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15.4G: Paralysis-Causing Bacterial Neurotoxins - Biology

15.4G: Paralysis-Causing Bacterial Neurotoxins - Biology


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Botulinum toxin is a protein and neurotoxin, which blocks neuromuscular transmission through decreased acetylcholine release.

Learning Objectives

  • Describe the mechanism of action for botulinum toxin

Key Points

  • Botulinum toxin is produced by Clostridium botulinum, C. butyricum, C. baratii and C. argentinense.
  • The light chain of botulinum toxin is an enzyme (a protease ) that attacks one of the fusion proteins (SNAP-25, syntaxin or synaptobrevin) at a neuromuscular junction, preventing vesicles from anchoring to the membrane and releasing acetylcholine.
  • The heavy chain of the toxin is particularly important for targeting the toxin to specific types of axon terminals.

Key Terms

  • neurotoxin: A toxin that specifically acts upon neurons, their synapses, or the nervous system in its entirety.
  • acetylcholine: A neurotransmitter in humans and other animals. It is an ester of acetic acid and choline with chemical formula CH3COOCH2CH2N+(CH3)3.
  • axon: A nerve fibre, which is a long slender projection of a nerve cell, and which conducts nerve impulses away from the body of the cell to a synapse.

Botulinum toxin is a protein and neurotoxin produced by Clostridium botulinum, C. argentinense. Botulinum toxin can cause botulism, a serious and life-threatening illness in humans and animals. In 1949, Arnold Burgen’s group discovered, through an elegant experiment, that botulinum toxin blocks neuromuscular transmission through decreased acetylcholine release. In 1973, Alan Scott used botulinum toxin type A (BTX-A) in monkey experiments. In 1980, he officially used BTX-A for the first time in humans to treat “crossed eyes” (strabismus), a condition in which the eyes are not properly aligned with each other, as well as “uncontrollable blinking” (blepharospasm). In 1993, Pasricha and colleagues showed that botulinum toxin could be used for the treatment of achalasia, a spasm of the lower esophageal sphincter. In 1994, Bushara showed botulinum toxin injections inhibit sweating; this was the first demonstration of non-muscular use of BTX-A in humans. The cosmetic effect of BTX-A on wrinkles was first reported by J. D. and J. A. Carruthers in a 1992 study on BTX-A for the treatment of glabellar frown lines. The acceptance of BTX-A use for the treatment of muscle pain disorders is growing, with approvals pending in many European countries. The efficacy of BTX-A in treating a variety of other medical conditions (including prostatic dysfunction, asthma, and others) is an area of continued study.

Foodborne botulism can be transmitted through food that has not been heated correctly prior to being canned, or food from a can that has not been cooked correctly. Most infant botulism cases cannot be prevented because the bacteria that cause this disease are in soil and dust. The bacteria can also be found inside homes on floors, carpet, and countertops, even after cleaning. Honey can contain the bacteria that cause infant botulism, so children less than 12 months old should not be fed honey.

Botulinum toxin is a two-chain polypeptide with a 100-kDa heavy chain joined by a disulfide bond to a 50-kDa light chain. This light chain is an enzyme (a protease) that attacks one of the fusion proteins (SNAP-25, syntaxin or synaptobrevin) at a neuromuscular junction, preventing vesicles from anchoring to the membrane to release acetylcholine. By inhibiting acetylcholine release, the toxin interferes with nerve impulses and causes flaccid (sagging) paralysis of muscles in botulism, as opposed to the spastic paralysis seen in tetanus. The heavy chain of the toxin is particularly important for targeting the toxin to specific types of axon terminals. The toxin must get inside the axon terminals to cause paralysis. Following the attachment of the toxin heavy chain to proteins on the surface of axon terminals, the toxin can be taken into neurons by endocytosis. The light chain is able to cleave endocytotic vesicles and reach the cytoplasm. The light chain of the toxin has protease activity. The type A toxin proteolytically degrades the SNAP-25 protein, a type of SNARE protein. The SNAP-25 protein is required for vesicle fusion that releases neurotransmitters from the axon endings (in particular acetylcholine). Botulinum toxin specifically cleaves these SNAREs, and so prevents neurosecretory vesicles from docking/fusing with the nerve synapse plasma membrane and releasing their neurotransmitters.


Examples of toxin in the following topics:

Paralysis-Causing Bacterial Neurotoxins

  • In 1973, Alan Scott used botulinum toxin type A (BTX-A) in monkey experiments.
  • The heavy chain of the toxin is particularly important for targeting the toxin to specific types of axon terminals.
  • The toxin must get inside the axon terminals to cause paralysis.
  • Following the attachment of the toxin heavy chain to proteins on the surface of axon terminals, the toxin can be taken into neurons by endocytosis.
  • The light chain of the toxin has protease activity.

Toxins

  • Some of the major types of toxins include, but are not limited to, environmental, marine, and microbial toxins.
  • Microbial toxins may include those produced by the microorganisms bacteria (i.e. bacterial toxins) and fungi (i.e. mycotoxins).
  • Mycotoxins are the classes of toxins produced by fungi.
  • The most potent toxin is AFB1 and it is associated with carcinogenic effects.
  • Describe the major toxin types (bacterial toxins and mycotoxins) and their mechanisms of action

Diphtheria

  • The toxin can cause damage to the heart that affects its ability to pump blood or the kidneys' ability to clear wastes.
  • Diphtheria toxin is produced by C. diphtheriae only when it is infected with a bacteriophage that integrates the toxin-encoding genetic elements into the bacteria.
  • This signals the cell to internalize the toxin within an endosome via receptor-mediated endocytosis.
  • Inside the endosome, the toxin is split by a trypsin-like protease into its individual A and B fragments.
  • Corynebacterium diphtheriae produces toxins that can affect the skin by causing skin lesions, as shown here.

Botulism

  • Botulism is a rare, but sometimes fatal, paralytic illness caused by botulinum toxin.
  • This toxin is a protein produced under anaerobic conditions by the bacterium Clostridium botulinum.
  • Botulinium toxin is one of the most powerful known toxins: about one microgram is lethal to humans.
  • Three main modes of entry for the toxin are known.
  • The bacterium then releases the toxin into the intestine, which is absorbed into the bloodstream.

Staphylococcal Food Poisoning

  • Staphylococcal toxins are a common cause of food poisoning, as they can be produced in improperly-stored food.
  • Foodborne disease can also be caused by a large variety of toxins that affect the environment such as pesticides or medicines in food and naturally toxic substances such as poisonous mushrooms or reef fish.
  • Staphylococcus is a Gram-positive bacteria which includes several species that can cause a wide variety of infections in humans and other animals through infection or the production of toxins.
  • Staphylococcal toxins are a common cause of food poisoning, as they can be produced in improperly-stored food.
  • Toxins for bacterial infections are delayed because the bacteria need time to multiply.

Human Health and Biodiversity

  • Maintaining biodiversity ultimately helps maintain of human health many medicines are derived from plants and, recently, animal toxins.
  • Most plants produce secondary plant compounds, which are toxins used to protect the plants from insects and other animals that eat them, but some of which also work as medicines.
  • By 2007, the FDA had approved five drugs based on animal toxins to treat diseases such as hypertension, chronic pain, and diabetes.
  • Other toxins under investigation come from mammals, snakes, lizards, various amphibians, fish, snails, octopuses, and scorpions.

Direct Damage

  • Upon the use of host nutrients for its own cellular processes, the bacteria may also produce toxins or enzymes that will infiltrate and destroy the host cell.
  • Examples of bacteria that will damage tissue by producing toxins, include, Corynebacterium diphtheriae and Streptococcus pyogenes.
  • It produces a toxin, diphtheria toxin, which alters host protein function.
  • The toxin can then result in damage to additional tissues including the heart, liver, and nerves.

Genetically Modified Organisms (GMOs)

  • Bt toxin has to be ingested by insects for the toxin to be activated.
  • Insects that have eaten Bt toxin stop feeding on the plants within a few hours.
  • After the toxin is activated in the intestines of the insects, death occurs within a couple of days.
  • Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects.
  • The crystal toxin genes have been cloned from Bt and introduced into plants.

Peroxisomes

  • Peroxisomes neutralize harmful toxins and carry out lipid metabolism and oxidation reactions that break down fatty acids and amino acids.
  • Like miniature sewage treatment plants, peroxisomes neutralize harmful toxins so that they do not cause damage in the cells.

Scarlet Fever

  • Scarlet fever is caused by an erythrogenic toxin, a substance produced by the bacterium Streptococcus pyogenes (group A strep. ) when it is infected by a certain bacteriophage.
  • Exotoxin A (speA) is probably the best studied of these toxins.
  • It is carried by the bacteriophage T12, which integrates into the Streptococcal genome, from where the toxin is transcribed.
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We found at least 10 Websites Listing below when search with toxins that cause paralysis on Search Engine

Toxicants that Cause Paresis or Paralysis IVIS

Ivis.org DA: 12 PA: 50 MOZ Rank: 62

  • Botulism is the disease caused by any one of 7 serotypically different, but functionally similar toxins produced by strains of Clostridium botulinum
  • They are among the most potent toxins known
  • Of these 7 serotypes (A-G), types C and D have historically been the most commonly implicated in domestic mammals and poultry.

15.4G: Paralysis-Causing Bacterial Neurotoxins

  • By inhibiting acetylcholine release, the toxin interferes with nerve impulses and causes flaccid (sagging) paralysis of muscles in botulism, as opposed to the spastic paralysis seen in tetanus
  • The heavy chain of the toxin is particularly important for …

Is there a poison that causes a temporary paralysis

  • Belladonna and Hemlock are neuromuscular toxins and can cause paralysis
  • Here and here you can read the treatments
  • Finally, wolfsbane and foxglove also cause symptons similar to paralysis
  • Both are very pretty flowers you could have in your garden without raising suspicion.

Paralysis: Causes, Symptoms, Diagnosis & Treatment

  • Toxins/poisons ALS (Lou Gehrig’s disease) In certain cases, some or all muscle control and feeling returns on its own or after treatment of the cause for the paralysis
  • For example, spontaneous recovery often occurs in cases of Bell’s palsy, a temporary paralysis of the face
  • It might also occur to some extent with treatment after a

Toxin From Tick Bite Paralyses Girl, Stumps Physicians

Webmd.com DA: 13 PA: 50 MOZ Rank: 67

  • 12, 2000 (Washington) -- While it is known that certain ticks carry Lyme disease, the common wood tick can cause paralysis and even death if it is not promptly removed.

Dangerous toxin, potentially causing paralysis, closes

Tvnz.co.nz DA: 14 PA: 50 MOZ Rank: 69

  • It comes as a dangerous toxin that can cause paralysis and respiratory failure has been found in shellfish in the region - including mussels, kina, cockles and tuatua

What toxins can paralyze humans but not kill them

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  • Poisoning with Clostridium botulinum toxin - this induces a gradual (a few hours) descending paralysis (difficulty keeping eyelids open, then smiling, swallowing and eventually breathing)
  • Likely to be fatal without extreme interventions like a heart lung machine, recovery is possible, but takes a few months and may be incomplete.

What are the Common Causes of Paralysis in Dogs

  • Exposure to toxins, pesticides and rodenticides can be a common cause of paralysis
  • Some ticks carry a salivary toxin that can cause life-threatening paralysis in dogs
  • Poisoning with botulinum toxin can also lead to life-threatening canine paralysis
  • Dogs are most likely to ingest botulinum toxin in contaminated food.

Life-Threatening Side Effect of Chemical Exposure

  • The inhalation of chemicals can lead to breathing problems that range from minor to severe
  • In the most severe cases, the sufferer may stop breathing altogether
  • Chemical exposure can cause a person to be unable to move in serious cases
  • In some instances, the paralysis only affects part of the body.

Cone Snail Toxins and Paralysis

  • This animation shows how cone snail toxins cause paralysis by blocking signal transmission at the synapse between neuron and muscle
  • Predatory cone snails use venom to paralyze their prey, such as fish
  • This animation illustrates how multiple toxins in the venom work together to induce paralysis.

Some plant foods causing paralysis, death, in malnourished

Neurologic disease caused by food toxins is probably more common than that caused by food contamination with manmade chemicals like the infamous Minamata poisonings of …

Toxicants that Cause Primary Muscle Dysfunction and/or

Ivis.org DA: 12 PA: 50 MOZ Rank: 73

  • (See Toxicants that Cause Paralysis) Avocado (Persea americana) [See Galey F
  • In: Colgate SM and Dorling PR (eds.) Plant Associated Toxins: Agricultural, Phytochemical and Ecological Aspects
  • - Available from amazon.com -] (See Toxicants with Mixed Effects on the Central Nervous System) Thermopsis montana

When Edible Plants Turn Their Defenses On Us : The Salt : NPR

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  • The grass pea contains β-ODAP, a toxin that can cause partial paralysis when eaten too often
  • Rae Ellen Bichell/NPR Paralysis Pea: Because it's …

Toxic Neuropathy Treatment Signs, Symptoms, Treatment

  • Toxins, poisons and chemicals can cause peripheral neuropathy
  • This can happen through drug or chemical abuse or through exposure to industrial chemicals in the workplace or in the environment (after either limited or long-term exposure)
  • Common toxins that cause neuropathy include: exposure to lead, mercury, arsenic and thalium.

Mold Toxicity: A Common Cause of Psychiatric Symptoms

Toxic mold based illness is a very prevalent and under diagnosed condition that can manifest in many different ways, including with symptoms …

Pesticides and Polio: A Critique of Scientific Literature

  • These four chemicals were not selected arbitrarily
  • These are representative of the major pesticides in use during the last major polio epidemic
  • They persist in the environment as neurotoxins that cause polio-like symptoms, polio-like physiology, and were dumped onto and into human food at dosage levels far above that approved by the FDA.

Tick Paralysis: Causes, Symptoms, Diagnosis, Treatment

Webmd.com DA: 13 PA: 50 MOZ Rank: 79

  • The American dog tick, Rocky Mountain wood tick, and dermacentor ticks are most likely to cause tick paralysis
  • But 40 kinds of ticks can cause tick paralysis, and the symptoms depend on the

New Strategy May Help Reverse Paralysis From The Worlds

Iflscience.com DA: 18 PA: 50 MOZ Rank: 85

Dong, who headed the second research team, clarified the modified version of the toxin Ichtchenko's team created was still able to cause paralysis

Dog Paralysis: Common Causes & Treatment Canna-Pet

Canna-pet.com DA: 13 PA: 39 MOZ Rank: 70

  • Surprisingly, the leading cause of paralysis in dogs is tick bites
  • Certain species of ticks can inject a neurotoxin into your dog’s bloodstream when they bite
  • This toxin can cause a sudden neuron paralysis, which in some cases, may result in sudden paralysis
  • When this happens, you need to address the issue as quickly as possible.

Tetanus toxin and botulinum toxin a utilize unique

  • Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most toxic proteins for humans
  • While BoNTs cause flaccid paralysis, TeNT causes spastic paralysis
  • Characterized BoNT serotypes enter neurons upon binding dual receptors, a ganglioside and a neuron-specific protein, either synaptic …

Botulinum toxins--cause of botulism and systemic diseases

  • Toxins of Clostridium botulinum (types A-G) are known as 'neurotoxins', causing the clinically well-known picture of flaccid muscular paralysis
  • The molecular biological background is the blocking of acetylcholine secretion in neuromuscular junctions by enzymatic cleavage of molecules forming the machinery of exocytosis.

Cat Paralysis: Causes, Symptoms, & Management Canna-Pet®

Canna-pet.com DA: 13 PA: 42 MOZ Rank: 76

  • Paralysis can take place anywhere throughout the body, and can either be partial (paresis) or complete (paralysis)
  • The symptoms of paralysis may vary from subtle to obvious, depending on the root cause
  • Symptoms of cat paralysis may occur suddenly—referred to as acute paralysis—or escalate over a period of time.

