RESISTANCE of VETERINARY PARASITES - fleas, ticks, flies, mites, lice, etc. - to PARASITICIDES

A document of FAO (Guidelines Resistance Management and Integrated Parasite Control in Ruminants, 2004) defined resistance to parasiticides as the "significant increase in the number of individuals within a single population of a species of parasites that can tolerate doses of drug(s) that have proved to be lethal for most individuals of the same species."

Another document (WHO Document Expert Committee on Ectoparasiticides, 1957) defines it as "the development of an ability in a strain of insect (mite or tick) to tolerate doses of a toxicant which would prove lethal to the majority of individuals in a normal population of the same species". This definition can also be applied to resistance of worms to anthelmintics. This acquired resistance is inheritable, i.e. it is usually transmitted to the offspring.

From a practical point of view, for a farmer or producer, resistance usually means that a product that provided good parasite control in the past repeatedly fails to achieve it, i.e. it is no more capable of reducing the parasite population, even after slightly increasing the administered dose.

In most cases resistance affects "active ingredients" that have been used for years in the same property (or pet). However, resistance can also affect active ingredients that were never used in the past in a given property, because active ingredients of the same chemical class often show so-called cross-resistance.

So far resistance has been reported for more than 500 arthropod (flies, lice, fleas, ticks, mites, etc.) and helminth (roundworms, flukes) species, that affect crops, livestock, pets or humans. For some of these species resistance was induced in laboratory trials and has not been reported in the field.

It is useful to know that the different development stages (e.g. larvae, nymphs, pupae, adults) may show a different degree of resistance to parasiticides. This is quite typical for insects with a complete metamorphosis (e.g. flies, fleas, mosquitoes). It can very well happen that whereas larvae are resistant against some parasiticides, adults are not, or vice-versa. In ectoparasites with incomplete metamorphosis (e.g. lice, ticks, mites) if adults are resistant against a given parasiticide, larvae and nymphs are often resistant too, but maybe with a different RF than the adults.

Mammals, including livestock, pets and humans, can also acquire resistance to parasites. In fact, a lot of research is devoted to investigating and developing livestock breeds that are resistant to parasites (ticks, worms, flies, etc.). But this kind of resistance has to do mainly with the immune system of mammals, and not with the mechanisms that drive resistance development to parasiticides by insects and helminths.

Insect eggs and pupae are often also resistant to parasiticides (and to other poisons), but in the sense that they withstand the action of mostly all. The reason is that they have protective envelopes that do not let the toxic molecules get inside the egg or the pupae. They do not develop resistance, but are resistant, regardless of whether the emerging adult or the preceding larvae are resistant to a given parasiticide or not.

Resistant species

Not all parasite species that affect livestock, dogs and cats have developed resistance. In fact, only a few ones have done it so far. The species with the most serious resistance problems worldwide are the following ones:

INSECTS

• Houseflies, Musca domestica, in any kind of livestock operation, mainly in dairy farms, cattle feedlots, piggeries and poultry houses. 
• Horn flies and buffalo flies, Haematobia irritans, mainly in grazing in cattle.
• Fleas, mainly Ctenocephalides spp on dogs, cats and livestock.
• Lice, mainly Damalinia ovis in sheep.
• Blowflies, mainly Lucilia spp in sheep.

TICKS & MITES

• Cattle ticks, Boophilus (=Rhipicephalus) microplus in cattle.
• Scab mites, Psoroptes ovis, in sheep.
• Poultry mites, Dermanyssus gallinae in chicken.

HELMINTHS

• Gastrointestinal roundworms (mainly Haemonchus spp, Ostertagia spp, Trichostrongylus spp, Cooperia spp and Nematodirus spp) in sheep, goats and cattle. 
• Liver flukes, Fasciola hepatica in sheep and cattle.

Resistance factor

When talking about resistance of parasites to parasiticides the resistance factor (=RF) describes how strong it is. It is calculated by dividing the lethal dose for killing a population of the resistant parasite strain by the lethal dose for a susceptible reference strain. Several lethal doses (=LD) may be used: LD50, LD90, LD100, indicating the LD for killing 50, 90, or 100 % of the population.

A RF of 10 means that 10 times more parasiticide is needed to kill resistant parasites that to kill susceptible ones. A RF of 100 means that 100 times more is needed.

Resistance factors are not determined for an individual parasite, but for sample of a population (such populations may become a strain and get an own name if they are maintained in a laboratory).

Resistant populations in different locations often have different RFs, even in neighboring locations.

Resistance factors of 2 to 5 are often considered as tolerance, not yet resistance. They are often found for some contact, non-systemic ectoparasiticides (organophosphates, synthetic pyrethroids, amitraz, etc.) used for dipping or spraying livestock, in pour-ons, insecticide-impregnated ear-tags, etc. Such low resistance factors are usually not noticed by farmers or producers. The reason is that most contact insecticides and acaricides are used at a recommended dose that is much higher than 2 to 5 times the minimum effective concentration to kill the parasites. This means that most products will continue to work "well enough", probably with a shorter protection period (= residual effect). The farmers may need to re-treat livestock at shorter intervals. For most systemic insecticides and/or acaricides (e.g. macrocyclic lactones, insect development inhibitors) and for anthelmintics (e.g. benzimidazoles, levamisole, etc), RF of 2 to 5 are already a serious problem because their efficacy at the recommended dose is usually close to the minimum effective concentration to kill the parasites (for several reasons related to product safety, tolerance residues, etc.). In this case a RF of 2 to 5 may already result in product failure.

