence there is less effect on normal parietal cell function. The reasons for the difference are not known. With Ostertagia and, to a lesser extent, Trichostrongylus infections (but generally not with Haemonchus - some later experiments have found it does and some earlier ones did not!), the metaplasia - which reduces the number of acid-secreting (parietal) cells - is accompanied by a rise in abomasal pH, sometimes to pH 5-6 or higher. Gastrin secretion is increased - probably partly because of the decreased acid production, partly because of the inflammation that is occurring and because of other unknown factors. Gastrin has a trophic effect on the mucosa so may be involved in the mucosal hyperplasia and increased cell turnover rates. Gastrin release may also be stimulated by parasite secretions. The change in pH decreases activation of pepsinogen (this is of minor importance in ruminants as pepsins key role is with digesting animal protein) and also allows rumen bacteria to survive and multiply - they are normally killed by the acid pH. While these changes are occurring, there is loosening of the tight-junctions between the mucosal cells so that the mucosa becomes more permeable - probably in part due to some mediators released by mast cells. The inflammation will also damage cells and the basement membrane beneath the epithelial cell layer. This has several important consequences: • It allows water and electrolytes to pass from the mucosa into the gut lumen. • It allows plasma proteins to leak into the gut lumen. This is discussed further later. • It may allow entry of bacteria and other noxious substances into the lamina propria, increasing inflammation. • It is thought to allow pepsinogen to leak into the blood stream - this is not harmful but is used diagnostically. However, some recent experiments suggest the rise in plasma pepsinogen may not be due to increased mucosal permeability per se but be caused by direct release into the lamina propria. Whatever the cause, the circulating levels of pepsinogen rise and can be detected. The osmotic effects of the electrolytes drawing water into the gut lumen together with the effects of inflammation and inflammatory mediators on smooth muscle are the cause of diarrhoea that usually occurs with Ostertagia and Trichostrongylus infections. Interestingly, with haemonchosis, where no pH change occurs, diarrhoea is not a clinical sign. (b) Small intestine (revise normal histology) The mucosal changes here are quite different but similar changes occur with virtually all helminth infections and many protozoal ones. It should be noted that trichostrongylid nematodes are generally confined to the proximal 1/3 to 1/2 of the small intestine. The most obvious changes are in the structure of villi in the parasitised region which become stunted, irregular and commonly fused. These changes are often referred to as "villus atrophy". They are accompanied by increased mitotic and cell-turnover rates as in the abomasum/true stomach. At the same time as the villi are being affected the mucosa is still undergoing hyperplasia and becoming thicker reduced villi but thicker mucosa overall. There is also an increase in the numbers of goblet cells and mucus secretion. The other epithelial cells in affected areas of the intestine have reduced metabolic activity and impaired absorptive capacity. However, in parasitic infections in ruminants, this is compensated for to a reasonable extent, by increased activity further down the tract so that there is very limited net effect on absorption (except with respect to some minerals in some circumstances). This is not necessarily so in malabsorption syndromes resulting from other causes or, perhaps, in monogastric species. There is some evidence to suggest that parasitised animals may have decreased ability to absorb minerals such as Cu, Co, P and Fe but this is not usually significant unless intake is marginal. The exception may be P where hypophosphataemia results in slower skeletal growth. The affected mucosa also becomes "leaky" because of loosening of tight-junctions (as in the abomasum/true stomach), allowing water, electrolytes and host proteins to pass into the gut lumen. The diarrhoea which usually accompanies intestinal parasitism is probably caused by these changes as outlined above and the actions of inflammatory mediators on smooth muscle (c) Large intestine This region has been comparatively less well-studied. Many large intestine parasites feed directly on the mucosa causing trauma and sometimes ulceration. There is also mucosal hyperplasia, increased mucus secretion and increased mucosal permeability with consequences as outlined above. Some strongyloid parasites of the colon induce inflammatory nodules at the L4 stage. This involves immunological responses

Host-parasite (H-P) relationships

Host-parasite (H-P) relationships are complex and dynamic. The epidemiology of parasitic infections depends on interactions between the host itself, its parasites and any intermediate or paratenic hosts involved in transmission, and the environment. For many helminth parasites, the stage that leaves the infected definitive host (usually in its faeces) is not immediately infective to new hosts. Thus animals become infected not so much by exposure to other infected animals (or their faeces), but by living in a (faecally) contaminated environment.

Develop a Worm Control Plan - Avoid "excessive" or "unnecessary" drenching.

If farmers take the time to plan their worm control plan and consider the pros and cons of each treatment then they should be making progress in avoiding unnecessary treatments. Obviously, one cannot define "excessive" or "unnecessary" in any dogmatic way as the circumstances and production objectives of individual farms differ and affect what level of parasite control is needed and what strategies are appropriate. As a general principle, however, animals should not be drenched unless there are definite production benefits to be gained and drenching should be kept to the minimum needed to meet production objectives. Grazing management, including grazing with alternative species (sheep/goats vs. cattle - NOT sheep vs. goats as they share the same parasites), should be used to reduce reliance on drenching for parasite control. However, simply reducing drenching frequency does not necessarily reduce selection pressure for resistance as grazing management is also an important factor. Some types of grazing management will increase the selection pressure, eg "Drench and Move"- this is a good policy for parasite control but a bad policy for selecting for anthelmintic resistance - the clean pasture the stock is moved onto may only be contaminated with survivors of the anthelmintic i.e. survivors can only mate with survivors. In general, routine drenching of adult animals with a fully developed immune response to nematodes should be avoided unless there is a diagnosed need to do so. Adult ruminants do require drenching from time to time so a blanket ban is not sensible but then routine treatment when it is not necessary is unwise from the perspective of selecting for anthelmintic resistance

Quarantine drench all bought in stock

If possible drench stock on the farm of origin (usually difficult) but if not then on arrival. Use anthelmintics, preferably more than one, that are likely to be effective. The principle is to use a combination of anthelmintics what will kill the "hardest to kill" parasites currently known i.e. you don't let these survive on the new farm. How to implement this strategy is not as easy as it initially seems. The problem is where to put the treated animals on arrival on the new farm? Animals should then be grazed on pasture that has high levels of larvae - any surviving eggs/larvae will be diluted. Do not quarantine drench and then graze on "clean" pasture that does not have any resident larvae as this is a recipe for rapid development of resistance. Given our knowledge of prevalence of resistance a sensible quarantine drench for sheep should be at least a 3 way combination anthelmintic or more likely one of the two new action families recently released - monepantel ("Zolvix") or derquantel+abamectin ("Startect"). Neither of these two new anthelmintics is currently licensed for use in cattle so a triple combination is the preferred choice in that host. The question can only be considered in relation to the returns that may be expected from the use of anthelmintics. It is simplest to look at this in sheep because we have most information on sheep, and on an industry basis because financial returns are well documented. It is necessary, of course, to make many assumptions and generalisations but they are not unreasonable ones.

Effects on Growing Animals

If we closely examine lambs that are taking in moderate numbers of nematode larvae over a period of weeks and compare them with uninfected animals, we would find that: 1. Growth of muscle, fat and bone is impaired. 2. Faeces are usually soft or diarrhoeic. 3. Serum albumin levels are often depressed, though globulins may be raised. 4. A degree of anaemia may be present (which will be more severe if blood-sucking parasites are involved). 5. Appetite is usually depressed. 6. The efficiency of utilisation of metabolisable energy (a measure of feed conversion efficiency) is decreased markedly. Changes such as these can be seen in animals that appear clinically normal and healthy. If our lambs are exposed to higher levels of infection which produce clinical signs of disease, the above changes become more severe so that: • decreased growth progresses to loss of weight • decreased albumin levels lead to oedema and transudate formation • anaemia may decrease activity and threaten life (if blood-sucking parasites are numerous) • food intake may be reduced to low levels • diarrhoea leads to dehydration. Having described the effects of parasites on gut physiology and the immune responses of the host, we already have some clues as to the causes of the changes we can observe in the animals. These will now be examined in more detail.

Toxocara canis and Toxocara cati in dogs and cats

In addition to the traditional means of transmission (ingestion of infective eggs) the ascarids of dogs and cats have found a particular way of ensuring infection of very young permissive hosts. They have done this by utilising the dog (T. canis) or cat (T. cati) not just as a definitive host, but also as a paratenic host. As ascarids, these nematodes typically need to undergo hepatic-tracheal migration following egg ingestion to establish patency, but do so best in the first few weeks of an animal's life. When older dogs and cats ingest infective eggs, the L3 emerge from the eggs and commence their migration, moving at first through the liver as per normal, but once they reach the lungs they are less able to break out into the airways to ascend the trachea and instead they may continue in the bloodstream to eventually reach a variety of somatic tissues (hence somatic migration). Here they attempt to hide encysted in the tissues, and although some are likely killed, some nevertheless survive and in potentially large numbers. The dog or cat is now capable of acting as a paratenic host. A similar thing happens in other animals such as mice that can also act as paratenic hosts. When a mouse harbouring encysted T. canis or T. cati larvae is eaten by a dog or a cat, the larvae may then be able to establish and reach patency (assuming the right carnivore ate the mouse!). But how do older dogs and cats fulfil their roles as paratenic hosts in the absence of predation/cannibalism? Only the female dog or cat can do this, and only when she becomes pregnant and/or suckles her young. Late in the pregnancy of a female dog, (in the last third, from around day 42 on) tissue larvae re-emerge from their hiding places and re-enter the bloodstream. They may eventually make it to the uterus, where they cross the placenta and penetrate the livers of the unborn pups (trans-placental infection), whereas other larvae make it to the mammary glands and are shed in the milk for the first few weeks of lactation (trans-mammary infection). In cats, few larvae migrate via the placenta and the transmammary route is much more important. By these mechanisms it is thought that nearly 100% of puppies and kittens become infected. As with the other ascarids mentioned earlier, puppies and kittens do not tend to re-infect themselves when they are still permissive of hepatic-tracheal migration, but later egg infections will generate a quantity of larvae hiding in the young animals' own tissues and will also top up the number of larvae in their mothers. Adult dogs and cats are theoretically still capable of harbouring patent infections of these ascarid nematodes (cats arguably more so), but by and large, such infections are rare - especially if effective anthelmintics are given at any point. Patent infections can arise after egg infection or ingestion of paratenic hosts such as mice, but can also arise, typically in the female, when larval ascarids are shed in the vomitus or faeces of infected pups/kittens and the mother then consumes the voided material.