Food-borne toxins Flashcards Quizlet

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rare since modern sterilization methods, ingestion of toxin causes paralysis of motor and autonomic nerves, high percentage die of respiratory failure- long term respiratory damage is a risk, most frequent source is home canned goods, infant botulism is infection by spores, followed by production of toxin in the digestive tract- honey is a reservoir of spores,


HARMFUL ALGAL TOXINS OF PRIMARY CONCERN

The most prevalent marine HAB toxins are known by the way they affect public health through seafood-borne intoxication, usually from filter-feeding shellfish (clams, mussels, and oysters) but also from fish (e.g., ciguatera poisoning). Ciguatera poisoning has been recognized since the 1500s and is widely distributed throughout tropical regions [ 16 ]. Responsible for more human intoxications from seafood than any other HAB, ingestion of as little as 0.1 μg of ciguatoxin can cause illness in an adult human. Symptoms of ciguatoxin poisoning are abdominal cramps, nausea, diarrhea, paresthesia of the lips and extremities, reversal of hot and cold sensation, weakness, dizziness, and, in severe cases, acute respiratory failure and coma [ 14, 17-19 ].

Ciguatoxins consist of a suite of polyether toxins produced by tropical benthic dinoflagellates (Fig. 1). Maitotoxins are water-soluble polyether toxins associated with ciguatoxin, originally found in fish in association with ciguatera poisonings but now known to be produced by the dinoflagellate [ 12, 18, 20, 21 ]. Dinoflagellates of the genus Gambierdiscus have been implicated and species from the genera Prorocentrum and Ostreopsis are suspect in ciguatoxic poisonings [ 20, 21 ]. These toxins bioaccumulate in the food chain, rendering large carnivorous fishes poisonous in ciguatera endemic areas. Ciguatoxins act on voltage-sensitive sodium channels in nerve cells, activating the sodium channel that depolarizes the cell potential, resulting in repetitive, uncontrolled firing of nerve impulses [ 11, 14, 19, 22 ].

Basic structures for representative toxin molecules [ 11, 16, 27 ]. Brevetoxin (suite of polyether toxins responsible for neurotoxic shellfish poisoning) type-1 = PbTx-1, 7, 10 Type-2 = PbTx-2, 3, 5, 6, 8, 9 okadaic acid (toxins causing diarrhetic shellfish poisoning) includes dinophysistoxins ciguatoxin (primary toxin responsible for ciguatera poisoning) domoic acid (neurotoxic amino acid responsible for amnesic shellfish poisoning) saxitoxin (primary structure for suite of toxins causing paralytic shellfish poisoning) includes neosaxitoxin and a suite of gonyautoxins.

Paralytic shellfish poisoning (PSP) is another historically significant shellfish toxin recognized as a human health problem since the mid-1700s. The PSP toxins consist of a suite of neurotoxins, known as saxitoxins, neosaxitoxin, and gonyau-toxins (Fig. 1), that affect voltage-gated sodium channels in nerve cell membranes. These toxins are sodium channel block-ers, interfering with nerve signal transmission. The PSP occurs worldwide in temperate coastal regions, causing severe public health problems from ingestion of contaminated shellfish.

Symptoms of PSP poisoning begin with paresthesia of the mouth and face progressing to the extremities, followed by nausea, cramps, diarrhea, dizziness, weakness, and respiratory paralysis, causing death [ 9, 11, 16 ]. Saxitoxins also have been implicated in the deaths of marine mammals as a result of bioaccumulation in the food chain [ 8, 23 ]. Three genera of marine dinoflagellates have been implicated in the production of PSP toxins, Alexandrium, Pyrodinium, and Gymnodinium [ 23, 24 ]. Recent studies have found that cyanobacteria also produce saxitoxins [ 3, 25 ], and evidence for production of sax-itoxins by marine bacteria is also mounting [ 26 ].

The dinoflagellate responsible for the Florida red tide, Gymnodinium breve, produces a suite of polyether neurotoxins [ 11, 27 ]. Adverse effects from G. breve blooms were reported in the mid-1800s, with documented massive fish kills, poisonous clams, and associated respiratory irritation. Found primarily in the Gulf of Mexico, G. breve has been transported by ocean currents to the Atlantic seacoast [ 28 ]. Gymnodinium breve produces a suite of at least nine closely related polyether neurotoxins, called brevetoxins [ 27, 29 ] (Fig. 1). These compounds produce neurotoxic effects in the same way as the ciguatoxins by activating the sodium channel in nerve cell membranes, thus depolarizing cell potential [ 11, 30, 31 ]. The resulting intoxication from ingesting brevetoxin-contaminated shellfish is called neurotoxic shellfish poisoning, with symptoms similar to those from ciguatoxin poisoning [ 9, 11 ]. Brevetoxins were implicated in the deaths of more than 150 manatees in Florida, USA, in 1996 [ 5, 32 ] and more than 100 dolphins from 1999 to 2000 (B. Mase, personal communication).

A unique characteristic of G. breve blooms is the associated airborne (aerosolized) toxin component [ 10 ]. Because these dinoflagellates are unarmored, the cells lyse, releasing brevetoxins into the water column (www.utas.edu.au/docs/plants_science/HAB2000/abstracts/index.html) [ 33 ]. When breaking waves are present, the toxins become incorporated into marine aerosol by bubble-mediated transport, causing severe respiratory irritation to humans and other mammals [ 10 ]. Recent evidence implicates another species of microalgae found in the coastal waters of the Gulf of Mexico and off the coast of Japan (the Raphidophycean, Chattenonella) as a producer of brevetoxin-like neurotoxins [ 34 ].

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness that was first reported in the 1960s [ 12, 35 ]. Caused by ingestion of shellfish that have consumed toxic dinoflagellates from the genera Dinophysis and Prorocentrum, the primary toxins are okadaic acid, a polyether toxin, and its derivatives, the dinophysistoxins (Fig. 1) [ 20, 36 ]. The DSP toxins inhibit protein phosphatase enzyme systems, having profound effects on smooth muscle contraction [ 35, 37 ]. Two additional types of toxins that have been implicated in association with DSP are pectenotoxins and yessotoxins, found in both shellfish and DSP-causing dinoflagellates [ 38-40 ]. Although associated with DSP and PSP toxic episodes, the molecular structure of yessotoxins is a polycyclic ether similar to that of ciguatoxins and brevetoxins [ 39 ].

More recent HAB toxin discoveries have resulted from newly observed human intoxication and environmental effects. Amnesic shellfish poisoning was first recorded in Canada in 1987, causing short-term memory loss in people who had eaten clams collected from certain areas around Prince Edward Island. It has since been observed along the Pacific coast, in Europe, and in Japan. The causative agent is domoic acid (Fig. 1), produced by the diatom Pseudo-nitzschia sp. [ 11 ]. Amnesic shellfish poisoning intoxication is manifest as an excitatory neurotransmitter and has been found to cause lesions in human brain [ 11, 41 ].

Pfiesteria piscicida was only recently discovered as the causative organism for fish kills in estuaries of North Carolina, Maryland, and Delaware, USA [ 42-45 ]. It is an ambush-predator dinoflagellate with an unusual life cycle including many life stages of zoospores, amoebas, and cysts along with the motile dinoflagellate stage, which apparently produces toxins in the presence of certain stimuli [ 43 ]. Public health effects are reported to be cognitive function impairments that are reversible three to six months after exposure. Considerable effort is being directed toward isolation and identification of the toxins however, the complex life cycle and uncertain conditions surrounding the toxin-producing stage have hindered isolation and identification of the toxin(s) [ 43, 45 ].

A new toxic shellfish poisoning surfaced in Europe in 1995, leading to the identification of a new toxic syndrome called azaspiracid poisoning, which produces symptoms similar to shellfish neurotoxic poisoning. The causative agent is a novel spiro ring compound thought to be produced by a dinoflagellate [ 46 ].


CA2752820A1 - Electrotaxis methods and devices - Google Patents

[0001] The invention generally relates to methods which utilize electrotaxis, and in particular, relates to methods of controlling the movement of nematodes in a microfluidic environment, and devices useful to conduct such methods.

BACKGROUND OF THE INVENTION

6-OHDA is also endogenously produced by the dopamine neurons as a byproduct of dopamine and inhibits the mitochondrial respiratory enzyme complex I and IV. MPP+ is the active toxic product of MPTP that inactivates the mitochondrial enzyme complex I of respiratory chain.

Toxin exposed worms have been shown to serve as effective PD models to study the basis of neurodegeneration. These models also facilitate screening of genes and compounds that protect neurons from toxin-induced damage.

[0006] Among the various features of C. elegans, its small size and the ability to grow in liquid media have facilitated high throughput screenings (HTS) for chemicals.
Chemicals which affect physiological processes, thus, may serve as potential drug candidates for a variety of medical applications. Conventional methods of chemical and animal screening involve exposure of a certain population of synchronized-age or -mutant model animals to thousands of chemical compounds individually and inside multi-well plate dishes, while monitoring the subsequent effects of the drugs on animals' growth, fertility, and other biological processes by immobilization and visual inspection. The above-mentioned methods are either manual and hence prone to human errors and time-consuming, or robotically automated and hence expensive and inaccessible to the majority of researchers. In addition, most conventional plate-based methods are currently focused on the cellular level analysis and tend to ignore the movement behaviour of C. elegans as one the most important parameters (especially for movement disorder diseases). Cellular level analysis is mostly done through immobilization and GFP imaging.
Methods for immobilizing worms (anesthesia or gluing) are fatal, non-reversible and not suited for post-experimentation studies. The glue or anaesthetic chemical compositions' effect on C.
elegans biological processes is also not well understood.

[0007] Microsystem engineering has played a critical role in providing the necessary technologies to tackle the challenges of small organisms' manipulation and analysis. C. elegans worms survive in liquid environment and due to their matching size scale with microfluidics (submicron to hundreds of micrometers), they have recently been studied in such devices. This has resulted in a dramatic increase in the experimental accuracy, consistency (by removing human interferences) and decrease in the cost of automation. Recently, microfluidic devices have been used for more precise and quantitative analysis of C. elegans development and behaviour.
These devices have also been used for analyzing nematodes behaviour and mechanical characteristics in response to diverse stimuli, in-vivo imaging of their neuronal activity, culturing, sorting and screening, and in-vivo studies of neuronal regeneration after laser nano and micro-surgery on individual animals. These microfluidic devices have significantly facilitated assays on worms in an automated high throughput manner. In order to visualize and manipulate animals within these environments, their natural movement is eliminated by the use of hydraulic and pneumatic flows and forces. Accordingly, these devices are complicated in terms of fabrication (multilayers of PDMS microstructures aligned and bonded together) and operation (computer-controlled pneumatics). Also, despite being advantageous in phenotypic, cellular and sub-cellular studies, these devices are not suitable for performing movement-related behavioural studies on worms.

[0010] In another aspect, a method of sorting nematodes based on a selected parameter is provided comprising the step of exposing nematodes to an electric field that induces a differential response among the nematodes based on the selected parameter, wherein the differential response functions to separate the nematodes based on the selected parameter.

[00111 In another aspect, a microfluidic sorting device useful to sort nematodes is provided comprising:

a nematode reservoir that feeds into a separation channel having a proximal end and a distal end

a series of collection channels that extend perpendicularly from the separation channel along the length of the separation channel, wherein each collection channels houses a collection electrode

an accumulation electrode adjacent to the reservoir at the proximal end of the separation channel and a separation electrode at the distal end of the separation channel.

[0012] In another aspect, a field flow fractionation device is provided comprising at least one separation channel having an inlet at its proximal end and a plurality of collection channels at its distal end, wherein the separation channel comprises a plurality of micropillars spaced throughout the separation channel which function to maintain non-responsive nematodes moving towards the collection channels, and separation electrodes positioned along each side of the separation channel to provide an electric field within the separation channel perpendicular to a flow from the inlet to the collection channels.

[0013] A continuous flow sorter device is provided in a further aspect comprising:
multiple parallel microchannels, each of said microchannels comprising an inlet at a proximal end and a collection chamber at a distal end thereof, wherein each of said microchannels is separated by an array of linearly spaced micropillars, wherein the micropillars are equidistantly spaced between each microchannel and wherein the spacing between micropillars decreases from the first microchannel and for each subsequent microchannel and an electrode adjacent to the first and last microchannels of the multiple microchannels for the application of an electric field across the width of the device.
[0014] A nematode storage microchamber device is provided in another aspect.
The device comprises a central chamber comprising an inlet channel through which worms can be loaded into the device and an outlet channel for removal of the worms and an entropic trap comprising an electrode connecting each of said inlet and outlet channels to the central chamber, wherein said entropic trap prevents the movement of worms therethrough except on application of an electric field across the trap.

[0015] In a further aspect, a microfluidic channel array is provided comprising: an array of parallel microchannels a worm storage unit at a proximal end of each microchannel an electrode-based worm detection unit at the proximal and distal ends of each microchannel and an injection channel and fluid circulating means connected to each microchannel.

[0016] In another aspect, a method of screening candidate compounds that affect nematodes is provided comprising:

i) measuring the movement characteristics of one or more nematodes
ii) exposing the nematodes to a candidate compound and iii) measuring the movement characteristics of the nematode following exposure to the compound, wherein a change in the movement characteristics of the nematode is indicative that the compound affects the nematode.

[0017] These and other aspects of the invention are described in the detailed description by reference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 illustrates a DC electrotaxis device (a) and a microchannel of the device (b)

[0019] Figure 2 graphically illustrates the effect of electric field on movement speed of different developmental stages of C. elegans

[0020] Figure 3 illustrates separation of two C. elegans animals at different developmental stages on application of 4 V/cm electric field

[0021] Figure 4 graphically illustrates the average speed of various C.
elegans animals in a microchannel on application of an electric field

[0022] Figure 5 graphically illustrates DC electrotaxis speed of C. briggsae at various electric field strengths

[0023] Figure 6 illustrates an electrotaxis device useful to study electrotactic response to AC electric field

[0024] Figure 7 graphically illustrates the AC electric field response of various C.
elegans animals

[0025] Figure 8 shows the Pulse DC electric field signal wave-shape

[0026] Figure 9 graphically illustrates the effect of duty cycle on C. elegans pulse DC
electrotaxis forward motion speed

[0027] Figure 10 shows the effect of duty cycle on C. elegans pulse DC
electrotaxis turn response time (a) and percentage of responders (b)

[0028] Figure 11 graphically illustrates the effect of frequency on C. elegans and C.
briggsae pulse DC electrotaxis forward motion speed

[0029] Figure 12 shows the percentage of responding C. elegans in less than (a) 40 s, (b) 20 s, (c) 10s, and (d) 5 s, to pulse DC electric field at different frequencies and duty cycles
[0030] Figure 13 shows the average response time of adult worms to pulse DC
electric fields at different frequencies and duty cycles

[0031] Figure 14 shows an electrotaxis-based separation device for selecting C. elegans worms with identical electrotactic response

[0032] Figure 15 shows a field flow fractionation device for C. elegans
[0033] Figure 16 shows an electric trap device for electrotactic separation

[0034] Figure 17 shows an electric trap device (a) and electrotaxis response of C. elegans in the electric trap device (b)

[0035] Figure 18 shows a continuous flow worm sorter

[0036] Figure 19 shows the separation of adult worms from a mixture of adult and L3 stage worms in continuous worm sorter device at various flow rates and electric fields (a), as well as the population distribution among 4 output channels in the device with no electric field and a flow rate of 10 L/min (b) and with an electric field of 4V/cm and 10 L/min flow rate (c)

[0037] Figure 20 shows a semi-continuous electrotactic sorter

[0038] Figure 21 shows electrotactic behaviour and sorting of YA/L4 (a) and YA/L3 (b) mixed C. elegans samples in semi-continuous sorter device

[0039] Figure 22 shows electrotactic sorting of YA and neuronal mutant (a) and YA and muscle mutant (b) mixed C. elegans samples in semi-continuous sorter device

[0040] Figure 23 shows electrotactic sorting of young and old adult C. elegans mixed sample in semi-continuous sorter device

[00411 Figure 24 shows a storage ring channel for collection of separated worm with narrow distribution of electrotactic response

[0042] Figure 25 illustrates a microchannel array for parallel experimentation on multiple worms

[0043] Figure 26 shows an optical imaging chamber for worms

[0044] Figure 27 graphically illustrates movement behavior of worms treated with 6-OHDA using an electrotaxis assay

[0045] Figure 28 shows electrotactic analysis of movement behavior of worms exposed to Rotenone

[0046] Figure 29 shows electrotactic analysis of movement behavior of worms exposed to MPTP

[0047] Figure 30 shows plate level analysis of 6-OHDA exposed worms
[0048] Figure 31 shows plate level analysis of MPTP exposed worms
[0049] Figure 32 shows plate level analysis of Rotenone exposed worms

[0050] Figure33 graphically illustrates YFP expression in a transgenic animal that expresses the DA transporter (a) and YFP expression following toxin-induced degeneration of DA neurons (b) and [0051] Figure 34 shows analysis of movement behavior of Acetaminophen pre-exposed worms against 6-OHDA, MPTP and Roteneone using electrotaxis assay.