Resistance factors of 10 to 100 usually result in product failure both for systemic and for non-systemic ectoparasiticides and anthelmintics, regardless of the delivery form. Farmers or producers will quickly notice that treated animals still carry parasites (ticks, flies, etc.). In the case of internal parasites (worms, flukes, etc.) they will notice that weight gains are not OK, many animals show clinical symptoms (e.g. anemia, diarrhea, weakness, etc.).

RF factors >100 mean that the compound has become completely useless.

RF of parasite field strains against organophosphates and amitraz are often <50. The practical consequence is that such products may still provide some level of control, although visibly insufficient. RF of field strains against synthetic pyrethroids are often >100, and can be reached in a few years. In fact, resistance to synthetic pyrethroids is worldwide the highest and most widespread among all ectoparasiticides.

Regarding most anthelmintics, it is quite unusual to determine RF for suspected resistant worm populations found in the field. The reason is that it is more laborious and expensive than for external parasites. As a general rule, RF for these compounds are relatively low (5 to 25) compared with RFs for some ectoparasiticides. But, as already mentioned, even low resistance factors often result in product failure for anthelmintics. An additional difficulty when dealing with anthelmintics is that worms are not "directly visible", in contrast with ticks or flies. Farmers and producers do not "see" that an anthelmintic does not work, at least not quickly, perhaps only months or years after resistance had already developed.

Resistance types

Side-resistance

If a parasite population becomes resistant to an active ingredient, it is most likely that becomes resistant to other active ingredients of the same chemical class as well. This is due to the fact that most active ingredients of the same chemical class have the same mechanism of action at the molecular level. If the parasites "learn" to overcome this mechanism, they will become resistant to all chemicals that have the same mechanism of action. This is usually called side-resistance (sometimes also cross-resistance). But exceptions are known to this. There are e.g. strains of the cattle tick Rhipicephalus (Boophilus) microplus in Australia that are resistant to most synthetic pyrethroids (e.g. cypermethrin, deltamethrin) but are susceptible to flumethrin, another synthetic pyrethroid. This seems to be due to the fact that flumethrin has a somehow different mode of action than other pyrethroids.

Nevertheless it is known that within each chemical class, resistance to some compounds is often stronger than against other ones. For practical purposes this means that such compounds with a weaker resistance can still be used for a certain time. There are cases of cattle ticks that became resistant to coumaphos, but could be controlled with chlorfenvinphos for years, both organophosphates. Housefly (Musca domestica) strains are also known that were resistant to topically applied organophosphates (e.g. dichlorvos, diazinon) but were controlled by azamethiphos, an orally administered organophosphate. And it has also been reported on blowfly larvae (Lucilia spp) that were resistant to diazinon but could be controlled with coumaphos, both organophosphates. 

Cross-resistance

Since there are different chemical classes with the same or similar mechanism of action, it is very likely that parasites resistant to active ingredients of one of these chemical classes will also be resistant to active ingredients of the other chemical classes with the same mechanism of action. This is usually called cross-resistance. It happens e.g. between organophosphates and carbamates, or between some organochlorines and synthetic pyrethroids. Sometimes the term cross-resistance is also used when talking about the previously mentioned side-resistance.

Multiple resistance

A parasite population can become simultaneously resistant to two or more chemical classes with different mechanisms of action. This is known as multiple resistance and the parasites are said do be multi-resistant. In most cases such parasites have developed more than one mechanism of resistance. Within external parasites, multiple resistance has been reported on cattle ticks (Rhipicephalus = Boophilus microplus and R. decoloratus), houseflies (Musca domestica), blowflies (Lucilia spp), red fowl mites (Dermanyssus gallinae), fleas (Ctenocephalides spp) and mosquitoes. It is also a serious problem on gastrointestinal roundworms (e.g. Haemonchus spp, Ostertagia spp, Trichostrongylus spp) of livestock, mainly in sheep and goats.

It seems that once a parasite population has developed resistance to a first chemical class, it is likely that it will develop resistance to second different chemical class faster than to the first one. However, research findings on this issue are not yet conclusive.

Metabolic or biochemical resistance

Parasites can develop resistance to an active ingredient by breaking down the toxic compound (so-called detoxification) into other molecules that are no more toxic to them. This is often achieved through specific enzymes. This mechanism often includes the acquired capacity of the parasites to produce much more quantities of such enzymes. Basically the toxic active ingredient is metabolized through biochemical mechanisms.

Physiological resistance

Several resistance mechanisms do not break down the toxic molecules. But the parasites change their normal physiological processes in order to make the pesticide harmless. This can be achieved by reducing the penetration through the cuticle, modifying the target sites of pesticides at the molecular level, increasing the excretion of the parasiticide, etc.