Arrested larval development

In many nematode life cycles, under certain conditions, the prepatent period can be prolonged, sometimes for several months, when larval stages become temporarily inhibited in development rather than progress through the lifecycle as normal. Larvae in this state are referred to as ARRESTED, INHIBITED or HYPOBIOTIC LARVAE. Inhibition usually occurs at the early L4 stage, sometimes earlier depending on the parasite concerned. Hypobiosis is still a poorly understood phenomenon but is generally considered to result either from climatic effects on developing free-living larval stages prior to infection, or host immunity - probably a combination of both. One purpose of this phenomenon is a mechanism for the parasite to avoid harsh environmental conditions that may be detrimental for survival and development of free-living stages. Such conditions include regular dry summer droughts or very cold winter conditions. In New Zealand most nematodes have adapted their inhibition to enhance survival over winter, i.e. most inhibited larvae are present over the winter period before resuming development in spring. They can then commence egg-laying in the springtime at a time when eggs and larvae will face better conditions in the external environment.

Significance of anorexia

In severe, acute gastrointestinal parasitism reduced food intake is responsible for around 40-50% of the effect on body weight. Obviously the effect of decreased intake will vary with the quality of food available and the animal's nutritional requirements. Terminally ill animals are usually completely anorectous. At lower levels of infection (subclinical), there may be temporary and intermittent losses of appetite but some have estimated this is still equivalent to a 10% reduction in intake. With continuing infection, the animal tends to adapt so that food intake becomes approximately proportional to body weight.

Eosinophils

Increased numbers of eosinophils in either tissues and/or blood are commonly associated with parasitic infection. These cells contain a number of inflammatory mediators which have been shown to be toxic to helminths, especially where this degranulation occurs directly onto the surface of these parasites. Such degranulation will follow cross-linking of IgE or IgG on the eosinophil surface. Experimentally, eosinophils have been shown to kill tissue dwelling or migrating stages of helminths. Their role against gastrointestinal nematodes is more controversial. It is well known that increased numbers can be found in the parasitised gastrointestinal mucosa. In general, the nematodes in the lumen are unlikely to come into physical contact with the parasite suggesting they have a limited direct effector role, rather they may play some, as yet not understood, regulatory role. However, some recent experimental work with Trichostrongylus in sheep has shown they are present around newly acquired parasites in immune sheep and appear to be playing a direct antiparasitic role in at least some instances.

Rotate action families on an annual basis.

It has been held that prevention is most likely to be achieved by slow rotation of "action families", preferably on a yearly basis rather than rapid alternation. The idea is that if there is any selection for resistance against Family A this year, as there is no cross-resistance between "action families", a switch to "action family" B next year will allow less-fit survivors from Family A to die out and the gene frequency for resistance against A to return to pre-use levels. This recommendation has been promoted for many years but the basis for it is unproven and it now seems likely that it achieves very little, if anything, in delaying emergence of resistance. It has also been thought that alternating "action families" rapidly, e.g. at monthly or two monthly intervals, is likely to induce multiple resistance because different parts of a single parasite generation are exposed to different drugs. Slow rotation was specifically intended to avoid this. Again however there is no evidence to support this view. Computer modelling has not shown annual rotation to be effective in slowing down selection for anthelmintic resistance. It should be noted that it does not do any harm from a resistance perspective but historically farmers believed this would prevent resistance developing and that is not the case.

Niclosamide

It is a chlorinated salicylanilide which was introduced in 1960. It is almost not absorbed by the host. It acts by disturbing mitochondrial energy metabolism of the cestode. It is effective (although not necessarily 100%) against most cestodes of domestic animals including Taenia spp., Moniezia spp. and Dipylidium caninum but not effective against E. granulosus. No longer available within New Zealand.

Allocation of Nutrient Resources

It is considered that animals can partition available nutrients towards particular needs, especially when those nutrients are scarce. The following table is a possible ordering of the priorities (1 highest and 4 lowest) given by a growing or reproducing animal to its various body functions when partitioning a scarce food resource - from Coop and Kyriazakis Vet Parasitology 84, 187- 204 1999. This framework suggests that improved nutrition will always lead to an increase in host "resilience", especially in young hosts. There have been a series of experiments in recent years that have shown that this resilience was maintained by protein supplementation and not by energy supplementation (more discussions in the sheep lectures)

Calicophoron calicophorum:

It is interesting to note that there is also a difference in susceptibility to treatment between adult paramphistomes and young migrating stages. The adults in the rumen and reticulum are harmless but easily removed with a variety of drugs but unfortunately none are available in New Zealand. The immature stages, which are highly pathogenic in large numbers, are very difficult to kill. At present there are no anthelmintics available in New Zealand which are effective. Historically in New Zealand there was niclosamide (which was primarily used against cestodes), oxyclozanide (was available as "Nilzan" which was a combination of levamisole and oxyclozanide but only the latter was active against trematodes) and rafoxanide. The absence of an effective treatment indicates the limited occurrence of clinical paramphistomosis.

OUTLINE OF THE EPIDEMIOLOGY OF ASCARID INFECTIONS (Order Ascaridida)

It is thought that the ascarid nematodes evolved in definitive hosts that were carnivores, and originally had indirect life cycles with prey animals as intermediate/paratenic hosts. The ability of eggs to hatch and for larvae to migrate in the tissues of a wide variety of vertebrates and even some invertebrates, clearly favoured indirect life cycles. Some of the ascarids later acquired the ability to infect the definitive host directly so the indirect life cycle, though still possible, became optional. This is the situation with Toxocara cati and T. canis (see later). The ascarid nematodes found in animals such as pigs and horses have evolved in a different direction. The pig or the horse may originally have been the intermediate host with some carnivore that preyed upon them being the definitive host. Eventually these ascarids developed the ability to grow to patency in the intermediate host. A major characteristic of the ascarids is that the infective stage (the L3) remains inside the egg until it is ingested by a potential new host. With a thick egg shell, ascarid eggs are very hardy and resistant to adverse environmental conditions (and disinfectants) and may also be sticky (and therefore hard to remove). The ascarids also tend to be very prolific egg layers, the females of some species producing over half a million eggs per day. Obviously, eggs cannot move the way larvae can, and hence egg numbers can become very unevenly distributed. Eggs can also become highly concentrated in areas where faecal material accumulates (e.g. back yards for households with dogs!). Most ascarid nematodes migrate in the definitive host and so stimulate a good acquired resistance - this is in addition to the age resistance many animals possess (see earlier). Animals that are very young are most easily infected to generate a patent infection. As animals age, and with the possibility of re-infection severely curtailed, adult ascarids start to disappear from the small intestine after most infected hosts have reached 6 months of age. Small numbers of egg-laying adults may persist for longer. Whether the loss of adults is due to the old-age of the worms or host immunity is not clear. Mature animals may have patent infections, but these usually involve few parasites and produce relatively few eggs. This "carrier" state may be of epidemiological importance in some cases. Some ascarids have developed particular means of ensuring infections of very young animals

Ascaris suum in pigs

Largely because of its resistant egg Ascaris suum is one of the few nematodes of the pig that can still complete its lifecycle when pigs are taken off pasture and housed indoors. Under these conditions, parasites with a pronounced environmental phase of development, such as the strongylids, struggle to complete their lifecycles. A. suum may still however thrive even when pigs are kept in conditions of reasonable hygiene. The sticky, resistant eggs take some time to become infective, but by then they may have already been smeared over the interior surfaces of the pig pens and are hard to remove with routine washing and disinfectants. Highest egg outputs are seen in young pigs and as with all ascarids, egg output rapidly declines as pigs get older, but even adult pigs may still harbour patent infections, albeit with diminishing egg output. Sows entering the farrowing accommodation may thus be shedding small numbers of eggs. These may then infect the young piglets and infective eggs have even been recovered from the skin of a sow's teats! The PPP for A. suum is about 8 weeks and piglets are typically weaned well before this period is completed so that piglets infected in the farrowing accommodation do not contaminate it themselves3 . They will however heavily contaminate accommodation they subsequently enter and given the hardiness of the eggs, the risk of infection persists for successive waves of piglets, even if they were not themselves infected prior to weaning. Ascaris suum is of course eminently capable of completing its lifecycle under free-range conditions. The resistant eggs persist in soil and on pasture and young piglets become infected as soon as they start picking up food off the ground, licking surfaces or rooting through soil.