DETAILED DESCRIPTION OF THE INVENTION

[0052] A method of inducing a response in a nematode in a microfluidic environment is provided comprising exposing the nematode to an electric field that induces the selected nematode response. The response induced by an electric field, such as a movement-induced response i.e. electrotaxis, has been determined to be reliable and highly reproducible. The electric field can be used as a powerful stimulus to efficiently control and orient nematodes as desired. In addition, exposure to the electric field is not harmful to nematodes since they continue to live normally and remain fertile following exposure to an electric field.

[0053] The term "nematode " is used herein to refers to organisms of the animal phylum "Nematoda", e.g. round worms, and particularly, to any round worm with amphid neurons that is expected to generate a response on exposure to an electric field. Examples of nematodes include, but are not limited to, Caenorhabditis elegans, C. briggsae and Oesophagotomum dentatum.

[0054] An electrotaxis device may be used to practice the method of inducing a response in a nematode. The device uses an electrical signal to manipulate nematode movement. The device comprises a microchannel having first and second reservoirs formed at first and second ends of the microchannel. The first reservoir includes an inlet for input of a sample into the microchannel and the second reservoir includes an outlet for removal of fluid and/or sample.
Each reservoir includes an electrode for use to establish an electric field between the reservoirs on connection to a power supply selected based on the electric field to be supplied (e.g. direct current vs. alternating current). As one of skill in the art will appreciate, any suitable electrode may be used such as metal electrodes, e.g. gold, copper or platinum wires, gel electrodes and liquid microchannel electrodes. Flow of sample into the microchannel via the inlet may be achieved by a suction pump connected to the outlet. The microchannel may be made of any suitable material that does not affect the application of an electric field, including polymeric materials such as organosilicon compounds. The use of Polydimethylsiloxane (PDMS) is preferred to provide a controlled environment in which the electric field is uniformly axial along the entire channel.

[0055] The present electrotaxis method may be used to sort nematodes based on a differential response between different nematodes to a given electric field.
The differential response between nematodes results from a distinguishing characteristic or parameter that exists between nematodes. The parameter may be, for example, developmental stage (age), size (length), a mutation affecting function of a neuronal or muscle gene and exposure to a chemical compound or drug that affects movement. Thus, nematodes of different ages may respond differently to a given electric field. Generally, adult nematodes exhibit a greater response on exposure to an electric field when compared to non-adult nematodes (e.g. L3-L4 stage nematodes), and shorter nematodes exhibit a greater response when compared with the response of longer nematodes. The term "response" is used herein to refer to one or more movement characteristics such as direction of travel, speed of travel, paralysis, body bends, turning time, extent of head movement and sinusoidal motion path, pauses and reversals.

[0056] Thus, a method of separating nematodes based on a selected parameter is provided comprising exposing nematodes to an electric field that induces a differential response among the nematodes based on the selected parameter, wherein the differential response functions to separate the nematodes. The differential response is induced by an electric field which is selected to promote a differential response based on a given parameter. If the nematodes are to be separated based on developmental stage, then an electric field of 2 V/cm or less may be selected, which is an electric field to which early stage nematodes do not respond and to which later stage nematodes do respond. If nematodes are to be separated based on length, an electric field of greater than 4 V/cm may be selected as nematodes shorter than wildtype exhibit a stronger response when exposed to this electric field than nematodes which are longer than wildtype.

[0057] The electric field may be applied as a direct current (DC), alternating current (AC), a pulsed DC current and any other variations thereof useful for sorting purposes. The application of AC is effective to localize nematodes, holding them in position by a non-physical means, for collection or until movement by application of DC is desired. The application of pulsed DC is effective to induce a turning response in nematodes.

[0058] Various methods and microfluidic devices useful to achieve sorting by electrotaxis are also provided.

[0059] In one embodiment, a method of sorting is provided comprising the steps of accumulating nematodes at the proximal end of a separation channel on application of an accumulating electric field, inducing the nematodes to move along a separation channel by application of a separating electric field, and collection of separated nematodes in separate collection channels by application of a collecting electric field in a direction perpendicular to the separating electric field. The strength of each of the accumulating electric field, separating electric field and collecting electric field will be based on the strength of field suitable to achieve the desired action. Thus, the strength of the accumulating electric field may be in the range of about 2-16 V/cm, and the strength of the collecting electric field may be in the range of about 2-16 V/cm. The strength of the separating electric field will depend on the characteristics of the organisms to be separated, as discussed above, and in more detail herein.

[0060] A microfluidic sorting device useful to conduct the sorting method is provided.
The sorting device comprises a nematode reservoir that feeds into a separation channel. A series of collection channels extend perpendicularly from the separation channel along the length of the separation channel. An accumulation electrode is positioned adjacent to the reservoir at the proximal end of the separation channel, a separation electrode is positioned at the distal end of the separation channel and a collection electrode is positioned within each collection channel. A
cathodic potential is applied to the accumulation electrode to accumulate the nematodes.
Following accumulation of the nematodes, a cathodic potential is then applied to the separation electrode to induce movement of the nematodes along the separation channel towards the separation electrode. Following sufficient separation of the nematodes within the separation channel, a cathodic potential is applied to the collection electrodes to generate an electric field that is perpendicular to the separation channel (in the direction of the collection channels) to move separated nematodes into separate collection channels.

[0061] A continuous field flow fractionation-type separation method is also provided. In this method, electric field is applied in a direction perpendicular to the direction of nematode movement in a pressure driven flow. Depending on the strength of electric field applied and the electrotactic response of the nematodes, they are fractionated into sub-groups continuously.
Thus, the method comprises the steps of flowing nematodes through a separation channel in a first direction, applying an electric field to the separation channel in a direction perpendicular to the first direction to cause separation of the nematodes based on their response to the electric field and collecting the separated nematodes. As one of skill in the art will appreciate, the strength of the electric field is selected to achieve separation of a target nematode population from a mixture of nematodes.

[0062] A continuous field flow fractionation device suitable to conduct continuous field flow fractionation is provided which comprises a separation channel having an inlet at its proximal end and a plurality of collection channels at its distal end.
Separation electrodes are positioned along each side of the separation channel to provide an electric field on actuation of a power supply across the separation channel and perpendicular to nematode flow from the inlet to the collection channels. The separation channel may comprise a plurality of micropillars which function to maintain non-responsive nematodes (e.g. nematodes that do not exhibit electrotaxis) moving in a straight line towards the collection channels to facilitate separation. The micropillars may be equidistantly spaced in straight lines throughout the separation channel. Thus, nematodes are input into the device at the inlet by a pressurized flow towards the proximal collection channels. An electric field applied perpendicular to this flow between separation electrodes causes separation of the moving nematodes based on their response to the selected electric field, followed by collection of the separated nematodes in the collection channels.

[0063] In a further embodiment, an electric trap device is provided comprising a microchannel having a reservoir formed at first and second ends thereof which are connected by a channel comprising a narrowed electric trap portion. Each reservoir includes an electrode for connection to a DC power supply to generate an electric field across the microchannel from one reservoir to the other reservoir. The strength of the electric field is increased within the electric trap portion from the field applied to the microchannel. A first reservoir includes an inlet for input of nematodes, while the second reservoir includes an outlet for removal of fluids/sample. In use, nematodes are delivered into the first reservoir via the inlet and a constant electric field is applied between electrodes to induce electrotaxis of the nematodes towards the narrow electric trap. As the narrowed electric trap has an increased electric field, it functions to separate nematodes based on their ability to withstand the increased electric field, e.g. are not paralyzed by the increase in electric field strength. Nematodes that can withstand the increase in the electric field within the electric trap (and are not paralyzed by it), will pass through the trap to the second reservoir however, if the electric field within the trap is such that it will paralyze the nematode (this is discernable by the nematode), the nematode will not pass through the trap.

[0064] Thus, a method of separation which utilizes electric trapping is provided. The method comprises the steps of inducing nematodes by application of a first electric field to move through a channel towards a narrow trap portion of the channel which exhibits a second electric field of greater strength than the first electric field such that it either permits or prevents passage of a target population of nematodes through the trap portion. The narrow trap portion of the channel exhibits a second electric field of increased strength such that certain populations of nematodes will not pass through the trap because the increased electric field within the trap is paralysis-causing.

[0065] A continuous flow sorter device is also provided. The device comprises multiple parallel microchannels with an array of spaced micropillars between each microchannel. The micropillars are equidistantly spaced between each microchannel, with decreasing spacing between micropillars from the first microchannel to each subsequent microchannel (e.g. spacing between micropillars decreases from the spacing of micropillars at the first microchannel to the spacing between micropillars of each subsequent microchannel, e.g. micropillar gaps between the first set of micropillars= 100 um, micropillar gaps between the second set of micropillars= 70 um, and micropillar gaps between the third set of micropillars= 40 m), thereby forming electric trap arrays of decreasing width which exhibit increasing electric field strength as width decreases. Inlets are provided at the proximal end of each microchannel through which a flow of liquid/sample may be input via a pump into the device, and a series of collection chambers exist at the distal end of each microchannel to collect sorted worms. Electrodes are situated along the length of the device adjacent to the first and last microchannels of the multiple microchannels for the application of an electric field across the width of the device.

[0066] A method of sorting using the continuous flow sorter comprises input of nematodes at the inlet of the first microchannel at a continuous flow rate, application of an electric field in a direction perpendicular to nematode flow sufficient to induce electrotaxis in select target nematodes. The select nematodes are then subjected to electric traps of increasing electric field strength such that separation of nematodes occurs when the field strength of an electric trap allows passage of some nematodes and prevents passage of other nematodes.

[0067] A semi-continuous sorting device is also provided. The device comprises a plurality of parallel electric traps which connect a loading chamber or reservoir to a separation chamber or reservoir. Electrodes situated at each of the loading and separation chambers provide a constant electric field from the loading to the separation chambers along the length of the electric traps on actuation (e.g. connection to a power source). Sorting of nematodes using this sorting device is similar to sorting achieved in a device with a single electric trap. Sorting is achieved by selection of an electric field that induces select nematodes to move from the loading chamber through the increased electric field within the electric traps to the separation chamber, while other nematodes are prevented from passing through the electric traps due to the increase field strength that occurs therein.

[0068] A worm storage microchamber device is provided in another aspect of the invention. The storage device comprises a central chamber (e.g. 1-20 mm), which may be of any shape, for example, ring-shaped or an irregular ring-like shape, and includes a microfluidic inlet channel through which worms can be loaded into the device, and a microfluidic outlet channel for removal of the worms from the device. A narrow entropic trap (e.g. a width of about 10-200 um) comprising an electrode connects the inlet and outlet channels to the central chamber and prevents the movement of worms from the chamber into the channels. Worms inherently prefer to move without confinement. The entropic trap is of a size that restricts worm flexibility of movement. This reduction in entropy of movement leads to the worm to remain in the chamber.
However, application of an electric field across the trap leads to a motive force that overrides the confinement effects, allowing the worm to progress through the trap at the inlet or outlet.

[0069] A microfluidic channel array is also provided in a further aspect in order to analyse the movement of multiple single worms at one time. Worms can be arrayed into individual microchannels and their movement analysed on exposure to a drug or other chemical compound. Entropic traps at the mouth of each microchannel along with application of an electric field serve as a valve to allow movement of worms into individual microchannels. A
electrical impedance sensor at the mouth of the microchannels near the trap will sense the arrival of the worm and cut off the trap electric field preventing any further worms from entering the microchannel. This feedback mechanism allow automatic arraying of several microchannels in parallel and ensure that each microchannel has a single worm. These microchannels also integrate electrical or optical sensors that can initiate electrotaxis and measure the speed of the worm. Regions in which drugs of various types and concentrations can be dosed to determine their effect on the electrotactic speed of the worm are incorporated as described in detail herein.
[0070] The present electrotaxis method may be used in movement-based high-throughput screening methods to identify candidate compounds that affect movement in selected nematode populations that may, for example, be representative of disease. Thus, a method of screening candidate compounds that affect nematode movement is provided comprising measuring the initial or control movement characteristics of one or more nematodes, and then following exposure of the nematodes to a candidate compound, measuring the compound-induced movement characteristics of the nematode. A change in the movement characteristics of the nematode is indicative that the compound affects the nematode, e.g. to improve movement characteristics or to hamper movement characteristics.

[0071] For example, a combination of null mutations in dystrophin (dys-1) and MyoD
(hlh-1) genes in C. elegans has been shown to cause progressive muscle degeneration similar to human DMD (Duchenne's Muscular Dystrophy) that impairs movement. The present electric field-based microfluidic channels may be used to screen for compounds that improve/restore movement in this mutants, thereby identifying potential candidates to test in human DMD
patients. Additionally, electric field-based assays may be used to study the mechanism of electrotaxis, as well as how the nervous system processes extracellular signals. Because movement is a complex behaviour that is controlled by many genes, the present methods will be useful to study the function of genes and pathways that mediate this behaviour.

[0072] In another example, the present methods may be used to study neurodegeneration in C. elegans. Though the molecular mechanism of electrotaxis is yet to be elucidated, it is known to be mediated by neurons, most of which are located in the head region (e.g. amphid sensory neurons). Thus, an electrotaxis method of screening for compounds having neuroprotective properties in certain nematode populations (mutants). In this regard, the effect of a known neuroprotective compound, acetaminophen, was determined to be neuroprotective to nematodes exposed to neuro-toxic compounds such as 6-OHDA, MPTP and Rotenone.-induced.
These results demonstrate that the present microfluidics electrotaxis-based assay system is a powerful and sensitive way to study neurodegeneration and identify neuroprotective chemicals in C. elegans.

[0073] Embodiments of the invention are described by reference to the following detailed examples which are not to be construed as limiting.