Behavioral resistance

Some parasites become resistant by changing their behavior in a way that it results in reduced contact or exposure to the parasiticide. This has been observed e.g. in houseflies that avoid scatter-baits containing sugar, or in horn flies that landed on the belly of cattle instead of doing it on the back where the insecticide concentration was highest.

Obviously this does not mean that single parasites "learn" to avoid a parasiticide perceiving that it is something dangerous, as a human person could do. What happens is that within a parasite population there is often a very small percentage of individuals that behave differently. If these individuals are the only ones that survive exposure to a parasiticide (i.e. are selected by the parasiticide), their offspring will inherit such behavior. If the selection by the pesticide is maintained, this different behavior will become dominant in the population after several generations.

Resistance Mechanisms

Metabolic and physiologic resistance is achieved through several mechanisms at the cellular or molecular level, the major ones being:

• Enhanced detoxification
• Enhanced excretion or sequestration
• Target-size insensitivity
• Decreased penetration through the cuticle

Enhanced detoxification

Most living organisms can break down (metabolize) pesticides (and many other molecules) that get into their organism and make them harmless. Specific enzymes do this by oxidizing, hydrolyzing or otherwise degrading the intruding molecules. Parasites, even non-resistant ones can do this as well. The problem for parasites is often that the speed at which this happens is not fast enough to keep the concentration of the toxic parasiticide in their body below the damage threshold, because parasiticides are usually administered at massive concentrations.

But some resistant parasites are capable of detoxifying substantially higher quantities of pesticides. They produce much more quantities of such detoxifying enzymes (e.g. because the gene that codes for these enzymes has multiple copies in their genome), or they produce varieties of such enzymes that are much more effective or can break down additional types of molecules, etc. The bottom line is that such resistant parasites are capable of breaking down the parasiticide before it reaches harmful concentrations in their bodies. As a consequence the harmful concentration of a parasiticide is substantially higher for resistant parasites than for susceptible ones.

Cytochrome P450 and mixed-function oxidases (MFOs) are two enzyme families that are often involved in increased detoxification by resistant insects and ticks.

The oldest reported case of metabolic resistance is housefly resistance to DDT, an organochlorine insecticide.

Enhance excretion or sequestration

It has be found that some roundworms resistant to benzimidazoles and macrocyclic lactones (e.g. ivermectin) are capable of pumping out of their cells the toxic molecules very quickly and efficiently. This is the case in some resistant strains of Haemonchus spp, the barber's pole worms of cattle and sheep. In worms of these resistant strains the P-glycoprotein is much more abundant than in susceptible strains. This protein transports many natural molecules across the cell membrane, and also anthelmintic molecules. The result is that the toxic anthelmintic active ingredients are very quickly excreted out of the worm cells, before they can cause harm to the cell organelles.

Another related mechanism observed in some insects resistant to DDT consisted in sequestrating the toxic DDT molecules into the fat bodies, storage organs of many insects. This way they prevented the toxic molecules from reaching their target site in the nervous system.

 

 

Target site-insensitivity

Most parasiticidal molecules have a "target site" inside the parasite's organism where they dock to. Such target sites are often receptors in specific cell structures. These receptors bind usually to other molecules called ligands in order to accomplish a particular function. These receptors accomplish essential vital functions in the parasite (e.g. transmitting nervous signals). Parasites bind to some of these receptors as well, blocking the natural ligands. This way the parasiticide interrupts the essential function, which usually kills the parasite.

Both receptors and ligands are complex proteins that work like a key in a lock. Some parasites are resistant to parasiticides because the receptors (i.e. the target sites) to which the parasiticides should bind to become toxic are slightly different. The lock has been changed and the key can't open it any more. The consequence is that the parasiticide doesn't work at all.

A well-known example of resistance due to target-site insensitivity is altered acetylcholinesterase in many insects and ticks. This enzyme is involved in the transmission of nervous signals and is blocked by organophosphates and carbamates.

This mechanism has also been found in roundworms. The target site of most anthelmintic benzimidazoles is tubulin, a protein that is the major constituent of microtubuli. Microtubuli are cell organelles essential for many cellular functions (e.g. motility, food intake, cell division, etc.). It has been shown that changing a single amino acid in the structure of the tubulin molecule is enough to substantially reduce its affinity for benzimidazoles without impairing its normal functioning in the cell.

A comparable target size modification has been found in roundworms resistant to ivermectin. In this case the modified receptor is a molecule in the so-called glutamate-gated chloride channels in the cell membrane, the normal target site for macrocyclic lactones. In such resistant roundworms ivermectin does not block this channel anymore, or only at a lower degree.

Decreased penetration

Most insecticides and acaricides work by contact. When animals that carry parasites are sprayed or dipped, the ticks, lice or mites they carry are virtually immersed in the parasiticide. Most parasiticidal active ingredients are lipophilic molecules that dissolve in the waxy layers of the cuticle of arthropods. Afterwards they are absorbed inside the parasite's body where they exert their toxic action. It has been found that some resistant houseflies have a modified cuticle that does not let the toxic parasiticide into the parasite's body.