Amino-acetonitrile derivatives (AADs)

Monepantel was released in 2009 as an oral drench for sheep ("Zolvix"). There is no indication so far of any further applications for other hosts such as cattle or horses - I would suggest this is a "watch this space" situation. Mode of action: It is a cholinergic agonist with its activity directed through a unique AcH receptor which is different to the one targeted by levamisole/morantel/pyrantel. This receptor appears to be unique to nematodes which implies it is safer than some other cholinergic agonists. Spectrum: against all GI strongylid nematodes but poor against Dictyocaulus and has no activity against Trichuris. Mechanism of resistance: Unfortunately, at least 3 cases of anthelmintic resistance have already been confirmed against this molecule in New Zealand but the mechanisms are not yet understood.

Non-immunological resistance - non-permissiveness

Most parasites are more or less host-specific reflecting the adaptation of particular parasites to particular hosts. This adaptation is essentially physiological and parasites often will not develop in an abnormal host because it is physiologically unsuitable: immune responses are not involved. We make use of this, for example, by using cattle to clean up paddocks infected with horse or even sheep parasites. (Another example of physiological unsuitability is the preference of nematodes to occupy just one site within an animal eg H. contortus only in the abomasum.)

Host-specificity

Most parasites are more or less host-specific, reflecting the adaptation of particular parasites to particular hosts. 1 This adaptation is essentially physiological and parasites often will not develop in an abnormal host because it is physiologically unsuitable: immune responses are assumed not to be involved. We make use of hostspecificity, for example, by using sheep to clean up paddocks infected with horse or even cattle parasites.

Fasciola hepatica.

Of all the trematodes of domesticated animals, Fasciola is the most important. For many years carbon tetrachloride for sheep and hexachloroethane for cattle were the principle drugs used in its treatment but disappeared from general use several decades ago. Fasciola is particularly vulnerable to molecules containing halogen atoms (Cl, Br, I) and it is noteworthy that some of the compounds effective against cestodes are also halogenated. Over the years a large number of drugs effective against Fasciola have appeared on the market and these essentially comprise two series of closely related halogenated molecules namely the substituted phenols (nitroxynil) and the salicylanilides (oxyclozanide, rafoxanide and closantel). All these bind to plasma proteins and appear to act by uncoupling oxidative phosphorylation and interfere with mitochondrial energy metabolism. Because they bind to plasma proteins they are generally only available to bloodfeeding parasites. Most of these are only highly effective against flukes >6-8 weeks old i.e. in the bile ducts and not younger stages, as these are more actively blood feeding stages. Of these only closantel is currently available and then only in combination with other antnematode chemicals. Closantel toxicity in sheep and goats is a potential problem as the safety index is small. It is associated with retinal degeneration and optic neuropathy. Both rafoxanide and closantel are effective against Oestrus ovis and Haemonchus, closantel having a persistent effect (4 weeks +) against the latter as it is firmly bound to plasma proteins. A persistent problem in synthesising drugs effective against Fasciola has been to find substances that are effective against both adults in the bile ducts and immature flukes in the liver parenchyma

Effect of season on larval development and survival

One would expect larval development rates to improve as temperatures rise in the summer but survival to be better in cooler months. Optimum development will occur in warm, wet seasons. Dry weather in summer may reduce the numbers of infective larvae produced. The importance of these effects varies with the extremes of climate that occur in different seasons and regions. For example, in much of Australia and other tropical or sub-tropical countries, temperatures may be high enough for much of the year, but rainfall is typically restricted to fewer months, occurring in either summer or winter. In temperate maritime climates such as New Zealand or the U.K., rainfall tends to be more evenly distributed throughout the year; temperatures tend to be not very extreme but are affected by altitude as well as latitude.

Albendazole

Other benzimidazoles do have limited activity against adult fluke as well as nematodes and possibly cestodes. Albendazole is effective against adult fluke but at higher dose rates than used for nematodes. Some other BZs, such as oxfendazole have some activity but not enough to warrant an efficacy claim.

DISTRIBUTION OF FASCIOLA IN N.Z.

Over the last 40-50 years considerable spread of fluke has occurred, especially in the North Island. Prior to 1950, it was largely confined to the Hawkes Bay/Poverty Bay areas in the North Island and several small areas of the South Island. By 1969 it had spread to parts of Northland and North Taranaki; and Western Bay of Plenty. Infected farms now occur in all livestock rearing areas and in all counties in the North Island. In the South Island, infection has also spread but to a more limited extent; the greatest change has occurred in Westland where Fasciola is now widely distributed with a high prevalence rate (over 30% in slaughtered sheep and cattle) - in 1969 it was very restricted and of little importance. Infected areas also occur in Nelson/Golden Bay, Nelson Lakes area, Marlborough, Kaikoura, Central Otago (irrigation areas) and near Timaru.

Black Disease (= Infectious necrotic hepatitis)

Pathogenesis: Liver damage caused by migrating fluke can allow spores of Clostridium novyi Type B to proliferate in the necrotic tissue which is virtually anaerobic. Areas of tissue necrosis caused by the bacteria result but death is caused by the bacterial toxins produced. Black Disease does not necessarily require large numbers of migrating fluke to set it off. [Note: It has also been reported in association with the migration in the liver of the larval stage of Taenia hydatigena before it emerges and develops in the abdominal cavity into the metacestode, Cysticercus tenuicollis.] Prevention: Where it occurs, vaccination is a very effective prophylaxis. It was recorded commonly in "traditional" fluke areas so preventive vaccination is routine there. Nothing is known of the situation in "newer" fluke areas. Usually included in the clostridial vaccinations that most ruminants receive in NZ and consequently is now rare. Diagnosis: A characteristic post-mortem lesion is a clearly demarcated, pale, necrotic area in the liver. See microbiology notes for specifics on diagnosis of clostridial infections.

Sheep and goats - Acute disease

Pathogenesis: This is likely to occur following ingestion of around 1000-3000+ metacercariae over a relatively short period (say 1-2 weeks). The young flukes wander through the liver, feeding on the tissues and growing. They cause extensive and severe traumatic damage which, together with haemorrhage into the liver parenchyma, is likely to cause sudden illness and death from liver failure. Deaths usually occur 5-6 weeks after infection when the flukes are about to enter the bile ducts. With somewhat smaller numbers of fluke, deaths may occur 8-12 weeks after infection because of virtually complete blockage of the bile duct system with mature and maturing fluke superimposed on extensive parenchymal damage. This is sometimes referred to as "subacute" disease. Diagnosis: Acute fasciolosis is rarely diagnosed ante-mortem except in the middle of an outbreak that has been already diagnosed from earlier deaths. Severe depression, inappetance and anaemia may be seen with evidence of abdominal pain. At post-mortem the liver is enlarged and inflamed with numerous migration tracks and early fibrosis; the liver capsule is often oedematous and covered with fibrinous material; subcapsular haemorrhages may be present. The abdominal cavity usually contains bloody, serous exudate. The liver tissue is readily broken up in water to reveal large numbers of young flukes, usually about 5-10 mm long; variable numbers are found in the bile ducts - much depends on the age of the infection and previous infection history. Acute disease is rarely diagnosed in New Zealand. It is most likely to occur in the late summer/autumn period. Presumably the interaction between snail, climate and definitive hosts means that there is rarely a high concentration of metacercariae for this syndrome to develop