Example 1 - Electrotaxis Device [0074] An electrotaxis device suitable to study nematode response to an electric field is provided, as shown in Fig. I a. The device 100 comprises a microchannel 110, as shown in Fig.
lb, having a reservoir 106 formed at both ends thereof. The reservoirs 106 are connected via an assay channel 107 along the edge of which is an optional length scale 105. One reservoir includes an inlet 102 for input of a sample into the microchannel, and the other reservoir includes an outlet 104 for the removal of fluid and/or sample. Each reservoir 106 also includes an electrode 108 to establish an electric field from one reservoir to the other reservoir by connection to a power supply 112. Flow of sample into the microchannel 110 via inlet 102 may be achieved by a suction pump 114 connected to outlet 104.

[0075] In one embodiment, the device may comprise: (i) a worm handling unit (syringe pump, sample container, inlet, and outlet pipes), (ii) a monitoring unit (digital camera and microscope lenses), (iii) an actuation unit (power source and electrodes), and (iv) a microchannel device (sealed PDMS microchannel with embedded electrodes in reservoir areas).
The microdevice consists of a simple microchannel instrumented with electrodes (5 cm apart) within the reservoirs.

[0076] The microchannel 110 of the device 100 may be any appropriate size, including, e.g. 300 m-wide, 80 m-deep, and 5 cm-long. In one embodiment, the microchannel 110 was fabricated using soft lithography as described (Xia et al. Annu. Rev. Mater.
Sci., 1998. 28: p.
153-184). The mask layout was designed in AutoCAD (Autodesk Inc., San Francisco, USA) and printed using ultra high-resolution laser photo plotting on transparency sheet. SU8-100 (80 pm-thick) negative photoresist (MicroChem Corp., MA, USA) was used to lithographically pattern a master mold of the device. Polydimethylsiloxane (PDMS) pre-polymer mixture (Sylgard 184 kit, Dow Corning Corp., MI, USA 10:1 ratio of the base and cross-linker) was then cast on the master mold, and cured at room temperature for 24 hours. The PDMS replica was then peeled off the master mold and cut into pieces containing individual channels. The inlet and outlet access ports were punched out at the reservoir areas. The top surface of the PDMS
replica and a bare PDMS piece of the same size were plasma oxidized (50 W for 30 s), micro-contact printed with PDMS pre-polymer, and bonded, sealing the microchannel. Inlet and outlet capillary glass tube tips (VWR International, USA, catalog number CA14672-380, 1.5 mm outer diameter, 20 mm long) were connected to the punched areas. Plastic tubes (Saint-Gobain Performance Plastics, OH, USA, TYGON R-3603, 2.4mm outer diameter and 10 cm long) were connected to the inlet and outlet glass tubes. Metal electrodes (Arcor Electronics, USA, C24, copper 0.5 mm diameter) were inserted into the reservoir areas by punching through the PDMS elastomer from the side.
Liquid PDMS pre-polymer was then used to seal the surrounding areas of the electrodes and the device was placed on a hot plate (120 C) to cure. The device was then attached to a glass cover slip again using PDMS pre-polymer and cured.

[0077] The inlet tube of the microchannel was placed in a petri dish containing worms and a syringe pump (KD Scientific 14-831) connected to the outlet was turned on (flow-rate =
200 pl/min) to aspirate worms (one at a time) into the channel. As soon as a worm reached the middle of the microchannel, the syringe pump was turned off and the outlet pipe was disconnected from it. The inlet and outlet pipes were then levelled to the same height preventing the possibility of pressure-induced flow. The electric field (generated using KEITHLEY 2410 as a power source) was then applied and the response of the worm was recorded.
Electrical resistance of the channel filled with the buffer M9 solution in all tests was -0.68 M. The videos were recorded to obtain raw data.

Nematode Strains And Culturing [0078] Worms were grown at room temperature (20 C) on standard NG agar plates seeded with OP50 E. coli bacteria as previously described by Brenner (Genetics, 1974. 77(1): p.
71-94).

[0079] The strains used in this study are: wildtype N2, BC347 unc-54(s74), CB78 unc-6(e78), PS55 Ion-2(e678), and PS250 dpy-5(e61). The PS55 strain also carries a him-5(e1490) mutation that increases frequency of males in the progeny. The N2 strain was used as a wild-type reference in all assays. All strains are available at Caenorhabditis Genetics Center (Minnesota, USA).

[0080] Nearly all experiments were done with synchronized stages of animals.
Gravid hermaphrodites were washed off culture plates using M9 buffer (3 g K142PO4, 6 g Na2HPO4, 5 g NaCl, and l ml 1 M MgSO4 in 1 liter). They were centrifuged and washed twice with M9 to remove excess bacteria and debris. A 2 ml of bleach solution (800 L of 4 N
NaOH and 1,200 L of commercial bleach) was added to 4 ml of worms. The mixture was incubated at room temperature for 3 minutes and then centrifuged and washed with M9 (at least 3x). The eggs were allowed to hatch in M9 for 24 hrs. They were subsequently transferred to NG
agar plates seeded with OP50 bacteria and were allowed to grow further. When required for testing, the worms were washed again with M9 and loaded into microchannels.

[0081] For electrokinetic flow tests dead animals were obtained as below. Wild type C.
elegans were synchronized using the above bleach protocol and kept in M9. The animals were left in the absence of food for one week at room temperature causing them to die. The dead animals appeared rod-shaped with no visible body bending or movement.

Data Analysis [0082] A length measurement scale was microfabricated alongside the microchannel as shown in Figure lb. The response of worms to different ranges of electric fields was recorded by a camera (Nikon Coolpix P5100, NY, USA) and analyzed by ImageJ software (http://rsbweb.nih.gov/ij/) and AutoCAD (http://www.autodesk.com). The ImageJ
software was used to analyze videos and obtain the snapshots (every 0.07 s) of recorded movies of worm movements. The sequenced images were used to measure the distance travelled by the worm inside the channel. For this purpose, the initial and final snapshots were imported into the AutoCAD software and the travelled length was measured by superimposing lines on the worms' pathway and comparing the line lengths to a reference length bar in the image.
Snapshots obtained from ImageJ software was used to measure the worm movement distance between length scales fabricated on the device. The worms' lengths were also measured by linear approximation method using AutoCAD software. One image of each tested worm was imported to the software. A total of 15 lines were drawn on the worms' body image. The lengths of the lines were added and the total length was compared to the reference value to determine each worm's length. This process was repeated three times for each worm and an accuracy of about m was obtained.

Results and Discussion [0083] An electrotaxis device incorporating a microchannel format, as shown if Fig. 1, allows electric streamlines to be confined and directed along the axis of the channel and provides a simple well-controlled format to study and understand the electrotaxis of C.
elegans. Straight microchannels (e.g. 5 cm long and 80 m deep) with varying widths of 2 mm, 1 mm, 500 m, 300 m, and 150 m were utilized.

[0084] Synchronized C. elegans of various age and size, from Ll (-250 m-long) to young adult (-I mm-long), were loaded individually into the microchannels filled with M9 buffer and positioned in the central section (2.5 cm away from each electrode) using a syringe pump. In the absence of a stimulus, the animals had random movement in the microchannel (n =
20). In some instances, it was observed that after travelling a certain distance in one direction, the animals turned and moved in the opposite direction. In two cases the animals exited the channel after spending 5 minutes inside the channel. The device having a 300 m-wide microchannel appeared to guide movement along the channel axis without any obvious physical confinement and eliminated any perpendicular motion of the worms. The 150 m-wide microchannel appeared to interfere with' normal swimming behaviour of the worms.

[0085] In the presence of low-voltage electric fields, worms exhibited electrotaxis and moved in a directed manner towards the negative pole. To rule out the influence of electrokinetic flows (electrophoresis as well as electro-osmosis), dead worms were loaded individually into the channels filled with M9 solution and positioned in the middle section (2.5 cm away from the electrodes) using a syringe pump. A wide range of electric field strengths (1-20 V/cm) was applied across the channel that showed that electrokinetic flow above 13 V/cm was able to move dead worms towards the anode. No change in the morphology of dead worms was observed during this process. Furthermore, electrokinetic effects (electrophoresis of the worm and electro-osmosis of fluid) had no significant role in the movement of worms below 13 V/cm in these confined geometries.

[0086] To characterize the electrotactic behaviour in more detail, synchronized animals of different stages (from L l to young adult) were introduced into the microchannel. A wide range of electric fields (1-12 V/cm) was applied across the channel and movement of animals was monitored. Early stage animals (L1 and L2) displayed no obvious response to the feasible electric field (1-12 V/cm) since they continued to swim randomly regardless of the direction and presence of the field. At later stages (L3 onwards), animals responded robustly to the electric field within a certain range that was different for each stage and exhibited directed movement towards the negative pole. A careful examination revealed that their swimming pattern was typical of unexposed animals in a liquid environment except that the response was directional.
This suggests that the electric field does not distort body bends, but rather induces swimming behaviour. Below the minimum threshold, the movement was found to be random whereas above the maximum threshold, worms appeared paralyzed although they resumed swimming upon the removal of the electric field indicating that the effect was reversible. Using a length scale, fabricated alongside the microchannel, the speed under an effective electric field range of response was measured. The finding that only older worms responded to the electric field suggests that this behaviour is developmentally regulated and is likely to be mediated by certain differentiated cell types that may be absent (or immature) at earlier L1 and L2 stages. It is also possible that early stage animals respond to electric fields above 12 V/cm that is beyond the feasible range of the present device. These results demonstrate for the first time that older larvae and young adults of C. elegans respond to electric field in a liquid environment inside the microchannel and move in a directed manner.

Effect of Age on Electrotaxis [0087] The developmental response of worms to the electric field stimulus led us to characterize it in further detail.

[0088] At the minimum threshold electric field (2 V/cm for young adults), worms showed robust movement and oriented themselves towards the cathode. The movement of larvae and young adults was characterized as shown in Fig. 2. L3 stage worms (385-528 m-long, dark rectangles) responded to electric fields above 4 V/cm with a speed range of 100-216 m/s. L4 stage worms (534-725 m-long, clear rhombuses) responded to the electric fields between 4 and V/cm with a speed range of 220-340 m/s. Due to the partial paralysis at 12 V/cm, the speed of L4 stage worms was reduced. The young adults (920-1,050 gm-long, dark circles) had the lowest effective electric field range (2-4 V/cm) since they were paralyzed above 4 V/cm. Within the effective range, their speed ranged between 296 and 471 pm/s. The upper threshold electric field was not observed for L3 stage worms due to the upper limit of allowable field without electrokinetic flow.

[0089] The results showed that the speed of individual worms at any one particular stage does not change significantly with variations in the applied electric field
however, older worms had a higher speed when compared with the younger ones (-ྌ% increase between L3 and L4, and -35% increase between L4 and adult). This difference in speed was effective in separating worms of two different stages as shown in Fig. 3. Separation of two animals (530 m-long L3 stage and 1,000 gm-long young adult) within 6 seconds upon application of 4 V/cm electric field was achieved. The thin and thick white arrows mark the anterior ends of the L3 stage and young adult worms, respectively. The scale bar is 1 mm.

[0090] L3 stage animals responded to the electric field robustly starting from 4 V/cm (minimum threshold). The maximum threshold (defined by the paralysis phenotype) could not be observed because animals continued to swim normally without a change in speed even at the maximum allowable field (12 V/cm with no electrokinetic flow effect) in the channel. At later stages, animals appeared more sensitive to the electric field. Thus, while L4 stage animals were partially paralyzed at 10 V/cm (revealed by occasional abnormal body bends and reduced speed), the young adults exhibited this effect at 4 V/cm. The minimum threshold response at these two stages was 4 V/cm (L4) and 2 V/cm (young adult). Reversing the applied electric field resulted in the reversal of the worm's movement. These results demonstrate that adult animals are more sensitive to the electric field and possess the shortest response range compared to L3 and L4 larvae.

[00911 Undulatory motion of worms of different sizes was studied to determine the frequency of body bending under various electric fields using a no-field application as the witness model. It was observed that in the presence of electric field the average bending frequency of worms of different sizes (450-1,000 .tm-long) (n = 15) ranged between 1.7 and 2.6 Hz. The bending frequency for each worm did not change significantly (<5%
variation at maximum) with a change in the electric field strength and/or direction, and it was in close approximation of the no-field movement frequency (1.78-2.52 Hz for different size animals) demonstrating that the electric field had only a minor effect on the worm's natural body motion.
Cellular Basis of Electric Field Responses In Microchannels [0092] Mutant worms with defects in specific cell types were used to explore the cellular basis of electrotaxis using methods as described by Gabel et al. (J Neurosci, 2007. 27(28): p.
7586-96) and unc-6(e78) mutant animals that exhibit defects in neuronal differentiation. Studies on unc-6 have shown that it encodes a netrin-like secreted protein that plays a crucial role in neuronal growth cone migrations. The unc-6 mutant animals are uncoordinated due to defects in dorsal and ventral nerve cords. The average speed of various animals in a 5 cm-long, 300 m-wide and 80 m-deep microchannel was determined. The analysis of unc-6(e78) young adults in the microchannel revealed no response to the electrical stimulus and showed no obvious sign of orientation and speed change following the application of electric fields (Fig. 4).

[0093] A muscle mutant unc-54(s74) was also tested in a similar setting to determine the contribution of muscles in electric field-driven swimming behaviour. The unc-54 gene is necessary for the proper differentiation of the muscle myosin class II heavy chain (MHC B). The unc-54 mutant animals exhibit disorganized muscles and are severely uncoordinated (paralyzed).
The unc-54(s74) young adult worms responded to the electric field in a manner similar to that of wild-type worms (2-4 V/cm range) although their speed was significantly slower (unc-54: 88 pm/s, wild type: 380 gm/s, average speeds) (Fig. 4). This is interesting considering that unc-54(s74) worms on standard culture plates (NG agar) exhibit almost complete paralysis. These results indicate that the electrosensory response in microchannels is primarily mediated by neuronal activity.

Sensitivity of Electric Field Response: Size vs. Development [0094] The experiments revealed that adult worms respond to electrical fields much more robustly compared to younger (L3 stage) animals (Fig. 2). Considering that electrosensory behaviour is mediated by neurons, it is possible that adult worms have a mature nervous system and are therefore capable of processing neuronal signals more efficiently than the younger developing worms. Such a difference may also result from a change in the length of worms since adult worms are significantly larger compared to L3 stage animals (-1,000 +/-100 m and -450 +/- 100 m, respectively).

[0095] To distinguish between these possibilities, two different mutant strains that are shorter (dpy-5(e61)) and longer (Ion-2(e678)) as compared to wild type were studied. The dpy-5 gene encodes a collagen that is necessary for the cuticle formation in developing larvae whereas lon-2 encodes a glypican family of heparan sulfate proteoglycans that negatively regulates DBL-1BMP signaling to control body length. Mutations in these two genes give rise to opposite phenotypes. Thus, while dpy-5 mutant animals are approximately 60% shorter compared to the wild type (400 +/- 100 m and 1,000 +/- 100 m at 62 hours, respectively), the lon-2 mutant animals are roughly 30% longer (1,300 m +/- 100 m at 62 hours) than wild type. Both these mutant animals are otherwise healthy and active.

[0096] The analysis of dpy-5(e61) animals in the microchannel revealed that, unlike wild type animals (effective response range of 2-4 V/cm), these animals responded to the electric field robustly starting from 4 V/cm and showed no sign of paralysis at the highest possible field tested (12 V/cm). Their average speed (106 m/s) did not change in response to an alteration in the electric field strength and/or direction. In contrast, the Ion-2(e678) animals did respond to the lowest threshold electric field like wild type (2 V/cm). However, these animals appeared extremely sensitive and were paralyzed under the influence of electric fields higher than 3 V/cm.
Due to their larger size, they were unable to move freely in the microchannel and exhibited abnormal movements. This precluded measure of their speed. These results demonstrate that longer worms are more sensitive to electric field than shorter worms indicating that size is a major determinant of the sensitivity of C. elegans to the electric field. This was most likely due to differences in the potential drop across the entire body that is greater in Ion mutants compared to wild type (-30%). This may also explain the variability in responses observed for different stages of wild type animals since they are not exactly alike.

Post-Exposure Effect of Electric Field [0097] Considering that worms appeared paralyzed when exposed to electric fields greater than their response range, the post-exposure effect was determined by examining behaviour, fertility and viability. For this, 10 adult animals were aspired into the channel individually and a constant electric field (2-4 V/cm) was applied across the channel for duration of 10 minutes. During this period, the polarity of the field was reversed every minute (while keeping the field strength constant) in order to keep the worm inside the channel and to prevent it from getting in direct contact with the electrodes. In one case, a young adult worm was exposed to 12 V/cm electric field (three times that of maximum threshold electric field of wild type animals) for a duration of 10 minutes. Following electric field exposure, worms were removed and grown on standard culture plates. In all cases (n = 11), animals recovered successfully within a few hours, exhibited normal sinusoidal pattern of movement (i.e. no uncoordinated movement), did not die prematurely (-18 days average age), and were fertile for 3-4 days (similar to unexposed wild type worms). This demonstrates that the electric field stimulus causes no visible harm to C. elegans and that there are no long-term developmental and behavioural changes following exposure.

[0098] Thus, C. elegans behaviour in a microfluidic environment was studied in the presence of a low voltage electric field and was determined to be useful as an attractant to guide their movement without physiological and behavioural side effects. Application of electric field in worms induces forward movement towards the cathode that is robust, highly reproducible, and sensitive.

[0099] The electric field response was measured in microchannels at a range of electric field strengths (with minimum and maximum thresholds) within which an optimum electrotactic response was observed. Not all stages of animals responded equally well within the same threshold range. Thus, while the effective range for L4 larvae was 4-10 V/cm, adults appeared significantly more sensitive and had a lower response threshold (2-4 V/cm).
Within the optimum range at any given stage, the speed of movement of animals remained unchanged suggesting that the electric field response is a binary phenomenon (all or none). All responding stages of animals (L3, L4, and young adult), when exposed to the electric field above the maximum threshold, exhibited paralysis as judged by their near rod-like shape and abnormal body bends. This effect was reversible since the animals resumed normal movement upon lowering the electric field.
Furthermore, it was demonstrated that the electric field manipulation of C.
elegans does not appear to be harmful since the exposed animals, when placed on standard culture plates, resumed normal movement and feeding behaviour and continued to reproduce normally.

[00100] The cellular basis of electrotaxis in a liquid environment was also found to be neuron-dependent. Thus, unlike previously described pneumatic microdevices that rely on forced liquid flow to move worms, the use of the electric field stimulus in the present assay induces a very precise and sensitive innate movement response.

Example 2 - DC Electrotaxis of C. briggsae [00101] It was also determined whether or not electrotaxis is a conserved process between C. elegans and its cousin species C. briggsae. C. briggsae is very similar to C. elegans in terms of morphology, development, and genetic makeup. C. briggsae worms (n=5, 845 40 m length, 62 hr YA) were tested under DC electric fields (1-6 V/cm) in a similar manner to C. elegans. All tested animals responded to the electric field and moved towards the cathode spontaneously after the application of the signal. The entire movement response was recorded and analyzed as described previously (Rezai et al. Appl Phys Lett, 2010. 96(15): p. 153702.6).

[00102] It was observed (as shown in Fig. 5) that C. briggsae responded to electric fields in a range of 1-4 V/cm and showed partial paralysis starting at 5 V/cm.
Consistent with this, the electrotactic movement speed at 5 and 6 V/cm was significantly lower compared to 1-4 V/cm (p < 0.01, ANOVA). The paralysis was initially observed in the tail region as animals appeared to gradually form coiled-shape configuration in the tail region which extended to the whole body if the signal persisted, eventually leading to coiling. Although the higher threshold of response (4 V/cm) of young adult C. briggsae was similar to that of C. elegans, it was observed that these animals, unlike C. elegans (which had 2 V/cm lower threshold), responded to DC
electric fields as low as 1 V/cm in a robust manner (100% responders, n=5). However, similar to C. elegans, swimming speed of C. briggsae did not vary significantly with electric field strengths in the active range (1-4 V/cm) (p > 0.01, ANOVA) and had an average value of 356 20 m/s. The C.
elegans (n=7, 719 37 m length, 62 hr young adult) DC electrotaxis experiment was repeated and an average speed of 296 43 m/s in the 2-4 V/cm range was observed. The body stroke frequency for both C. briggsae and C. elegans was on average 2 0.1 Hz, resulting in slightly higher speed for C. briggsae (Fig 5).

Example 3 - AC Electrotaxis of C. Elegans [00103] Although worms can be stimulated to travel along an intended direction using the DC electric field, they cannot be held in one place. As localization is an important control mechanism for high throughput automated handling of worms in a microfluidic channel, the use of alternating (AC) electric field to localize C. elegans in microchannels for an extended period of time without any physical confinement was tested. An AC electrotactic device 200 was used to study the effect of AC field on worms is illustrated in Fig. 6. The device 200 consists of a microchannel component 202 (e.g. 5 cm-long, 300 m-wide, and 80 m-deep) comprising a reservoir 204a, 204b formed at each end connected by a channel 205 marked with a length scale.
An inlet 206 feeds into one reservoir 204a from a sample holder (e.g. worm sample), while an outlet 207 extends from the second reservoir 204b and is connected to a syringe pump 208.
Electrodes 209 and 210 (cathode 209 and anode 210) were embedded at both reservoirs and connected to a power supply unit 212 (an electric signal generation, e.g.
amplifier and function generator) that applies different waveform AC electric fields. The microchannel 202 was constructed of polydimethylsiloxane (PDMS) pre-polymer using soft lithographic and contact printing methods as described above.

[00104] Worms were loaded individually into the microchannel 202 using a syringe pump 208 and placed in the middle (2.5 cm away from each electrode). A constant DC
electric field was applied across the channel to stimulate animals to initiate swimming towards the cathode for a short distance (3 mm). Afterwards, an AC electric field with square waveform (frequency between 20 mHz and 3 KHz) was applied and the response of animals was recorded using a camera. Results are shown in Fig. 7. Normalized traveled range (Traveled Range / Average stage length) decreases with frequency increase for all stages. Regions I, II, III correspond to DC-like electrotaxis, 1-directional movement, and localization frequency range respectively, illustrated for wild type (dashed double side arrowed lines) and muscle mutant nematodes (solid double side arrowed lines). Applied electric field was 10, 8, 6, and 3 V/cm for L2, L3, L4, and young adult (YA) stages respectively. Average length for each developmental stage is as follows:
L2 (320um), L3 (470um), L4 (680um) and YA (900um).

[00105] At low frequencies (between 20 and -100 mHz), L2 and older stage animals moved in one direction (towards the cathode) in the positive half of the cycle and reversed their direction (towards cathode) in the negative half of the cycle (portion I in Fig. 7). The distance travelled during each AC field half-cycle before direction reversal was measured and dubbed as "travelled range". The travelled range data (shown in Fig. 7) was normalized using the average length of nematodes tested at each developmental stage (n=10 for each stage).
At frequency ranges between -100 mHz and -1 Hz, the nematodes swam in the following pattern (termed "stop and go"). When the polarity of the AC electric field coincided with the direction in which they were initially travelling, the animals continued to move in that direction. Interestingly, field reversal (during the other half cycle) did not alter their direction of travel. The animals were either momentarily localized or their speed was severely reduced.
Subsequently, in the next cycle, they continued their forward motion. This demonstrates that although the nematodes are able to sense the field reversal, they are unable to reverse their movement direction (1D
movement shown as portion II in Fig. 7). At higher frequency ranges (-I Hz - 3 KHz), young adults were almost completely localized in the channel, with movement being restricted to an average distance of 0.41 of their body length (See portion III of Fig. 7). It appears that the frequency of signal switching is just enough for worms to sense the direction, but before they could initiate movement, the direction of the field had reversed.
Consequently, the animals were unable to move at all. The nematodes stayed localized in the channel for the entire duration of the AC electric field, with no or a few rotations. Subsequent application of the DC electric field induced them to move towards the cathode. To illustrate the localization behaviour inside the microfluidic channel, worms were transported using the DC electric field to a desired location inside a channel, localized using AC electric field for a specific duration of time (20 s) and then subsequently transported away using the DC field. These results demonstrate that a combination of DC and AC electric fields could be used to efficiently guide and localize worms in a microfluidic channel without the use of physical constraints (such as the vacuum suction).
[00106] Synchronized worms of various ages (L1 stage to young adult stage) were tested using the same square waveform and frequency range. The results are shown in Fig. 7. All developmental stages (except for L1 stage) responded to the AC electric field in the same manner as young adults but with a slight variation in their range of localization. All stages demonstrated decreased travelled range with increase in frequency. Frequencies above -1 Hz appeared to localize worms but as shown in Fig. 7, older animals responded more robustly to the AC electric field (average normalized travelled range of 0.41 for young adults and 0.91 for L2 stage for f >1 Hz). For the older animals, more instantaneous responses to the AC electric field compared to the younger ones was observed. For example, some L2 and L3 stages (n=4 out of 20 total) exposed to the frequencies above 1 KHz failed to respond instantaneously and travelled for a short distance (<1mm) before being localized. This phenomenon was not observed in L4 and young adult stages that were localized immediately upon AC electric field application. All stages that showed a response exhibited a few or no rotations at the point of localization, although shorter worms (L2 stage) preferred to orient their body perpendicular to the axis of the microchannel.

[00107] The effect of sinusoidal and triangular AC signals on young adult stage worms in frequency ranges above 1 Hz (localization range) was tested. The response of animals was found to be the same as with square waveforms. Square waveforms with varying duty cycles (1% up to 80 %) were also tested on young adults to minimize the electric field exposure time while preserving the localization effect (data not shown). Even 1% duty cycle signals localized the worms in the same manner as square waveforms reducing the exposure time by 99%.

[00108] In addition to wild type, young adult worms with defective body wall muscles (unc-54(s74) mutant) were tested for their response to square AC electric field. The animals showed AC and DC electrotaxis but since they had considerably lower mobility compared to wild-type animals, even less average normalized travelled range (0.25) and reduced localization frequency range (100 mHz-3 KHz) was observed.

[00109] Thus, control of the movement of C. elegans in a microfluidic environment using AC electric field as a stimulus has been exhibited. Since movement is controlled by neurons and muscles, this discovery holds promise in the development of microfluidic-based high throughput assays to study and manipulate these two cell types in worms. The ability to induce movement in a desired direction (using DC electric field) and to localize animals at a specific location (using AC electric field) can be very useful in various applications. Incorporating a method of movement-based microfluidic assays is useful to study diseases, screen for drugs, and examine their mechanism of action in C. elegans.

Example 4 - Pulse DC electrotaxis of C. elegans and C. briggsae [00110] The device 200 used to perform these electrotaxis experiments on worms in Figure 6 and comprised a PDMS microchannel 202 (e.g. PDMS channel 100 gm-deep, 300 gm-wide, and 50 mm-long) with wire electrodes 205 and fluidic inlet 206 and outlet 207 access tubes inserted at its end reservoirs. The outlet tube 207 was connected to a syringe pump 208. Soft lithography technique was utilized for the fabrication of the microchannel 202 in polydimethylsiloxane (PDMS) material. The electrodes 205 were connected to an electric signal generation unit 210 (comprising a function generator and amplifier) for the application of electric fields and hence currents in the channel. This unit consisted of a AFG3022B
function generator (Tektronix Inc., OR, USA) with a maximum voltage output of 5 V, a 677B
amplifier (TREK
Inc., NY, USA) with a 400 gain, a custom made simple switch to reverse the polarity of electrodes, and copper wires to connect the setup to the device electrodes.

Generation of Electric Field [00111] The function generator was set to output a pulse DC electric field with adjustable characteristics such as the frequency (f =1/tT ), maximum pulse electric field strength (EFmax), as well as the duty cycle calculated using equation (1). The waveform of the pulse DC signal is shown in Fig.8. The signal rises in a step-like manner from zero to a maximum set point for a controllable duration of time (duty cycle) and decays to zero for the rest of the signal cycle which is then repeated at a fixed frequency.

Duty Cycle (%) = f x t,õ x 100 (1) where tO11 is the on-portion time of the pulse signal. Since the maximum pulse electric field strength (EFm) output of the function generator did not exceed 1 V/cm, the signal was subsequently amplified and applied to the microdevice. The rise and decay time of the pulse signal were 50 .ts each, which restricted the experimental frequency range to less than 1 kHz and the duty cycle to more than 10% in order to produce a rectangular-shape pulse waveform.
Animal Culture And Maintenance [00112] The C. elegans N2 and C. briggsae AF16 strains were grown on standard NG agar plates previously seeded with OP50 strain of Eschericia coli and maintained at 20 C.
Synchronized worms were used in all the experiments. For this, gravid adult hermaphrodites were treated with bleach solution (commercial bleach and NaOH (4N) in the ratio of 3:2). After bleach treatment, dead worms were washed with M9 solution (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml MgSO4 (1M) in 1 litre). Embryos were hatched in M9 following 24 hours incubation such that hatched worms were arrested at L1 stage. The animals were plated on NG agar plates and grown till they reached adulthood.

[00113] For all experiments, synchronized worms were individually loaded into the microchannel from a reservoir of diluted worm suspension (in M9) and positioned at the center of the channel by applying suction at the outlet. The inlet and outlet tubes were then levelled to the same height to prevent any flow in the channel. The head orientation of the worm was then determined in the microscope. The worms moved randomly in either direction (left or right) or remained stationary within the channel. The electric field (either constant or pulse DC) with a specific duty cycle and frequency was then applied along the channel (parallel to worm's body) in a direction opposite to its head orientation. The resulting movement response was digitally recorded using a camera (Coolpix P6000, Nikon Inc., Tokyo, Japan) connected to the microscope for the entire duration of the experiment. The applied stimulus either forced the worm to turn inside the microchannel and move towards the cathode at its rear or did not generate any response. For the responding worms, forward motion speed under signal application and turning time under signal reversal were measured from the video as behavioural phenotypes.
Since there was a large variation in turning response time between individual worms in the case of pulse DC signal, and to identify the responding worms after each field reversal, the worm was allowed to sense the signal reversal and initiate a turn response for up to 40 s immediately after the signal reversal. The worm was considered either a responder, if it turned within the given time window, or non-responder, if this time elapsed without a turn. The aggregate population response time was then used for data analysis. The turning time data was recorded for different durational intervals (i.e. 5, 10, 20, and 40 s). The responder worms were allowed to swim towards the cathode for a distance of 5 mm after which the polarity of the electrodes across the channel was reversed and the worm's behaviour was recorded. This process was repeated thrice for each animal to rule out a random electrotactic turning response. In the case of non-responding worms in the 40 s time window after signal reversal, the field was switched off, the worm was delivered to the center of the channel again pneumatically, and the same experiment was repeated two more times. The signal characteristics (either frequency or duty cycle) were then set to a different value and the experiment on the same worm was repeated for the desired spectrum of frequency (1-1000 Hz) and duty cycle (10-90%). After this set of experiments, the worm was washed off the channel.

Results and Discussion [00114] Two different movement phenotypes i.e. turn time after signal reversal and forward motion speed during signal application, were studied. The results were compared to DC
electrotaxis of these animals and are described below.