Chronic Disease

Pathogenesis: The pathogenesis is complicated. Most damage is caused in the ventral (left) lobe of the liver and most adult flukes are found in bile ducts draining that area. (i) Flukes feed on bile duct lining causing inflammation of bile ducts, hyperplasia of lining epithelium and fibrosis of the duct walls. The ducts become enlarged and prominent and are usually visible on the visceral surface of the liver, especially the lower (left) lobe. This is a characteristic sign of liver fluke infection and is readily seen with the naked eye as the bile ducts become more prominent and visible on the surface of the liver to some extent. (ii) N.B: Flukes ingest blood and blood is lost from the damaged bile duct epithelium causing anaemia. There is also leakage of plasma proteins through the biliary mucosa; this and the blood loss leads to hypoalbuminaemia. These effects are most important in the pathogenesis of disease. (iii) Further localised damage to the liver parenchyma occurs as a result of inflammation and thrombosis of venules in areas adjacent to bile ducts containing flukes; interference in bile flow caused by the physical presence of the flukes and increased viscosity of the bile also causes hepatocyte damage in the region "upstream" from infected bile ducts. These effects result in progressive scarring and fibrosis, mainly of the lower lobe of the liver. (iv) Often there is depression of food intake (loss of appetite, anorexia). These effects are superimposed on damage caused by migrating fluke. The changes outlined above result in anaemia (through loss of blood), hypoalbuminaemia (through leakage of protein through inflamed duct linings and blood loss) and decreased liver function. The latter is, to a large extent, compensated for by hypertrophy of other parts of the liver. Liver weights are usually increased in infected animals. Plasma globulin levels are raised above normal. The anaemia can be made worse in long-standing or severe cases by impaired bone marrow function - probably as a result of decreased amino acid availability and loss of iron. Experiments have shown that low dietary protein intake further reduces the ability of the animal to absorb iron. As a consequence of iron loss, the anaemia becomes microcytic and hypochromic with time. Decreased food intake and poor nutrition exacerbate these changes Clinical signs: The resulting clinical signs are variable and depend on the size of the infection, how long it has been present and the adequacy of the animal's nutrition. The classic signs of chronic fasciolosis are: - poor growth and/or loss of condition - decreased wool-growth and fleece tenderness - anaemia with pallor of mucous membranes in severe cases - submandibular oedema and transudates in body cavities in severe cases, especially terminally - diarrhoea is sometimes seen but not consistently. N.B. jaundice does not occur in chronic fasciolosis. Production effects: 100-250 flukes can kill an adult sheep in 3-5 months: experiments indicate that around 30 flukes can cause measurable losses in wool production and 50-100 flukes significant losses in weight gain. Most experiments have been carried out with housed animals. A study carried out in New Zealand involving trickle-infections of grazing lambs with metacercariae, showed that up to 40% reductions in daily gain occurred before the animals developed obvious clinical disease. In this trial, a mean burden of 40 flukes (range 27-50) caused an 8% reduction in carcase weight and a 13% reduction in carcase value, 23 weeks from the first infection; with a mean of 116 (101-147) flukes, carcase weight was reduced by 18% and value by 35%. Most sheep infections in NZ usually involve 10 or so adult flukes at one time - it is a question as to the effect of this level of infection. Given that 10 flukes is considered the threshold for causing measurable milk production decreases in dairy cattle it is reasonable to presume at least a similar low number or even less for sheep. Sheep (and probably goats) develop no effective immunity to fluke infection and so can accumulate infection over months or even years. They do mount an antibody response which is measurable (and diagnostic) but doesn't influence the fluke in any meaningful way. Fluke will slowly die, presumably of old age after a few years. Fluke burdens display the typical overdispersed pattern of many parasite infection with some animals harbouring many flukes and most very few. This variation is not related to acquired immunity, i.e. is independent of previous infection. What this variation in susceptibility is due to is not known. Chronic disease is usually seen in the winter following the main infection period which coincides with the climatic stress of winter, pregnancy of ewes and, often, inadequate nutrition. It is most important to note that livers are a valuable by-product of the meat processing industry and 'flukey' livers are condemned an unfit for human consumption. This alone is a significant cost to the industry, even though it is not directly attributed to an animal's value and hence a penalty to the owner of the animal at this stage - it gets averaged out over all animals killed. Diagnosis: This is based on clinical signs and detection of eggs in faeces (sedimentation technique). Egg counts can be used to estimate fluke numbers but only give a rough guide to infection levels. Usually laboratories report egg numbers in terms of +, ++, +++ etc. More recently detection of coproantigens (fluke antigens detected in faeces) is available to be used to detect patent infections (see comments for cattle below). Estimation of serum gamma glutamyl transpeptidase (GGT) can be used as an aid to diagnosis as it is elevated by bile duct damage. However, it is not specific for fluke infection and is raised in facial eczema and other diseases associated with bile duct damage. For these reasons, GGT estimations are of relatively little value for diagnosing fluke infections under New Zealand conditions, particularly in areas where facial eczema occurs. Unfortunately the climatic conditions that are associated with facial eczema are also those favoured for liver fluke free-living development. It is used more widely and is more useful overseas. There are serological tests that can be used to detect current or past infection. They were developed for cattle and have been at least partly validated for sheep. Be careful interpreting titres on an individual animal basis as an interpretation of magnitude of infection. They are more useful for establishing prevalence of infection. Antibody levels wane 2-3 months after an infection is removed by anthelmintics. The circumstances and history of the farm are also important but remember that fluke is often being recorded on farms that were not known to be infected. At necropsy of severe cases, the carcase is usually thin and anaemic with transudates in body cavities, characteristic liver lesions and large numbers of fluke in bile ducts - in most cases 100+. In "subacute" disease 500+ flukes may be found in the ducts - this is the ONLY circumstance in which fasciolosis occasionally may cause a degree of clinical jaundice. Remember it is NOT a characteristic of chronic fasciolosis. N.B. It is very important to remember that the mere presence of fluke and liver lesions does not justify an assumption that fluke is the cause or even a major contributor to a condition under investigation. Many infected animals carry low fluke burdens with only minor effects if they are otherwise healthy and well-fed. Fluke infections are often incidental findings at post-mortem though, of course, they were not doing the animal any good! Furthermore, the lesions caused by infection (scarring, enlarged and fibrosed bile ducts) persist even though the infection has been removed.

Host permissiveness to parasite establishment

Permissiveness and non-permissiveness mean essentially the same things as susceptibility and resistance. Resistance however implies that hosts in which parasites fail to establish are actively interfering with parasite establishment. This may not always be the case and putting an infective-stage in the wrong host may simply fail to present the parasite with the right signals that would otherwise initiate changes (eg. egg hatching or larval exsheathment) that would allow the parasite to begin attempting to establish.

Refugia

Refugia refers to parasites on a farm that are able to cycle through livestock without being exposed to anthelmintics - there is no "selection pressure" towards anthelmintic resistance in this population. Refugia is not a new theory but one revived to explain the concept of "selection pressure" for developing anthelmintic resistance. These unselected nematodes are thus a repository of susceptible parasites that are able to dilute out the survivors of any anthelmintic given to other animals. Thus if an animal is treated with an anthelmintic and a small number of nematodes have survived, the general aim is not to have these resistant survivors being able to mate with other resistant survivors, rather newly ingested larvae that are susceptible can mate with them meaning the progeny are heterozygotes and if able to infect another animal are likely to still be killed by a standard dose of anthelmintic. If only these survivors are present then the progeny are likely to be resistant and some of the progeny may be more resistant than their parents and more likely to be homozygotes for resistance. An example here is for a standard sheep farm where ewes are not being regularly treated and are cycling low levels of infection whereas lambs are on a much more vigorous control programme. The ewes are cycling unexposed nematodes to dilute out any survivors from the lamb drenching programme. There is currently a debate about treating ewes at lambing and the influence this has on selection for resistance. A different example is a "bull beef" farm where only young bulls are farmed and sold at about 18-24 months of age - just as they develop a full level of immunity to GIT nematodes. In this situation the young bulls are regularly treated and there is little opportunity on this farm for cycling of susceptible nematodes. This is not a problem just confined to animals. Mass treatment programmes are being conducted in humans for parasites in developing countries and evidence is emerging of anthelmintic resistance in human parasites in these countries as well, particularly as in recent years these campaigns have been conducted more effectively with a very high participation rate. The aim of a parasite control programme (see later lectures) should be to prevent ingestion of infective larvae i.e. to have pasture with low levels of infective larvae. It is really too late to wait for animals to get infected and then treat them as this means there will be contamination of pasture with eggs which in turn will mean a high challenge with infective larvae subsequently. There is an immediate conceptual problem with maintaining a refugia population and a good parasite control programme - this is the challenge for vets to discuss with their farmer clients. Sensible compromises are required. Finding the balance between maintaining a refugia and keeping pasture larval levels low is a point of considerable confusion with regards anthelmintic resistance at present.

RESISTANCE TO PARASITES

Resistance to parasites (or, conversely, susceptibility) is a very important topic because it directly affects the epidemiology of infections, it is involved in the pathogenesis and pathology of parasitic disease, and it can be made use of in parasite control strategies. It is also a complicated subject and at this stage will be dealt with only in general terms and very superficially.

Percutaneous invasion

Skin invasion by infective larvae is seen in several strongylid nematodes, e.g. the hookworm Ancylostoma caninum, and is also seen in rhabditid nematodes such as Strongyloides westeri. Obviously it is not an option for species like the various ascarids and other egg-infective nematodes which have no 'free-wriggling' stage. Some biologists see per-cutaneous invasion as the ancestral infective behaviour for nematodes such as the strongylids, with oral infection developing later. Certainly, the ease with which grazing animals (grazing being a relatively modern behaviour) ingest infective [L3] off pasture would have lessened the need for skin invasion. Damp, warm and muddy conditions probably favour percutaneous invasion, providing the perfect milieu for larval development and contact with the skin, and in fact such conditions have occasionally been associated with temporary cutaneous parasitism by the essentially free-living nematode Pelodera (Rhabditis) strongyloides. Success of the percutaneous route of infection is variable. Whilst A. caninum is good at it, the other canine hookworm U. stenocephala is poor4 , and if its larvae attempt skin invasion, most die in the skin. Death of larvae in the skin may result in (hookworm) dermatitis as the larvae are destroyed. This can be a very itchy phenomenon and may be more likely in sensitised animals. Occasionally hookworm larvae target the wrong host. Cutaneous larva migrans (CLM) is a human condition typically associated with skin penetration by hookworm species such as A. caninum, and especially its more tropical relative Ancylostoma braziliense. CLM is sometimes called 'creeping eruption' reflecting the red wavy tracks that develop on sufferers as the larvae crawl within the dermal tissues before they eventually die. CLM is also variously called 'ground itch', sand itch' and 'plumbers itch' reflecting the association with the backyards of properties with hookworm infected pets and the fact that some people, like plumbers, face an elevated risk of getting this syndrome since they spend a lot of time crawling under people's houses.