[00115] With a pulse DC signal, the duration of the electric field stimulation of the worm can be adjusted by changing the duty cycle of the signal as shown in Fig. 9.
In addition, this method has the potential to provide information about the response time of a population of worms to electrical signals. For instance, if the pulse width of the signal is below the response time of the animal, then that signal will not elicit a movement response even when an indefinite number of cycles of this signal is applied. In this experiment, C. elegans and C. briggsae worms (62 hr young adults) were loaded individually into the channel. The time for the worms to turn around in response to reversal of the pulse DC signal, at various duty cycles, were recorded for a population of worms. Simultaneously, the speed of worms in response to pulse DC signal at various frequencies and duty cycles was also recorded. For all pulse DC
experiments, the maximum electric field of the pulse signal was set to EFm 3 V/cm.

[00116] In order to examine the effect of the duty cycle on electrotactic response, C.
elegans worms (n=14) were exposed to a range of duty cycles (from 10% to 90%) at a constant frequency of 1000 Hz and their forward motion speed was analyzed (Fig. 1Oa) and turning response time (Fig. 10b) to the directional change of the signal. The speed of the worms remained constant with average values between a minimum of 290 37 m/s and a maximum of 360 24 m/s at different duty cycles (30%-90%, robust response range shown in Fig. 10b) as shown in Fig. 10a. These average speeds were statistically independent (p >
0.1, using a single factor analysis of variance method, ANOVA) of duty cycle values and were averaged at 315 37 m/s. This result indicates that even when the electrical stimulus is not continuous, it is sufficient to induce movement response in the worm. The turning response of the worms to DC and pulse DC signals was examined when they were reversed suddenly. Here, the worms were exposed to a constant or pulsed DC signal (with specific duty cycle at a fixed frequency of 1000 Hz) that was reversed suddenly. The turning time of the worm was then measured as described in the experimental section. The signal reversal was repeated twice to confirm that the turning response was genuine. The average turning time for a 3V/cm DC electric field was measured to be 6.6 2.6 s for 100% of responder YA worms. However, the time for the worms to turn around in response to pulse DC signals varied significantly (Fig. 10a) for certain duty cycles even though the experiments were done on a homogenous population of young adults. This variation in response time was greatest when the duty cycle was the smallest (large standard deviations at low duty cycles in Fig 10a) and was reduced as the duty cycle increased and the signal approached constant DC. In addition to this, a lesser number of worms responded in the 40 s window at very low duty cycles. For instance, no worm responded at 10% duty cycle in less than 40 s and the turn response was first observed at 20% duty cycles. In order to understand this data better, population statistics was used as shown in Fig. 10b, which plots the percentage of worms that responded to the pulse DC signal (at 1000 Hz frequency) of a certain duty cycle in a specific time window (i.e. 5, 10, 20 and 40s). At lower duty cycles, comparatively fewer worms responded quickly to the reversal of the pulse DC signal. For instance, only 20% of the worms responded to 20% duty cycle signal with a turning response time of 5s or less.
However, 60% of them were able to respond to the change in direction within 40s. Thus, as the duty cycle of the signal was increased, an increasing proportion of the worms responded to the signal in a faster manner. For instance, the proportion of the worms responding to the reversal of signal within 5s increases from 20% at 20% duty cycle to -80% at 90% duty cycle. At lower duty cycles, the majority of worms responded later to the field reversal and their turning time was prolonged.
This phenomenon could be related to the time required for the polarization of the neurons that mediate the movement response. This variation in duty cycle of pulse signals and turning response time to field reversal together provide a unique method to elicit varying movement response to correspondingly varying electrical stimulus.

[00117] The effect of the frequency of pulse DC signal on the forward-motion electrotactic movement speed was also studied for C. elegans. A duty cycle of 50% that induced robust response in worms (more than 80% responders in less than 10 s in Fig.
10b) was used for the pulse DC field with frequencies ranging between 1-1000 Hz. The speed of the electrotactic forward movement is shown in Fig. 11. Each data point corresponds to an average of 7 worms (62 hr young adult, 719 37 m long) examined. Average DC electrotactic speed of these animals is also depicted in the same figure for comparison.

[00118] It can be seen clearly that the frequency does not have a significant effect on the pulse DC forward electrotactic movement speed (p > 0.1, using ANOVA) which was similar to their DC electrotactic movement speed. The C. briggsae worms (n = 7) responded in a similar fashion (Fig. 11), indicating that the mechanism of electrotactic response is conserved between the two species.

[00119] It was shown that for robust (>80% responders) pulse DC electrotaxis, a minimum duty cycle at 1000 Hz frequency level was used. The effect of different frequencies on turning behaviour of C. elegans was also studied to assess the number of responders at different times (Fig. 12, tR=40s (a), tR=20s (b), tR=10s (c) and tR=5s(d)) and their average response times (tR) (Fig. 13). According to Fig. 12, the number of worms responding at each pulse DC
frequency level increased with increase in duty cycle from 10% to 90%. On the other hand, at a specific duty cycle, the highest number of worms responded at higher frequencies. However, this increasing response was not linear. In fact, the number of responders decreased from f=1 Hz to f=5 Hz at all duty cycles and then increased as the frequency was raised. Even at 90% duty cycle at f=5 Hz, less than 10% response was observed in tR<10 s (Figs. 12c and 13).
At f--5 Hz, only -20% of the worms responded in between 10s to 20s to 80% duty cycles (Fig.
12b) and an additional 20% of them responded in 20-40 s (Fig. 12a). All other frequency levels at 80% duty cycle saturated to 100% worm response (Fig. 12) and an average response time of tR s 20 s (Fig.
13) to the field. It can also be seen that the response becomes more similar to DC electrotaxis as the frequency increases to 1 kHz (Fig. 13). In addition, fast and robust response happens either at high frequencies with low to high duty cycles or at f51 Hz but duty cycles of 50% and above.
[00120] This technique can be used to precisely study the turning behaviour of nematodes and the neurons and genes involved in this behaviour.

Example 5 - Microfluidic Components - Integrated Electrodes [001211 For the development of electrotaxis-based high-throughput screening (HTS) assays, integrated microelectrodes for actuation and control of worm transport and integrated sensors for position identification and feedback have been designed. The worms have short time adverse reaction to high electric field gradients and to contact with the electrode while exposure to ionic currents in the microchannel is benign.

[00122] The present integrated microelectrodes do not interfere with the normal behaviour of the worm. One approach is to microfabricate hydrogel coated metallic electrodes so that the worms are exposed to only ionic currents. Various methods to integrate the hydrogel with electrodes, such as photolithographic based microfabrication, and to construct gel-filled microchannels as electrodes are possible. In another approach, we can use liquid electrodes and its effect on the viability and behaviour of the worms. Liquid electrodes consist of microchannels filled with highly conductive liquids and gels such as polyelectrolytes, polyelectrolyte gels and agarose gel, and interfacing with the main microfluidic channels through a high density nanoporous membrane, e.g. a polycarbonate porous membrane, that effectively transmits ionic current and potential into the channel but prevent transport of biological material across it. The effect of pore size and pore density (e.g. a pore size of 1 to 5 micrometers and density of 2x 107 to 4X105 pores/cm2, respectively) on the electric field distribution may be optimized to design a suitable electrode that imposes electric field but does not affect the functioning of the worms or the assay in any way.

[00123] We have also designed electrical methods for detecting the position and location of worms that is simple and can be multiplexed and suited for HTS. This is necessary in a number of unit operations described below. The sensing will be done by measuring the impedance of the microchannel cross section using electrodes that are embedded in the bottom surface of the microchannel. The impedance measurement depends on the permittivity of the fluid filling the microchannel. When the worm enters the domain over which impedance is measured, it changes the overall permittivity of the gap and this event can be recognized by change in the impedance signal.

Example 6 - Microfluidic sorters [00124] A microfluidic sorter has been designed to obtain highly synchronized populations of animals with similar electrotactic response. The use of synchronized populations is necessary in chemical screening methods in order that chemical exposure is the primary determinant in the change in the electrotactic behaviour, thereby increasing the confidence and sensitivity of the assay.

[00125] As shown in Fig. 14, a microfluidic sorter 300 is provided. The sorter comprises a worm reservoir 310, a separation channel 312 and a series of collection channels 314 which extend perpendicularly from the separation channel at various lengths across the separation channel 312. An accumulation electrode 316 is positioned adjacent to the reservoir 310 at the proximal end of the separation channel 312, and a separation electrode 318 is positioned at the distal end of the separation channel 312.

[00126] The sorter 300 separates worms into sub-groups based on electrotactic response to a constant electric field. The worms were loaded into the reservoir 310. An electric field (cathodic potential) was applied to the accumulation electrode 316 to collect the worms. Once a sufficient number of worms accumulated, the polarity was switched and the separation electrode attained cathodic polarity. The worms then moved along the separation channel at various speeds based on their electrotactic response. Once sufficient separation was been achieved, electric field was applied in the perpendicular direction via collection electrodes within the collection chambers to move the iso-electrotactic sub-groups into individual collection channels 314. Separation of worms based on their size and electrotactic response in a microchannel was achieved using this sorter [00127] In another embodiment, as shown in Fig. 15, continuous field flow fractionation-type separation sorter 400 is provided. In this sorter 400, electric field is applied in a direction perpendicular to the direction of worm movement in a pressure driven flow.
Depending on the range of electric field applied and the electrotactic response of the worms, they are fractionated into sub-groups continuously. The sorter 400 comprises separation channel 412 having an inlet 410 at its proximal end and collection channels 414 at its distal end. Worms enter into the separation channel 412 at inlet 410 via a pressurized flow (10-15 l/min) Separation electrodes 416 and 418 are positioned at either side of separation channel 412 and function to provide an electric field across the channel 412, perpendicular to worm flow. The channel 412 comprises a plurality of micropillars 315 which function to provide confinement in the vertical direction, enabling worms that are non-responsive to the electric field to move along a straight line in the horizontal direction.

[00128] Worms were introduced at a constant small flow rate of 10-40 l/min at the inlet 410. A constant electric field of 4-5 V/cm was applied in the vertical direction between the two separation electrodes 416 and 418. Responsive worms, thus, moved horizontally with the flow and vertically based on their electrotactic response. After a significant residence time of 4-5 min in the separation channel, the worms of similar electrotactic response were separated vertically and collected in different collection channels 414. This format allowed for continuous separation of the worms based on their electrotactic response.

4-way microjunction [00129] In line with the continuous field flow fractionation-type separation sorter, electrotaxis was used to control the movement of C. elegan in a 4-way micro junction. The device comprised 4 microchannels, a,b,c and d meeting at a junction. Each microchannel had a reservoir at the other end with an electrode inserted in it. The worms in a diluted suspension were fed into the bottom reservoir (c) of the device with a 10 l/min flow rate moving towards the junction. Electric fields were applied desirably from a to b channels (linearly, from side a reservoir to side b reservoir), from a to d (from side to top reservoir) and a to d (from side to top reservoir). The worm arriving at the junction with the flow travels the cathodic channel, despite the fact that, at the junction, it could choose to move into the other two channels with no electric field or anodic bias.

Example 7 - Electric trap sorter [00130] An electric trap device 500 is also provided as shown in Fig. 16. The device 500 includes a single main PDMS microchannel 510 (e.g. 120 m deep, 300 m wide and 30 mm long in total) having a reservoir at both ends 511, 512 which gradually narrow to an electric trap center 514. The electric trap center 514 may be 100 m wide and 3mm in length.
The reservoirs 511, 512 each include an electrode 516 and 518 for connection to a DC power supply and generation of an electric field across the channel 510. One of the reservoirs 511 includes an inlet 515, while the other reservoir 512 includes an outlet 517.

[001311 The electric field (EF) across a microchannel with uniform cross sectional area is EF = VIL where V is the applied voltage and L is the length of the channel across which the voltage is being applied. Knowing that V = RI and R = pL/A , the electric field across each microchannel section (wide-narrow-wide) in series can be calculated from equation (1):

EF=V=pI
L wt (1) where R is the electrical resistance (f2), p is the electrical resistivity (92.m) of the media in the channel, I is the current (A), A(=wt) is the cross sectional area (m) of the channel, and w and t are the width and the thickness of the channel (m), respectively.
[00132] Equation (1) demonstrates that the ratio between electric fields of two microchannels (same thickness) arranged in series (same current passing through both) and different in width is inversely proportional to their width ratio (EFnarrow E
F > r i d e - wivide / wniuroir ).

[00133] For electrical trapping assays using the device of Fig. 16, synchronized wild type animals of various ages (n=10 each) were delivered via inlet 515 into reservoir 511 of the device.
A constant electric field (16.8 V potential) was applied across the axial direction of the channel to cause electrotaxis of the worm towards the narrow electric trap 514. Three different criteria were investigated, (i) the electric field required in the wide section to initiate electrotaxis, (ii) the electric field at which the worm started to demonstrate paralysis effects (tail coil and loss of control) in the reservoir but still able to move forward towards the electric trap, and (iii) the electric field in the electric trap section which inhibited the worm from entering this section.

[00134] A range of electric fields (black column in Fig. 17b) were found to induce electrotaxis and made the worms move towards trap 514. As illustrated, this range is different for different stages of the worm, e.g. YA (2-5 V/cm), L4 (4-11 V/cm), L3 (5-15 V/cm) and L2 (7-21 V/cm). The electric field within the electric trap 514 is however always 3 fold higher than the reservoir 511. Trap electric fields which cause partial paralysis of the worm when it enters into this section also vary with the stage of the worm (gray column in Fig.
17b) . However, it was observed that above the higher threshold of the gray column, worms avoid entering the trap section since they would become completely paralyzed.

[00135] Expecting that the worms do not like to be paralyzed and therefore will not intentionally move into the region that induces paralysis, a 16.8 V potential across the channel which contained a mixed population of L3 and YA stages was applied. The applied potential resulted in -6 V/cm and -18 V/cm electric fields in the wide reservoir 511 and the narrow electric trap 514, respectively. The electric field in the wide section was sufficient enough to induce electrotaxis for both L3 and YA stages (Fig. 17a). However, the electric field in the narrow section was sufficiently high to induce full paralysis for YA but not for L3 stage worms if they entered the trap 514. Thus, the YA worms did not enter the electric trap 514 but instead remained in reservoir 511, while L3 worms continued to move through trap 514 into reservoir 512. This behaviour led to automatic self sorting of the L3 worms (passed through the trap into the second reservoir) from the YA worms (did not pass through trap, but remained in first reservoir). In a 2-4 min time period, a mixed population sample of L3-YA
worms, more than n= 10 L3 stage worms were observed to traverse the trap in spite of the accumulation of a number of YA at the mouth of the trap.

Example 8 - Continuous Flow Sorter [00136] A continuous flow sorter device 600 as shown Fig. 18 is also provided.
The device 600 comprises multiple parallel main microchannels 610 (e.g. 500 m-wide and 80 m high each) with an array of micropillars 612 formed between each microchannel 610. The micropillars 612 between each microchannel equidistantly spaced, but are spaced differently from microchannel 610 to microchannel 610 (the spacing between micropillars decreases from the first microchannel of the multiple to the last, e.g. micropillar gaps between first set of micropillars=100, micropillar gaps between second set of micropillars= 70 and micropillar gaps between third set of micropillars= 40 m), thereby forming electric trap arrays. Inlets 614 are provided at the proximal end of each through which a flow of liquid/sample may be input via a pump into the device, and a series of collection chambers 615 exist at the distal end of each microchannel to collect sorted worms. Electrodes 616 and 618 are situated along the length of the device adjacent to the first and last microchannels of the multiple microchannels for the application of an electric field across the width of the device 600.

[00137] Electric field was applied perpendicular to the microchannels 610.
Worms were introduced into the device at inlet 614a (inlet of the first microchannel of the multiple) with a continuous flow in all four microchannels using syringe pumps. When a potential is applied perpendicular to the flow (between electrodes 616 and 618), the electric field between the micropillars 612 is greater than that in the microchannel regions. Since the spacing between micropillars 612 decreases from the first microchannel 612 (1) to the last microchannel 612 (4), the electric field between micropillars also increases as shown in Fig. 18 from El in each microchannel, to E2 in the first set of micropillars, E3 in the second set of micropillars and E4 in the last set of micropillars. Thus, El was set in a range of response for the desirable worms while providing micropillar electric fields so as to exclude other stages of worm.

[00138] To quantify the performance of the device 600 in terms of separation, an efficiency parameter was defined as:

Ili N = G' -H` x 100 G H (2) where Go and Ho represent the total number of adult and L3 worms fed into the device. G and H are the number of adult and L3 worms in output i (=1, 2, 3, or 4) in Fig.
18.

[00139] A mixture of adult YA (Go=100) and L3 (Ho=100) worms were used in all experiments. Electric fields and flow rates of 2<E1(V/cm)<5 and 5<Q(p.L/min)<30 were used in preliminary characterization experiments (Fig. 19a). Testing the mixture of L3 and YA with E1=4 V/cm electric field in the microchannels (leading to E2=8 V/cm, E3=11.5 V/cm, and E4=20 V/cm), L3 stages did not become separated from the input stream (H1=100 and H2=H3=H4=0) since El was less than the minimum response range of L3 stage. But the YA
stages were able to respond to El, pass through E2 trap (still lower than their threshold), but get trapped at E3 level (Fig. 19c).

[00140] Fig. 19a also shows the effect of different flow rates on separation efficiency for adult worms with either E1=4V/cm or no electric field. Efficiency increased from 14.5% to a maximum of 38% by decreasing the flow rate from 30 L/min to 5 L/min in the absence of any electric field. However, loading of the worms was difficult at lower flow rates.

[00141] Distribution of worms among the 4 outlet channels with no electric field and 10 pl/min flow rate is shown in Fig. 19b. Increasing the electric field enhanced the efficiency up to 72.9% for adult worms with an optimum flow rate and electric field of 10 L/min and 4 V/cm (Fig. 19c). The optimum represents the balance between the electrotactic response of the adult worm and its residence time in the microchannel dictated by flow velocity.
High electric field intensity in the second micropillar row (E3) prevented adult worms from passing from channel 2 to 3 in Fig. 19c. By lowering the El value and hence E2-4, worms can be collected from output 3 and 4 as well.

Example 9 - Semi-Continuous Sorter [00142] A semi-continuous sorting device 700 (120 m-deep) is shown in Fig. 20.
The device 700 consists of a plurality (e.g. 20) of parallel electric traps 710 as described above (e.g.
100 .tm-wide, 500 m-long, 400 m pitch) which connect a loading chamber 712 to a separation chamber 714 (e.g. each 8 mm by 1.5 mm). Electrodes 716, 718 were situated at each of the loading and separation chambers 712, 714 in order to maintain a constant electric field throughout the device 700 across the electric traps 710. In one embodiment, 100 m-diameter Pt wire was inserted inside the loading and separation chambers perpendicular to the axes of the electric traps 710 to produce the electric field. An inlet 720 is provided to input worms into the loading chamber 712 and an outlet 722 is provided to collect separated worms from the separation chamber 714.

Experimental [00143] A set of experiments were conducted to study the worms' behaviour in the absence and presence of a desirable electric field in these chambers. In the first experiment, YA
worms were introduced (N=10) into the loading chamber with no electric field applied across the traps. After 10 minutes, only one of the worms was observed passing through the traps into the separation chamber. This demonstrated that the worms have a preference to reside in the loading chamber in absence of any stimulating signal.

Sorting of C. Elegans By Developmental Stage [00144] Worms of two different developmental stages which were easily distinguishable by size (i.e. YA and L3 or YA and L4) were separated using this device 700. A
range of electric fields (e.g. 3.5 - 7.1 V/cm) were applied towards the separation chamber 714 to yield electric fields ranging from 14 - 28.4 V/cm within the electric traps 710, as shown in Fig. 21. The number of attempts that the worms made towards entering the electric traps as well as the number of successful passages through it towards the separation chamber 714 were counted for each developmental stage of C. elegans. The percentage of worms (out of attempted ones) passing through the trap is shown for YA/L4 and YA/L3 in Fig. 2l a/b, respectively. Sorting experiments were repeated 3 times. As shown in Fig. 21, greater than 65% of the YA worms passed the electric field traps until the field in the electric trap was 14 V/cm. However, upon increasing the electric trap field strength, the percentage traversing through the trap dropped quite significantly to under 5% at 28.4 V/cm. On the other hand, this increase in electric field across the trap did not significantly affect the percentage of L3 and L4 worms passing through the electric trap and the number passing through the trap remained consistently above 60%.
Between the L3 and L4 stages, the L3 worms had a higher percentage passing through the trap (-ྌ%) at 28.4 trap electric field as compared to L4 (-60%). High trap electric field significantly inhibits YA worms (< 5% pass through) followed by L4 stage worms (-60% pass through) and has virtually no effect on the L3 stage worms. L4 stage worms could be more effectively separated (80%) from YAs at 24.8 V/cm trap electric fields. It should be noted that the critical electric field needed to inhibit the worm movement for a particular stage was on an average higher than that obtained in single trap experiments (Fig. 17b) for all tested stages. This is believed to be due in most part to the shorter length (500 m, 80% shorter) of the electric traps in the sorter device 700, resulting in less exposure time of the worm to the high strength field while attempting to pass.

Electrotactic Sorting of Neuronal or Muscle Mutants from Wildtype [00145] Muscle (unc-54(s74)) or neuron (unc-6(e78)) mutant animals were mixed with YA stages in a 1:1 ratio. The wild type YA worms had GFP markers in them to distinguish them easily from the mutants. The sorting process was conducted for >50 worms at an electric field of response for adult animals (3.4 V/cm). The magnitude of the electric field in the trap was set so that the wild type YAs which respond to the electric field would be able to pass through the trap.
The mutants would generally not pass through the trap due to the electric field. The worms that were able to cross the electric trap were collected from the separation chamber in a Petri dish and counted under the florescent microscope. It was found that 89% of passed worms (n=64) were wild type and only -11 % were neuron mutants (Fig. 22a). Between wildtype and muscle mutants, 96% wild type passed through the trap, while only 4% of the muscle mutants passed through the trap (n= 52) (Fig. 22b).

Young and Old Adult Electrotactic Sorting [00146] A similar approach was used to separate young from old adult worms that are similar in size. It has been reported that the electrotaxis swimming speed of worms reduces by 70% as they age (for a week) following the YA stage. In order to demonstrate this type of sorting, a mixed population of old adult (OA) (4 days) and GFP-tagged YA worms (>64 worms) was prepared in 1:1 ratio. Since both stages are highly sensitive, a low electric field (1.8 V/cm) was utilized to perform this sorting experiment.

[00147] Surprisingly, it was observed that the older animals are even more sensitive to the electric field. From the sample extracted from the separation chamber, of more than 64 worms, approximately 77% were OA and 23% were YA (Fig. 23).

Example 10 - Design and development of microfluidic storage device for C.
elegans [00148] Worms are highly mobile. Even in the absence of an electric field, they continue to move in random directions. A microfluidic storage device 800, as shown in Fig. 24, is provided that stores worms and permits their continuous circulation. The device 800 comprises a ring-shaped microchannel reservoir 810 having an inlet 812 and an outlet 814 which include entropic traps 815 such that in the absence of an electric field the entropic variation between the reservoir 810 and the inlet 812/outlet 814 prevents escape of the worms.
However, in the presence of electric field, there is a strong motive stimuli that attracts the worms to overcome the entropic barrier and allows them to enter or exit the device.

[00149] Iso-electrotactic worms arrive at the inlet channel 812 and are prevented from entering into the device due to the entropic trap 815. Actuation of an injection electrode 816 at the inlet 812 and the let side control electrode 818 (cathode) creates an electric field that allows the worms to be injected into the storage ring 800. The dimensions of the ring are such that the worm under random motion will prefer to reside in the larger microchannel 810 rather than enter the narrow confinement of the entropic trap 815 at the inlet 812 or outlet 814. Sufficient food (M9 solution) and oxygen can be provided by recirculating the fluid in the storage ring using macroporous membranes (1- 10 micrometers pore size) that retain the worm within the device.
When worms need to be arrayed, an electric field is applied across the outlet 814 with the ejection electrode 817 at the cathode and this electrical stimuli will allow the worms to overcome the entropic barrier 815 to exit the device.

[00150] Various designs of such entropic traps may be used to optimize a protocol for efficiently capturing worms based on their age/size. The entropic traps may be integrated with the microchannel storage ring and optimized for worms of various age/size.
Nutrient delivery means may also be included within the entropic traps to grow worms in a physiological manner.
For example, macroporous membranes (pore sizes 1- 10 gm) may be integrated into a portion of the microchannel ring wall such that bacteria and oxygen can diffuse into the reservoir from an adjacent microchannel.

Example 11 - Automated arraying of C. elegans in microfluidic channels [00151] Methods to automatically array worms into microfluidic channels where they will be exposed to chemicals and tested for electrotaxis response have been developed. To conduct such a method, a microchannel array device 900, as shown in Fig. 25, is provided. The device 900 comprises a worm storage reservoir 902 which is connected to a single worm holding unit 904 at the base of each of a plurality of parallel assay microchannels 910.
Each assay channel 910 is to be populated by a single worm. The application of an electric field at an impedance-based entrance worm detector 906 simultaneously drives the motion of worms from the storage reservoir 902 to each assay channel 910 in parallel. The entrance of the worm into an individual assay channel 910 is detected electrically by the impedance based worm detector 906, and this feedback shuts off electric stimuli selectively in that channel, ensuring that only one worm is loaded into each channel. Storage rings, as described in Example 10, may be connected to individual assay channels via microfluidic connectors on the storage rings which have the same electrical resistance as the assay channel so that the electric field applied across the individual channels and hence the electric stimuli to the worms are identical. This will ensure that the worms populate the individual assay channels, simultaneously. Entropic traps 905 may be integrated at the entrance of the assay channel so that once one worm has entered and the electric field shut off, the trapped worm will not be allowed to exit. Since the arraying technique is electrical-based, an external circuit will multiplex and probe several thousands of these sensors in real-time to identify the event of a single worm entering an individual assay channel for thousands of channels in parallel.

[00152] Arrays of 100 microchannels, 1cm in length, are typical. The array may have a common inlet for connection to the storage ring when desired to access worms of similar electrotactic response. The assay entrance valve 905 permits loading and confinement of loaded worms inside the assay channel 910. The entrance worm detector 906 provides electrical feedback to ensure that only one worm is loaded in each assay channel, e.g.
via impedance or capacitance-based position detectors. As soon as one worm enters the region of detection, a feedback signal renders the detector to be anodically biased to prevent others worms from entering the channel, and the trapped worm from exiting. The fluid inlet 912 and outlet 914 channels are used to recirculate fluid (M9 solution) to maintain the physiological condition of the worm. The drug injection channel 915 comprises a nanoporous interface 916 for injection of defined quantities of drug compounds dynamically or in a pulsed fashion into the assay channel 910. The electrical drug injection method allows automated dosing of various combination and doses of drugs into a large number of microchannels simultaneously. Once the worms have been incubated with the drug, they are electrically collected at the entrance of each assay channel 910 and simultaneously made to traverse the channel 910 to determine movement characteristics. The worm detector 908 (similar to detector 906) at the end of the channel 910 detects the arrival of the worm. Worms that have been exposed to drugs that remedy their neurodegenerative behaviour will arrive at the end and these chemicals and their doses can be identified.

[00153] The worms exposed to chemicals for 3 days inside microchannels may be examined by electrotaxis. Depending upon the effect of chemicals, the speed of worms or the time of their arrival at a detector at the far end of the channel may differ from a control worm, e.g. a Htn-polyQ control worms.

[00154] In order to perform this assay, chemical compounds are dosed accurately into assay channels simultaneously and the worm is immobilized at a drug dosing region, e.g. a nanoporous interface 916 between the assay channel 910 and the drug injection channel 915, for a certain duration of time in order to obtain similar exposure profiles.
Subsequently, an electric field is applied simultaneously to cause movement of the worms to the worm detector simultaneously. A microchannel based assay system with electrical control of dosing, worm localization and exposure, worm motion and detection simultaneously in all microchannels is, thus, provided.

[00155] The drug dosing region 916 comprises a nanoporous region that interfaces the assay channel to a microfluidic network above it, capable of convectively transporting various drugs or drug combinations to this location. Electrophoretic pumping can be used to transport precise and defined quantities of drugs into the assay channel in order to expose the worms to the drug. Electrophoretic pumps formed in the nanopores of the membrane can effectively be used to achieve digital dosing control of macromolecules, proteins and DNA in a reliable and repeatable manner. This proven method can be used to dynamically control the dosage of drugs into the microchambers over a period of time.

[00156] Since the worms move randomly under normal conditions, they may be confined underneath the nanoporous membrane to ensure sufficient exposure. This confinement is achieved simultaneously across all assay channels. In addition, application of an AC electric field at certain frequency ranges may be used to immobilize the worms in certain locations.

[00157] After the assay, the worms may be isolated, few in number, that have been favourably or adversely affected by the screening and obtain high content information from them. Microfluidic chambers, as shown in Fig. 26, that provide optical fluorescence images at multiple angles are provided to study these selected organisms in detail.

Example 12 - Drug Screening Using Microfluidic-based Electrotactic Screening [00158] The toxins 6-OHDA (catalog no. 162957), Rotenone (catalog no. R8875) and Acetaminophen (catalog no. A7085) were obtained from Sigma Aldrich. MPTP
(M325913) was from Toronto Research Chemicals. Desired concentrations of 6-OHDA (100 M), MPTP (700 M) and Rotenone (25 M) were prepared in M9 one day before the assay and stored at -20 C.
6-OHDA solution is sensitive to light and therefore it was kept in the dark. A
stock solution of Acetaminophen (10mM) was prepared and stored at -20 C. At the time of toxin assay, the stock solution was diluted to 100 gM final concentration.

[00159] All assays with toxins were done using L 1 stage worms. Synchronized L
1 worms were exposed to toxin solutions for different time periods with mild shaking on a rotating platform. At the end of the time period, tubes were briefly centrifuged to remove drug solution and worm pellets were diluted with 100 gl M9 buffer and were plated on normal NG agar plates.
[00160] L1 animals treated with 5 mM 6-OHDA showed extreme sluggishness, uncoordinated movement, protruding vulva, growth arrest and early larval lethality. Some of these worms were loaded into the channel of a device as shown in Fig. 1 and found that they were practically immobile and unresponsive to the electric field stimulus.
Reducing 6-OHDA
exposure (100 gM for 4 hr) enabled a successful electrotaxis assay. Worms exposed to 100 gM

6-OHDA for as little as 30 min showed abnormal electrotactic response demonstrating sensitivity of the assay. The MPTP and Rotenone treatment protocols were also modified.
For MPTP, exposure of L1 worms to 1.4 mM toxin for 3 days resulted in worms that were almost completely uncoordinated and paralyzed and thus not suitable for electrotaxis assay. A
range of conditions were tested and it was found that worms exposed to 700 gM MPTP for up to 8 hrs showed obvious defects in the channel assay. Interestingly these animals appeared fairly active and healthy on NG agar plates, once again demonstrating that the present electrotactic assay is sensitive in detecting behavioral abnormalities. Finally, for rotenone a micromolar range of concentrations was tested and it was found that worms exposed to 25 gM of drug for less than 12 hrs showed morphological defects similar to MPTP in repeated trials. It wad determined that animals treated with 25 M of Rotenone for 1-8 hr appeared largely healthy on the plate but had abnormal electrotactic movement.