Organophosphates

Some of the organophosphate (OP) insecticides also have activity against some nematodes and are used as anthelmintics, although many OP compounds are either too toxic or ineffective to be used in this way. They tend to be narrow spectrum anthelmintics. OP compounds act as anticholinesterases. They have been superceded as anthelmintics by better compounds but the growing problem of anthelmintic resistance may lead to their re-introduction. There is one OP still marketed in Australia, for example, to help control some nematodes that are resistant to other anthelmintics. No OPs are licensed for use in New Zealand as anthelmintics. OP compounds will be discussed further in relation to ectoparasites and their control. In New Zealand in mid2016 there are moves to fully ban the use of OPs for any purpose.

Responses to drenching lambs

Some years ago, a survey has established that on average lambs (which remained on the farm for the whole season ie. Replacements) were being drenched 6.3 times in their first year. A recent survey has found a similar frequency of use in lambs. Analysis of trials at that time (n=45) showed an average response of 0.6 - 0.7 kg increase in liveweight/drench given. Most of this trial data was generated before the most modern and effective drenches were available and before preventive schemes were introduced although comparisons of a 6 drench preventive programme with no treatment arrived at a similar figure. In addition, in most trials, control and dosed animals are grazed together to avoid nutritional differences so the response to drenching is reduced by reinfection from the controls. The average liveweight response is thus an underestimate of what can be achieved. Assuming, nevertheless, a response of 0.6 - 0.7 kg/dose with a cost of a lamb drench (presume 30kg liveweight) varying from as little as $0.06 for levamisole to $0.65 for a monepantel the justification for drenching is obvious. With 6.3 doses, the liveweight gain would be increased by 3.95 kg on average. At 45% killing out percentage, this represents 1.8 kg increase in carcase weight. The value of this to an individual lamb is difficult to estimate but will be about $5-6. In drenching trials, mortalities in undosed animals have ranged up to 30% but it could be assumed that in the absence of drenching 5% of lambs would die and a large percentage would be permanently stunted thus affecting their future productive life. Although these figures are very crude and ignore labour inputs they do give an indication of the response for the investment. The average response in terms of wool in the same lamb dosing trials averaged about 0.05 - 0.1 kg/dose. Even allowing for the inevitable errors arising from assumptions made, the ratio of return/drug costs was 12.5X and 16X respectively. More recently, others have estimated that 1/3 of total sheep production results from parasite control, mainly by drenching. Given that perspective, drenches are very cheap even though farm returns have fallen. It is also an indicator (and underestimate) of the value of lost production that parasites can cause. In a report by Brundson (1988) he summarised the situation as: Trials undertaken in NZ in the 1960s and 70s experienced an average mortality of 23% attributed to nematode infection. Consequently if one assumes that under present day farming conditions lamb numbers would be reduced by 23% in the absence of drenches and additional production losses were at least 10% (an underestimate for sure) then 33% of current sheep production is drench-dependent. A similar statement can be made for cattle and deer.

FASCIOLOSIS - THE DISEASE, ITS PATHOGENESIS AND PATHOLOGY

The disease differs between sheep (and goats) and cattle so they will be considered separately though there are common features. It is usual to arbitrarily divide fasciolosis into "acute", "chronic" and "Black Disease" syndromes. Essentially "acute" disease is caused by immature fluke migrating in the liver parenchyma (which they do for 5-6 weeks), and chronic disease is caused by mature fluke in the bile ducts: the distinction is not absolute and an animal could be suffering from both at the same time! Nevertheless the distinction is useful in most circumstances. "Black Disease" is a secondary condition caused by clostridial bacteria multiplying in necrotic liver tissue and causing death from toxaemia. This is now a rare condition due to widespread vaccination.

Development of the infective stage (egg to [L3]) in the external environment in Order Strongylida

The following is typical of the development in the external environment of strongylid nematodes that have direct life cycles and are infective by the oral route: - the egg, containing an embryo (typically a morula) is passed in the faeces. - the first stage larva develops in the egg and hatches. The L1 and eventually the L2 feed on bacteria in the faeces, grow and enter an inactive period (lethargus) before moulting to the next stage. - The L2 undergoes an incomplete moult so that the L3 retains the L2 cuticle as a sheath around it - the ensheathed L3 or [L3]. The [L3] is the infective stage - Under optimum conditions, development to the [L3] can take as little as 6-7 days; but under field conditions it is usually longer than this and may take four weeks or more in cooler weather. - The [L3] cannot feed and depends for its survival on stored metabolites. It leaves its food source behind and migrates into the environment to await ingestion by a new host (in certain groups the L3 may penetrate the skin - e.g. the canine hookworm, Ancylostoma caninum). - For a small number of strongylids (e.g. some of the metastrongyloids) development from hatched L1 through to L3 occurs in the tissues of an intermediate host.

Overcrowding

The host is a finite resource and even though parasite biomass is typically small in comparison to the host, there is usually a limit to how many parasites can cram into a specific organ. Well described examples include Teladorsagia circumcincta in sheep for which female fecundity drops off dramatically at very high population densities, and ascarid infections in many hosts in which smaller parasites may be found in heavier infections. The basis of these differences may well be due to competition for the limited size of the niche (how many worms can fit into the space available) or for its finite resources, but may also include direct interactions between the parasites themselves. There is some evidence that an existing adult population can discourage the development of incoming larvae (see next section).

Macrocyclic Lactones (MLs=Avermectin/Milbemycins)

The macrocyclic lactones (MLs) first appeared in 1981 with the arrival of ivermectin but several other compounds are now marketed. They are all derived from related soil fungi or chemical modifications (=semi-synthetic) of these which are aimed to improve safety/efficacy of these original fungal products. They are effective against most nematodes and many arthropods but show no activity against cestodes or trematodes. The term endectocides was coined to describe them (end - endoparasites; ecto - ectoparasites; cides - to kill). Although the basic molecule is quite large small changes can result in changes to efficacy. The available members of this group do vary in the range of parasites they are effective against, especially for the arthropods. Currently available products include ivermectin, doramectin, abamectin, eprinomectin, moxidectin and selamectin as well as milbemycin oxime. Note that nearly all have the suffix -ectin. Milbemycin and selamectin are only used in small animals. Mode of Action: they bind to glutamate-gated chloride ion channels on nerves allow Clinto nerve hyperpolarise the nerve unable to generate an action-potential paralysis. Originally it was thought they bound to GABA receptors but this has since been found to be an incidental finding which is not the principal mode of action in nematodes although is important in killing arthropods. All are very lipophilic, and the chemical is absorbed into lipid depots and may then be released over a period of time giving persistent activity. This is particularly pronounced in moxidectin more than other MLs - see comments below. The consequence for moxidectin is that blood levels remain relatively high for much longer and consequently it has pronounced persistent activity (often many days or a few weeks) against the "easier to kill" parasites. In sheep these are Haemonchus, Ostertagia and Dictyocaulus. Mechanism of Resistance: this is not yet fully understood but current research is directed towards resistance occurring due to changes in various places. It has been observed that there is a change in P-glycoproteins between resistant and susceptible nematodes. The current hypothesis is that these P-glycoproteins are responsible for removing the active ingredient from its site of action in the nematode and hence in resistant nematodes the rate of removal of the drug from the nematode is much quicker. There may also be a role for changes in the actual receptor itself. At present it appears to involve several different genes which is a point of some relevance for development of resistance. MLs are available as oral, injectable and pour-on formulations for cattle (all 3) and sheep (oral and injectable only). Injectable formulations result in a depot of drug which also gives persistent activity for some time. Pouron formulations are invariably given at a higher dose rate than either oral or injectable formulation - they also result in persistent activity as the active ingredient pools in the cutaneous tissues. In addition there is a formulation for use in sheep that is delivered in an intraruminal bolus that releases about 10% of a standard oral dose/day for approx 100 days. For small animals milbemycin is available for oral use as a tablet ("Milbemax tablets for dogs/cats") in combination with praziquantel. Selamectin is available as a topical treatment ("Revolution for cats/puppies and kittens") for control of fleas, mites and Toxocara cati. Moxidectin is available in combination with imidacloprid as a topical treatment ("Advocate" for cats/dogs) and has a claim for control Toxocara, hookworms, Trichuris as well as a range of ectoparasites. Toxicity of Abamectin - compared to the other MLs abamectin has a small safety margin. It has caused toxicity in young animals with minimal fat reserves to absorb the chemical. Consequently care should be taken with any product containing abamectin in young animals. For example, there are now weight restrictions on the use of many abamectin compounds where cattle need to be over a minimum (varies between 100 and 110kg). This means that preweaning drenches and weaning drenches should probably not contain abamectin

Species differences in response to temperature

The minimum temperature and to a lesser extent the optimum temperature for development, vary with different nematode species. This has an effect on the geographical and seasonal distribution and importance of different species. For example, in New Zealand, the warm-climate preferring Haemonchus contortus is mostly a North Island problem, whereas the cool-climate preferring Nematodirus spp. may be more prevalent and important in the colder parts of the South Island. Humans transported sheep and cattle across the globe and took parasites with them. Climatic factors played a major part in deciding whether parasites established in the new environment. Many species that are common and important in tropical or subtropical countries would fail to establish or fail to become numerous if introduced into a temperate country such as New Zealand. The situation would obviously be quite different if an exotic parasite was introduced that was already adapted to local conditions, e.g. from Great Britain to New Zealand.Within any nematode species, strains may eventually become adapted to local conditions. Experiments comparing the minimum temperatures for development of isolates of the same species from Britain and the U.S.A. have shown marked differences (of the order of 3-5C) in the minimum temperature for development. learn figures on page 6

"Non-physical" effects

The most important effects of most parasites do not depend on the physical damage they do. These "non-physical" effects will be considered under three headings.

Effect of pasture composition etc. on larvae

The nature of the pasture itself has a variety of influences on larval ecology. Pasture length, density and composition affect the microclimate at or near soil level. Longer pasture provides more shade, higher humidity and less variation in temperature. Density of pasture has a similar influence. Newly sown pastures often have a lower density and hence may support fewer larvae. The composition of pasture has an influence through the proportion of broad-leaved plants in it, e.g. clovers. More clover produces more shade and protection from desiccation. Different herbage plants also vary in their ability to provide suitable moisture films for movement. In this respect infective larvae appear better adapted to grasses than to broad-leafed species. It does not necessarily follow that long or dense pastures with high proportions of clovers are more dangerous from the host's point of view. Pastures with more clover often provide better nutrition than those with less and this is important in relation to the host's ability to resist infection by the parasites. Periods of rapid pasture growth (e.g. in spring) may result in a drop in larval density on pasture - same number of larvae, but more grass. Pasture management can affect nematode larval populations. Keeping pasture short by hard grazing may expose the larvae to desiccation, but young susceptible animals forced to graze closely may take in more larvae/kg herbage than on longer pasture. Resting pasture for hay or silage production results in removal or death of most of the nematode larvae.

Non-permissiveness due to age (age immunity)

The older host can represent a greater challenge to establishment even if the host has never met the parasite before. This phenomenon is very poorly understood, but by definition does not involve the acquired immune response. An example is the poorer ability of ascarid nematodes to successfully complete hepatic-tracheal migrations in older animals. Toxocara canis, for instance, can complete its migratory path readily in a 3 week old pup, but struggles in even a 3 month old dog

The parasitic phase in the host

The parasitic phase of development in the definitve host typically begins with the inadvertent ingestion of the ensheathed L3. In response to chemical stimuli in the new host, the L3 exsheathes, i.e. discards the L2 cuticle; exsheathment usually occurs in the region of the digestive tract immediately anterior (cranial) to that in which the adult lives. Inside the host animal, the availability of water is no longer limiting and the warm temperatures nematodes encounter will allow rapid development at more predictable rates. The availability of oxygen is less clear cut. Some nematodes have been shown to respire anaerobically. Without a circulatory system, larger nematodes (e.g. ascarids) may be too big to allow oxygen to diffuse into them. Adult nematodes also commonly inhabit a niche (the lumen of the gut) in which O2 levels are very low. Other, smaller nematode species (or stages) may inhabit sites closer to the mucosal surface, or even within the tissues, where O2 levels are higher and almost certainly respire aerobically. Non-migratory species typically enter the mucosal glands in the organ in which they will mature soon after they have exsheathed. They do NOT usually penetrate through the epithelium and into the lamina propria, however, some do, and some will penetrate as far as the sub-mucosa, even the serosal surface of the gut. By occupying any of these tissue niches, larvae may also be able to access higher O2 levels, or alternative sources of nutrition. A few days, often as little as 2-3 days, after ingestion, the L3 moults to the L4 stage. Depending on the species of parasite and other factors, the L4 may emerge from its tissue niche to complete development or may delay emergence until the last moult has been completed. Later L4 stages begin to show signs of sexual differentiation, but maturation of the reproductive organs follows the last moult. Following copulation, the eggs pass out in the host animal's faeces and the cycle is complete. The larvae of some nematode species leave the gut shortly after infection and migrate through the tissues. This may well expose them to higher O2 levels than if they stayed in the gut. The appearance of eggs (sometimes hatched larvae) in the host's faeces marks the end of the PREPATENT PERIOD: the infection is now a PATENT infection (in the sense of being "obvious" or "detectable"). Migrating species follow a variety of routes outside the gut and moult in the organs they encounter en route. They may stay in these organs for periods ranging from days up to several months; the prepatent period is thus often longer for migratory than for non-migratory species. The prepatent period may range from as little as two weeks to almost a full year depending on the nematode species.

SLOW-RELEASE DEVICES for sheep

There are currently 2 controlled release capsules for use in sheep in NZ. These all involve the same technology which is plastic casings with "wings" that are released after the bolus has entered the rumen and which prevent regurgitation of the device. A spring inside the casing drives pellets/tablets containing the active ingredient down to a small opening where ruminal fluid slowly dissolves them Originally they held only tablets containing albendazole (a BZ - "Extender" capsules) and later ivermectin (a ML - "Ivomec Maximiser CR Capsules"). Two later variations have been added including one with alternating tablets of albendazole and ivermectin ("Extender Max" capsules) and the second albendazole and abamectin mixed together into the same tablets i.e. at the same time ("Bionic" capsules). Generally these are available at two dose rates - one size is for use in adult sheep and a lower one for lambs. They all provide continuous low-level release for 100 days - about 10% of a standard oral dose/day for the respective active ingredient. The theory is that continuous release of a low level of drug will kill existing nematodes and prevent establishment of incoming larvae. It is a different principle to a single treatment. The albendazole capsules have a nil withholding and those containing ivermectin have a 126 day withholding period (from time of administration) - so use of the latter, especially in ewes prelambing prevents sale of wet/dry ewes for slaughter until the WHP has passed.

Development of immunity to ascarids compared to gastrointestinal trichostrongyloids

There is an interesting contrast between how the immune response develops between these two different types of nematodes. As described above the immune response to gastrointestinal nematodes such as Teladorsagia or Trichostrongylus is very slow to turn on and only shows measurable effects on the numbers of nematodes from about 4-6 months of age and isn't fully mature until about 18 months of age. In contrast, there is a different pattern with ascaridoids such as Ascaris suum and Parascaris equorum. For A. suum in pigs there is a rapid development of immunity against the incoming L3 larvae which switches on in the first few days/weeks of life and very quickly limits the ability of larvae to complete hepatic tracheal migration i.e. they don't manage to complete migration through the liver, then lungs then back to the small intestine but are either rejected from starting or, more likely, killed in the liver en route. However, those larvae that do manage to complete their migration back to the small intestine and mature will then usually live until the pigs are about 6-9 months of age when the immune response finally rejects these adults from the gut as well. Thus there are some differences between the immune response for the different age groups. As with the trichostrongyloids, adult animals with a mature immune response may still carry small burdens of these nematodes but they are generally of limited clinical significance in most animals. There are slight differences between different ascaridoids but they generally follow this same pattern with a rapid ability of the definitive host to stop incoming larvae completing migration but then any successful adults get to live until about 6-9 months of age until they too are rejected. Toxocara canis (dogs) and Toxocara cati (cats) have a few more nuances around this immune response as they can also act as a paratenic host for the parasite and don't necessarily just reject the incoming larvae, rather they get diverted to somatic tissues where they remain until they get reactivated and pass to the pups across the placenta or milk (T. canis) or to kittens just in the milk (T. cati).

Use Anthelmintic combinations

These are specifically formulated combinations of anthelmintics belonging to two and three action families. Pretty well every permutation of combination with the older action families are available as specifically formulated combinations. There are several 3-in-one combinations including BZ+LEV+Abamectin (eg. "Matrix") and one BZ+LEV+moxidectin ("Trimox"). Their development was specifically directed at slowing down the emergence of drench resistance. A full therapeutic dose of each drug is used. Resistance to different action families is inherited independently. If we suppose that in a population of susceptible parasites the probability of any parasite having the genes for resistance is, for example, 1/1000, the probability of it having genes for resistance to both is 1/10002 = 1 in a million. This makes it extremely unlikely that such double-resistant parasites will mate with similar genotypes and produce double-resistant progeny. Parasites with resistance genes to only one of the two drugs should be eliminated by the other drug in the mixture. Published results of computer modelling suggests using combinations of action families is the best way to prolonging the effectiveness of anthelmintics rather than an annual rotation of anthelmintics. These combinations will be most effective on farms where there is no resistance but are still believed to be useful if some resistance is present. One caveat needs to be emphasised here. If animals are treated with a 3 way combination and then moved to "clean" pasture this could prove to be a very rapid way to generate multiple resistance - care needs to be taken there is a refugia available. In reality as drench resistance is now so common all farms should be using some form of combination product unless they are using one of the newer drenches such as Zolvix

Parascaris equorum in foals

This ascarid of horses has a lifecycle very similar to that of A. suum. The PPP is slightly longer at 10-12 weeks. The young foal is the most permissive of patent infections and egg counts can reach several thousand eggs per gram in clinically healthy foals of about 3-6 months of age. As stated earlier the long PPP of this parasite, with the addition of a few weeks for eggs to develop in the environment, means that foals do not generally reinfect themselves. Instead eggs will hang around in the environment and infect next year's foal crop - beware using the same paddocks every year for young foals! Adult horses, particularly young adults, may shed small numbers of eggs, but play a minimal role in the epidemiology of this parasite. In fact, patent infections in even yearling horses are exceedingly rare given the frequency of anthelmintic use in most horses.

Emodepside

This is a semi-synthetic derived from Mycelia sterile, a fungus that inhabits the leaves of Camellia japonica plants. It is the first in a class of anthelmintics known as the Octadepsipeptides. Its mode of action is believed to be by binding to a receptor called latrophilins. Binding to this stimulates a cascade of signals leading to disturbance of synaptic transmission, especially in the pharynx of nematodes inhibiting activity. Its activity is largely within the ascaridoids and in NZ is only licensed for use in cats as a topical application (in combination with praziquantel)

OUTLINE OF THE FACTORS INVOLVED IN THE EPIDEMIOLOGY OF INFECTIONS OF RUMINANTS WITH TRICHOSTRONGYLOID NEMATODES.

Trichostrongyloid nematodes, such as Teladorsagia circumcincta and Trichostrongylus colubriformis, are the most important group of nematode parasites of ruminants, capable of causing considerable losses of production and much clinical disease. The majority of infections with these nematodes follow a relatively similar seasonal pattern.

Morantel/Pyrantel (also referred to by their chemical structure as the Tetrahydropyrimidines)

Unrelated structurally to levamisole but they have the same general mode of action and are included with levamisole in the same action family. Both morantel and pyrantel exist as different salts which affects their solubility and efficacy against some nematodes. Pamoate salts are insoluble in water and are poorly absorbed whereas the tartrate and citrate salts are soluble and are absorbed. They all generally have broad spectrum activity against most GIT nematodes and some activity against some cestodes eg. Anoplocephala in horses. Mammalian safety is reasonably high Morantel is used in ruminants and horses in NZ; pyrantel in dogs and cats. A third member oxantel is primarily directed against Trichuris and used for this purpose in dogs (usually combined with pyrantel to achieve a broad spectrum). Little is known about the mechanism of anthelmintic resistance in this group but is presumed to mirror the situation with levamisole.

Effects of immune and inflammatory responses

When helminths enter the gut for the first time, there is a rapid influx of lymphocytes and plasma cells, and reactive hyperplasia of the regional lymph nodes. These changes are associated with local and systemic antibody responses. Local antibody responses involve the production of: (i) IgA (secretory antibody) which is secreted in mucus. (ii) IgE (cytophilic or reaginic antibody) which is the mediator of immediate hypersensitivity responses. The cross-linking of IgE by corresponding antigens occurs on the surface of mast-cells causing them to degranulate and release vasoactive amines (e.g. histamine, 5-hydroxy-tryptamine) and other chemical mediators ( e.g. proteases, leukotrienes) which cause vasodilation, increase capillary permeability and attract cells such as eosinophils. These reactions are an important part of the inflammatory response to parasites. One result of these reactions is passage of plasma proteins into the lamina propria. This coincides with the loosening of the tight-junctions in the mucosa which may be an indirect result of the local hypersensitivity responses. It allows plasma proteins, including albumin and globulins, to pass into the gut lumen. The globulins may include IgG humoral antibodies to the parasites. At this point note that increased cell-turnover in the mucosa, increased IgA secretion, increased mucus production and leakage of plasma proteins all involve the loss of host-protein into the gut. This is referred to as a protein-losing gastroenteropathy and it has important implications. The host can digest and recover some but usually not all of this lost protein but at some "cost" in terms of energy and protein to do so. Immune responses can also be important where larval nematodes, such as Oesophagostomum, spend some time in the mucosa. This can result in severe local hypersensitivity responses leading to tissue necrosis and severe inflammation - in itself this can be a significant problem. In effect there is a focal inflammatory lesion around individual larvae and this develops through the classical inflammatory cascade with a fibrous response endeavouring to wall off the pathogen (quite a different response to the hyperplasia/metaplasia that is seen in the abomasum with Teladorsagia/Ostertagia). This can also cause massive losses of plasma proteins and these inflammatory nodules may become pus-filled because of secondary bacterial infection. In severe cases the surrounding tissues may also become involved with oedema and accumulation of inflammatory cells. It will almost certainly be causing some abdominal pain/discomfort to the animals. As the larvae die or leave these nodules may heal by scarring or persist, filled with caseous purulent material that may calcify. The inflammatory reaction may kill the larvae in the nodules, though it does not always do so. As the nematodes leave such nodules they then allow gut contents to enter into the nodules to further exacerbate the inflammatory response. Overall, this inflammatory pathology can have severe effects on the host. Such nodules are a feature of some Oesophagostomum species in various hosts. Cyathostomes in horses also invade the mucosa, do induce the accumulation of some inflammatory cells but don't induce this same degree of dramatic inflammatory nodule formation - there is some inflammatory response and nodule formation but it is very much reduced compared to Oesophagostomum. The emergence of cyathostome larvae also allows influx of noxious gut contents into the mucosa/submucosa which can exacerbate the inflammatory response.

Pathogenesis and pathology of helminth infections of the gut

While a few parasites exert their main effects on the host by directly damaging or removing host tissues (e.g. Haemonchus sucking blood), for the majority of infections this is not so. Even in Haemonchus infections disease is not caused solely by blood loss. Most parasitic disease results from the host's response to the parasite's presence and activities rather than what the parasite is actually doing. It is, then, the interaction between host and parasite that results in disease. This interaction is complicated and only partly understood (see Fig. 1). Parasite disease is often divided into "clinical" disease where observable clinical signs occur, and "subclinical" disease when they do not. The distinction is arbitrary and of limited value as the severity of disease covers a continuous range and the detection of clinical signs depends on how hard you look! Nevertheless, at one extreme we find obvious signs such as diarrhoea, poor growth and death, and at the other what appears to be normal good health. While the former is more dramatic and can be costly to the individual farmer involved, it is "subclinical" disease that is of greatest economic importance by far. Outbreaks of clinical disease are sporadic. All grazing animals are affected by subclinical parasitism. In cases of parasitic gastroenteritis, regardless of the helminths involved, similar signs are usually seen though they may vary in intensity. Most important are anorexia, diarrhoea, decreased plasma albumin levels and anaemia (most severe with blood-sucking parasites but occasionally with others). The fact that these effects occur with a wide variety of infections indicates common underlying mechanisms. Before exploring these further, let us first consider what the parasites are actually doing in the gut. Larval nematodes invade gland crypts in the mucosa - in some species they may penetrate to the lamina propria. Some develop in nodules in the mucosa or submucosa. Adult nematodes may be free in the gut lumen (e.g. ascarids), on the surface of the mucosa (e.g. Haemonchus, Ostertagia, Strongylus) or burrowed into the superficial epithelium (e.g. Trichostrongylus, Trichuris). They may be feeding on soluble host tissue fluids or digesta, on blood or on the mucosa itself. Cestodes are attached by the scolex suckers and, often, hooks. Trematodes attach to the mucosa with the ventral sucker and usually feed on the mucosa with the other.

Effect of the faecal mass on larval development and survival

With a high surface area to volume ratio, sheep faecal pellets offer little protection against desiccation, whereas the cattle dung-pad does; thus, if drying occurs early on, a higher mortality of larvae would be expected in sheep faeces. In dry conditions, cattle faeces can in fact act as a reservoir of moisture; larvae are protected by the crust that forms on the top of the faeces; larvae are also prevented from moving out of the faeces by this crust until it is softened by rain, or is mechanically disrupted. On the other hand, in wet conditions particularly, sheep faeces break down more rapidly than cattle faeces and eggs and larvae are washed to soil level where they may be relatively protected. Watery, diarrhoeic faeces whilst wet initially, could dry out very quickly in dry conditions.

Competition for nutrients

With few exceptions, this is of very little importance. The biomass of parasites is invariably very small compared with that of the host and their nutritional requirements are proportionately small. Large nematodes such as ascarids which have very high metabolic rates may be exceptions as they may compete with the host for soluble energy sources in the digesta such as glucose. However, they also cause changes in the villi which result in decreased nutrient absorption and this is probably more important. Cestodes, though comparatively large, have low metabolic requirements. They absorb low molecular weight substances from the mucosal surface but this does not measurably affect the host in most circumstances. As they are parasites, by definition there will be some effects, but they are so small that experimental infections have generally failed to measure an effect A few parasites selectively accumulate specific nutrients (e.g. Diphyllobothrium latum in man which absorbs vitamin B12, and Ascaridia galli which absorbs vitamin A). This can be significant especially where dietary intake is inadequate.

INDIRECT LIFECYCLES

With indirect lifecycles, one has to consider not just the biology of the parasite and of the definitive host, but also that of the intermediate host. This can lead to widely differing patterns of epidemiology. The absence of suitable intermediate hosts will of course guarantee that the parasite lifecycle cannot be completed. A variety of indirect lifecycles use either vertebrate or invertebrate organisms as intermediate hosts. Vertebrate intermediate hosts can be long-lived, and if the parasite stages within them are as well, the risk of infection to the definitive host can be relatively constant over time and is typically a function of how often the definitive host comes in to contact with (ingests) infected versus uninfected intermediate hosts. In contrast, many invertebrate organisms show marked differences in seasonal abundance and activity and thus infection of the definitive host may also follow a seasonal pattern. As an example, the spirurid nematode Dirofilaria immitis (American heartworm) utilises a variety of mosquito species as intermediate hosts. In most areas with temperate climates, heartworm transmission only occurs in the warmer months of the year, when mosquitoes are actively biting and this can be important as it influences how much of the year dogs need to be given heartworm preventative medication. The seasonal pattern only applies to the acquisition of new infections by dogs. Heartworm is a long-lived parasite, so once a dog is infected, it could be infected for years to come and disease due to heartworm may be diagnosed at any time of the year.

Th Lymphocytes:

Work in murine models has demonstrated that response to infection can vary significantly depending upon the activation of different types of lymphokine-secreting cells. In mice (and humans) these two subsets are referred to as Th1 (T helper cell type 1) and Th2. These distinct subsets of T helper cells produce distinct arrays of lymphokines that drive immune responses into one of two defined patterns. The Th1 cells produce, among other lymphokines, Interleukin 2 (IL2) and -interferon (-IFN) and are responsible for immune responses that are referred to as Delayed Type Hypersensitivity. In contrast, the Th2 subset produces, among other lymphokines, IL3, IL4, IL5 and IL10 and the resulting immune response is termed an Immediate Type Hypersensitivity response. IL4 induces IgE and IgG1 production, IL5 induces eosinophilia and IL3+IL4 initiate mast cell proliferation. In addition to driving the immune response in these directions the two T helper subsets tend to inhibit each other so there is either one or the other operating. It is generally accepted the Th1 responses are necessary for protection against intracellular parasites, while Th2 responses are involved in protection to extracellular parasites, most notably helminths. Whether these two subsets are identically represented in all animals is still under debate. It appears that with ruminants it may be a little less clear-cut than in mice, but the general concept seems to hold

LIVER FLUKE (Fasciola hepatica)

You should be thoroughly familiar with the life cycle of this parasite as that is the basis for understanding the epidemiology and the pathogenesis of disease. For many years, liver fluke has been an increasingly important cause of decreased animal production and liver condemnations. It is most common and important in sheep and cattle but it can be a significant problem in goats and deer. Other animals including rabbits, hares, possums and, rarely, horses, pigs and man can be infected.

Triclabendazole,

benzimidazole which is highly effective at standard dose rates against all Fasciola older than 1 week. As the name suggests it is a halogenated BZ. This material is marketed in N.Z. under the trade name "Fasinex" plus some others. Although a benzimidazole, it is not effective against nematodes or cestodes or trematodes other than those in the family Fasciolidae. As such it is considered to be operating by a different mechanism than other benzimidazoles.

Clorsulon

chlorinated sulphonamide, is highly effective against adult fluke in cattle and is marketed (in New Zealand) combined with injectable ivermectin ("Ivomec Plus"). Note the halogenated molecule being involved with antifluke activity.

ANTHELMINTIC RESISTANCE

inherited ability of parasites to withstand doses of anthelmintic which would normally be expected to kill them, i.e. as established by efficacy trials. Resistance arises through selection of resistant genotypes. The key is frequency of genes for resistance in a population. As selection continues by exposure to the same drug action-family, the parasite population becomes more homozygous for resistance, the frequency of genes for resistance increases and the level of resistance increases to a maximum. You need to think in terms of factors that influence the "selection pressure" for resistance to develop. Worms resistant to one drug are likely to be resistant to other drugs having the same mode of action, i.e. in the same "action family" though the level of resistance may differ. If these drugs are chemically related, this is termed side-resistance (e.g. within the BZ group). If the drugs are chemically unrelated, it is cross-resistance (e.g. levamisole resistance may confer resistance to morantel). If nematodes are resistant to drugs having different modes of action (e.g. a BZ and levamisole), this is termed multiple-resistance: resistance to each "action family" is independently inherited. The rate at which resistance develops in a parasite population is very variable and affected by selection pressure (e.g. increased by more frequent drug use) the initial prevalence of resistant genotypes in the population and the complexity of the genetics involved. Another important issue is whether the gene/s which induce resistance are dominant or recessive or trend to one side or the other. Generally they have been recessive but not for all. By the time resistance is evident by obvious clinical treatment-failure, the situation is serious and it has been preceded by decreasing effectiveness of control and associated production losses for a variable period.

Non-permissiveness

mechanisms that are independent of immune processes

Black Disease

occurs less commonly than in sheep i.e. is also rare in NZ given the level of vaccination that occurs.

Incidence

reflects the number of new cases (of a disease) occurring in a particular period, is a more useful measure for short term, acute conditions, e.g. bacterial mastitis.

Chronic Disease in cattle

the basic pathogenesis is as in sheep but there are some significant differences. (a) Infected bile ducts usually become calcified. This will persist even when the flukes are no longer present. Killing sheets from abattoirs will often report their presence and should therefore be interpreted with care as the infection may have been some time previously (b) Cattle are considered to be able to acquire a degree of "resistance" to Fasciola though whether this is immunological or physiological incompatibility as a result of liver fibrosis and bile duct calcification is uncertain. Nevertheless, adult cattle still have live fluke and can get infected as adults. However, as a result, cattle may lose infection "spontaneously" or when reinfected; the migration of the flukes in the liver parenchyma is also prolonged after reinfection and reduced numbers reach the bile ducts and later than normal, and survive a relatively short time. As above, adult cattle can harbour burdens of fluke that can likely affect milk production in dairy cows. Studies in Europe have shown that cattle that harboured fluke over winter were still able to develop further infection the following grazing season. Much effort has been made to understand and harness this "immune" response with the development of vaccines but to date with no useful outcome. Because of these differences, lesions can occur and be quite extensive with few flukes in the bile ducts, sometimes none at all. Repeated infection can progressively damage the liver causing decreased productivity though relatively few flukes mature - and those that do may not survive long. Obviously this has important implications for diagnosis. Young susceptible animals can cope with larger numbers of fluke than sheep; it may take about 500+ adult fluke in the bile ducts to cause fatal chronic disease though much depends on the circumstances. In reality, such high burdens are rare in New Zealand and we are usually concerned with the potential impact of <50flukes. Experiments have clearly shown that the effect of infection on the growth of young cattle is very much affected by their level of nutrition and exposure to adverse environmental conditions. Well-fed housed animals may continue to grow whereas similarly infected animals kept outdoors may develop severe anaemia and die. In some trials decreased feed intake and feed conversion efficiency have been observed. Relatively little work has been done with grazed animals but it is likely that the effect of infection is greater than trials with housed animals suggest. More research is needed. Recent adhoc surveys have shown quite high levels of infection in dairy cows in Northland and the West Coast of the South Island. It is difficult to predict what this would mean for production except to indicate it will have an effect. Levels of infection as low as 10-40 flukes are considered to have a significant effect on milk production in dairy cows which is actually very few flukes. Infection of young animals which impairs their growth can lead to lowered fertility and extend the age to first calving. Recent research in Europe has considered 10 flukes in growing animals as the threshold for loss of production - both weight gain and milk yield. At this level fluke will be having an effect on dairy cattle throughout the endemic fluke areas of New Zealand. Note: Cattle livers are an important and valuable by-product and rejection of livers because of fluke damage is a major cost to the meat industry. Diagnosis: More difficult than in sheep or goats. Clinical signs are important - poor growth, loss of condition, signs of anaemia, hypoalbuminaemia and raised globulin levels are strongly suggestive. Constipation or diarrhoea can occur. Examining faeces for eggs is possible. The sensitivity of a standard sedimentation test is from 45-65% depending on how much faeces is examined. It is more likely to detect heavily infected animals than lightly infected animals. An egg count >5eggs/g is considered to be significant, but even lower counts are likely to be significant in many cases (quite a bit different to evaluating strongylid egg counts!). The reality is that egg counts are cumbersome, expensive for the information you receive and not very widely used at present. A new test that measure faecal antigen has been developed but at this time (mid-2015) has not been offered in New Zealand. A high correlation between faecal antigen and fluke burdens has been established and this will proved to be a useful test in the future. It is currently expensive as it will only be useful for one animal at a time and would be $20+ based on the cost of the kit to purchase. Estimation of gamma glutamyl transpeptidase (GGT) is useful in theory as it is specific for bile duct damage but as GGT levels are also raised in facial eczema, its practical value is limited under New Zealand conditions. It is widely used elsewhere, particularly as faecal examination for eggs is unreliable. Serological tests can be used and one, an ELISA test, is currently available. It can be used on individual animals (~$19/test) or on pooled serum samples (~$25/test). This same ELISA has been modified to be able to be used on whole milk (bulk or individual) at about ~$30/test but it is less sensitive than with blood. Nevertheless, for a dairy farm the bulk-milk tank test is the most easily adopted. A low pool result indicates <20% animals are infected or been recently exposed, a medium pool result indicates 20-50% of animals have been infected or recently exposed to fluke and a high pool result indicates at least 50% of animals have been infected or recently exposed. Antibody levels become negative about 3 months after animals have been treated. These values have not been correlated to milk production under New Zealand conditions. Farm history and location can be relevant also.

Prevalence

the percentage of a population affected by a particular disease/condition at any given point in time. Since helminth infections are generally chronic conditions affecting animals for months at a time, often subclinically, prevalence is a much more useful parameter to measure than incidence.

Epidemiology

the study of ALL the factors that determine the occurrence of a disease or infection. Understanding the epidemiology of parasite infections is essential for their successful control.

factors involved in parasite epidemiology

these include the effects of factors such as temperature and rainfall on the development of infective stages in the environment, and factors affecting the uptake of infective stages by potential hosts. The host is not a passive recipient of infection, however, and it exerts effects on parasite populations, chiefly through immune responses to infection, but also through other factors such as behaviour (to avoid parasites many animals will avoid grazing too close to faeces). The host can thus affect such things as the numbers of parasites that succeed in developing inside it, how long they survive and how many eggs they produce. All these interacting factors may also be affected by human interference, e.g. the choice of stocking rates, grazing management and dosing with anthelmintics.

Acute Disease for cattle

this is extremely rare anywhere and has not been recorded in NZ.