Electrotaxis Assay and Data Analysis [00161] Experimental design was as described Rezai et al., ibid. Electric field was fixed at 3V/cm. Each worm was allowed to move a minimum distance of 5000 m inside the channel, of the device as shown in Fig. 1, in one direction. Then, the direction of the electric field was changed and the worm was allowed to move the same distance in the opposite direction. The movement of each worm was recorded using a Nikon Coolpix P5100, NY, USA. The captured videos were analyzed using the ImageJ NIH image module. Three parameters - U-turn, head movement and speed - were used to characterize the behaviour of animals. For U-turn, the time (in seconds) that was taken by a moving worm to come to a stop and turn to the opposite direction after the electric field direction was reversed was recorded. Most wild type worms (-90%, see results) were able to change direction in less than 10 seconds.
Worms taking 10 sec or longer were considered to have an abnormal response. For the head movement analysis, the number of full sine waves (i.e., spanning up to three-fourth of the channel width) that a worm makes within a 30 sec. duration were determined. Wild-type worms make full sine waves in majority of cases with failures typically being less than 25%. Therefore, a threshold of 25% to place a worm in normal or abnormal category was used. In the present assay, 90% or more wild type worms were observed to be below the threshold (see results). To analyze speed, the time taken by a worm to move a distance of 5000 m in a given direction was used.
Speed was measured in both directions of movement and the average value was used as a true response.
Wild type animals typically moved with a speed of 215gm/sec and above.
Therefore, animals moving slower than 215gm/sec were considered to have an abnormal response.

Results [00162] Since wild type worms move towards the cathode when exposed to a low voltage DC electric field inside the microfluidic channel. This response, termed "electrotaxis", requires intact neuronal and muscular systems. Therefore, the role of these neurons in the microfluidic channel assay was tested.

[00163] In addition to testing genetic mutants with abnormal neuronal function, namely unc-6, osm-5, tax-6, cat-2, dop-1, dop-2, dop-3), the response of worms exposed to a very low concentration (10 M) of levamisole (an anthelmintic drug that acts as acetylecholine agonist in body-wall muscle) was tested. The tested worms had reduced movement (only 5 worms were tested). Thus, the assay system can reveal abnormalities in neuronal as well as muscular activities using movement as a read-out.

Electrotactic Response of Worms Exposed To Neurotoxins [00164] Having established that the assay system detects movement defects in worms with altered neuronal activity, the response of animals treated with certain toxins that cause degeneration of sensory neurons, namely, 6-OHDA (6-hydroxydopamine, a hydroxylated form of dopamine), MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) and Rotenone (an organic pesticide), all of which have been shown to cause preferential degeneration of dopaminergic neurons in a specific region of brain (substantia nigra) in vertebrates, was tested. These toxins also cause degeneration of DA neurons in C. elegans. Three parameters (U-turn, head movement and speed as described above) were used to characterize these defects in detail.

[00165] In the present assay, 6-OHDA exposure was found to cause U-turn defect in 30-80% of worms within 4 hour exposure (Fig. 27). The penetrance was similar in MPTP-treated worms with defect in 30%-70% of worms. U-turn response was most sensitive to Rotenone treatment with a gradual increase in the proportion of defective worms (50%
to 80%) within 1-8 hr exposure time (Fig. 28).

[00166] Similar to the U-turn, head movement was also highly defective in toxin-treated animals. In the case of 6-OHDA, about one-third of worms exhibited a defective response following 30 minutes toxin exposure. The proportion of defective animals gradually went up (to 50%) as the exposure time was increased to 1 hr (Fig. 27). By 4 hour exposure 61% of the worms were defective. Rotenone treatment caused a less severe head movement defect. Thus, only up to 30% worms showed abnormal head movement response even after 8 hrs of toxin exposure (Fig. 28). In comparison to 6-OHDA and Rotenone, MPTP exposure had a severe defect in head movement response. A 4 hr MPTP exposure caused defects in 80%
worms (Fig.
29). A longer toxin exposure did not make it worse suggesting that this duration was enough to cause maximum defect in movement in response to electric field stimulus.

[00167] Movement speed of toxin-treated animals was also tested. Treatment with 6-OHDA resulted in most defective response compared to the other 2 toxins (Fig.
27). On the other hand, MPTP showed a wild type response in all 3 conditions tested (Fig. 29).
Finally, Rotenone exposure caused 48% defect in the speed of animals (Fig. 28).

Morphological and Behavioral Analyses of Neurotoxin-Treated Animals [00168] Worms exposed to high doses and long exposure of 6-OHDA, MPTP and Rotenone exhibit uncoordinated movement, abnormal morphology, reduced size, and retarded or arrested growth. These phenotypes are in agreement with published studies. As described in the methods section lowering the concentration and exposure time of toxins resulted in significant reduction in the proportion of animals showing morphological and behavioral abnormalities.

[00169] Among the three toxins, 6-OHDA had the highest impact. A 4 hr exposure resulted in uncoordinated phenotype in most of the animals (86%). Occasionally animals also showed morphological defects such as protruding vulva (Pvl). Reducing the exposure time to just 30 min resulted in fewer worms (one-quarter) showing a weak uncoordinated phenotype (Fig.
30). Unlike 6-OHDA, exposure to MPTP and Rotenone had only subtle and low penetrant movement defects (Figs. 31 and 32). This was revealed by slow reaction of animals when touched on the head or tail. In no case was a reduced size and growth arrest phenotype observed, and except for the 4 hr 6-OHDA condition, all other animals appeared fairly healthy, active and fertile.

Exposure of Neurotoxins Causes Degeneration of Dopaminergic Neurons [00170] Previous work has demonstrated that dopaminergic neurons are highly sensitive to all three toxins noted above. Administration of 6-OHDA in a mammalian model produces various biological effect including increase level in reactive oxygen species (ROS) that leads to depletion of DA neurons. In the case of MPTP, it is metabolized into MPP+
which is the active toxic product that gets inside the DA neuron through the dopamine transporter.
Inside the DA
neuron, MPP+ has multiple targets including the inhibition of mitochondrial respiratory enzyme complex I that leads to increase level of ROS. Exposure to rotenone in rat and Drosophila melanogaster model has shown to cause apoptosis oxidative damage, and degeneration of dopamine neurons. Damage of dendritic process of DA neurons has been shown upon exposure to 6-OHDA. MPTP also cause depletion of DA neurons as the florescence in the neuron specific marker loss after treatment. Rotenone causes decrease in survival rate.

[00171] To visualize the toxin-induced degeneration of DA neurons in the present assay, dat-lp::yfp transgenic animals were generated (as described in Nass et al.
2002, PNAS, vol. 99 (5), 3264-3269. dat-1 encodes the DA transporter and is expressed in all DA
neurons. The analysis of dat-1p::yfp expression in bhExl20 animals revealed bright YFP
fluorescence in CEP
neurons (Fig. 33a). Exposure to 6-OHDA (100 M) for 4 hours did not cause an obvious degeneration of DA neurons, however a longer exposure (-71 hrs) resulted in defects in 15% of animals. In these cases the dendritic processes of CEPs showed variable degeneration such that YFP fluorescence had a spotted and blebbing appearance. In some cases, the entire dendritic processes were missing.

Channel-Based Drug-Screening Assay [00172] In order to establish a baseline to screen for neuroprotective compounds in a channel-based assay, the neuroprotective effect of acetaminophen (a known analgesic) against neurodegeneration caused by toxinsas tested . Previous studies in rats and C.
elegans had demonstrated the protection conferred by acetaminophen against MPP+, 6-OHDA
and glutamate toxicity in dopamine neurons.

[00173] Pre-treatment of worms with acetaminophen (100 uM, 24 hrs exposure during L1 stage) prior to treatment with 6-OHDA as above, conferred a significant protection on the worms as evidenced by the electrotactic response of worms using all 3 movement parameters described above. The protection ranged from 30% to 50% (Fig. 34). In the case of MPTP, acetaminophen pre-treatment had a similar effect. The defect in head movement was decreased by 40% and U-turn by 30%. The impact of Acetaminophen on Rotenone-induced neuronal toxicity and provide first evidence that Acetaminophen protects DA neurons from the toxic effects of Rotenone.
While the head movement defect was decreased by 20%, abnormalities in U-turn response and speed were lower by 50% and 38%, respectively.

[00174] In summary, the present microfluidics-based electrotaxis assay was effective to show that Acetaminophen effectively protects DA neurons against neurotoxins, and that the microfluidics-based assay is an effective drug screening tool for use to identify other protective compounds.


Castor Bean (Ricinus communis)

Widely grown as an ornamental, the castor bean is an attractive plant native to Africa. While the processed seeds are the source of castor oil, they naturally contain the poison ricin and are deadly in small amounts. It only takes one or two seeds to kill a child and up to eight to kill an adult. Ricin works by inhibiting the synthesis of proteins within cells and can cause severe vomiting, diarrhea, seizures, and even death. The poison was used in 1978 to assassinate Georgi Markov, a journalist who spoke out against the Bulgarian government, and has been mailed to several U.S. politicians in failed terrorism attempts. Most fatalities are the result of accidental ingestion by children and pets.


15.4G: Paralysis-Causing Bacterial Neurotoxins - Biology

Australia is justifiably famous as the island continent with the most venomous and poisonous animals. These include native animals like Australian venomous snakes and introduced species like the cane toad. Many of these species pose a significant health risk to companion animals and livestock and thus are of both veterinary and economic importance.

Animal venoms are used effectively for defense and predation poisons are used primarily for protection from predation. Both venoms and poisons are complicated cocktails, consisting of several hundred different components. Venom toxins are the primary actors for toxicity in animal venoms, particularly for invertebrate venoms [ 1 ]. Venom toxins are peptides, generally 3𠄶 kDa in size containing between 2 and 4 disulfide bonds, in a highly stable inhibitor cystine knot (ICK) motif [ 2 ]. ICK venom toxins can have a wide range of activities, including ion channel blockers (including neurotoxins), hemolytic agents, and antiviral or antibacterial agents. Toxins are distinct from enzymes, larger proteins, and nonpeptidic components like alkaloids and polyamines, and toxins are responsible for much of the biological activity and pharmacological interest around animal venoms and poisons.

Australia’s most dangerous venomous snakes are front-fanged elapids and their venoms are potent and diverse. Further, they are common in both rural and urban areas posing a significant health risk to domestic companion animals and livestock. Snake venoms primarily contain procoagulants, anticoagulants, neurotoxins, myotoxins, and nephrotoxins however, the locally acting necrotoxins generally found in non-Australian elapid and viper venoms are largely absent [ 3 ].

Cane toads are introduced amphibians that have been wreaking havoc on Australian ecosystems since their introduction in 1935 [ 4 ]. The cane toad has a highly toxic paratoid secretion that is particularly toxic to dogs [ 5 ]. Cane toad poison is composed primarily of biogenic amines, bufadienolides, alkaloids, and peptides and proteins [ 6 ]. Ontogenic variation in the cane toad poison has been reported, and the eggs contain higher concentrations and a wider range of active compounds than do adult toads [ 7 ]. The poison in the parotid glands induces neurologic or respiratory signs in dogs and cats when the toads are mouthed or ingested, and effects of poisoning can be so severe that death results despite treatment [ 8 ].

The Australian paralysis tick, Ixodes holocyclus (Acari: Ixodidae), contains toxins, particularly holocyclotoxin, in its saliva which can be lethal to companion animals and livestock [ 9 ] an antidote is available for paralysis ticks. For other invertebrate species, anaphylaxis or localized severe reactions are the primary concern for their bites and stings [ 10 ]. Insects cause clinical signs related to bites and stings, may cause anaphylaxis, and may be poisonous if ingested in the case of sawfly larvae or caterpillar species with urticating hairs or spines [ 11 ]. Australian tarantulas (Araneae: Theraphosidae) are unique in that they have been shown to be lethal to canids, but not to humans [ 12 ]. Scorpions are of clinical importance because of their neurotoxic venom, which affects both humans and animals [ 13 ], and no scorpion antivenom currently exists.

The diverse range of pathophysiological effects of the venoms and toxins from Australian venomous and poisonous animals present a major challenge for veterinary treatment. Further, for many of Australia’s venomous and poisonous animals no antivenom is available, and the clinical signs can only be treated symptomatically (including spider bites and cane toad poisoning). Venom and poison toxins can be a source of novel pharmaceutical agents, which is only recently being explored in humans [ 14 ]. The goal of this review is to provide an overview of venom and poison pathogenesis of veterinary import in Australia and discuss the potential for targeted compounds in drug discovery for animal therapeutics.

2. Venom Pathogenesis and Poisoning in Australia 2.1. Snakebite

Snake envenomation is an important presenting problem at veterinary clinics, with previous studies estimating the prevalence at 0.31% of clinical cases [ 15 ]. Another survey estimated up to 6,200 cases reported per annum, predominantly in dogs and cats, with 78% of cases occurring in rural versus 22% in urban areas [ 16 ]. Identifying the snake correctly is difficult in veterinary circumstances, given that the animal may be bitten in isolation (or while unsupervised) and the snake may not be presented with the animal for correct identification. A commercially available rapid freeze-dried sandwich enzyme immunoassay, the CSL snake venom detection kit (CSL Limited, Parkville, Victoria), is available for use in Australian animals. With significant treatment associated costs for hospitalization, often with intensive care and antivenom, most owners are reluctant to pay for the additional cost of a venom detection kit. In the late 1990s, the kit was estimated to be used in only 1% of cases [ 16 ]. If a snake venom detection kit is used, it is important to select the most appropriate test: a blood test, a urine test, a swab of the bite site, or a combination of all three.

A study of rapid immunoassay snake venom detection kits in an experimental model of tiger and brown snake envenomation in cats demonstrated that if envenomation occurred less than 8 hours previously, blood was the best sample however, after 8 hours it was essential that urine be sampled [ 17 ]. Notably, a horse envenomated by a tiger snake gave a negative result from a serum sample venom detection kit (SVDK) but was strongly positive when a urine sample was used [ 18 ]. Although bite site swabs can be used, bite sites are rarely identified in animals either in life or during a postmortem examination. False positives with SVDKs have been anecdotally reported however, a study on urine from 50 dogs and 25 cats presenting to veterinary clinics demonstrated no false positive reactions, so test specificity was estimated at 100% on urine as a test sample [ 19 ]. False negatives can occur with high venom concentration saturating binding antibodies in the kit (known as the “hook effect”), with venom levels below the limit of detection in subclinical envenomation, and insufficient time for venom to concentrate in the urine, or an extended period of time between envenomation and testing, which results in venom levels in urine below the level of detection [ 19 ].

The three most commonly encountered snakes causing envenomation of veterinary importance are the venomous brown snake, the tiger snake, and the red-bellied black snake. The latter two snakes are mostly localized near the coast, particularly the east coast, but the brown snake is ubiquitous throughout the continent the tiger snake is the only one recorded in Tasmania (Figure 1 ).

A map of the distribution of the three most commonly encountered Australian snakes of veterinary importance: the venomous brown snake ( Pseudonaja spp., 11,923 records, (a)), the tiger snake ( Notechis scutatus , 2,366 records, (b)), and the red-bellied black snake ( Pseudechis porphyriacus , 4,017 records, (c)). Relative density is indicated by the legend to the right of each map. Maps from [ 20 – 22 ].

(a) (b) (c) 2.1.1. Venomous Brown Snakes ( Pseudonaja spp., Elapidae)

Venomous brown snakes in the genus Pseudonaja are distinct from unrelated brown snakes whose habitats overlap, including the venomous king brown ( Pseudechis australis , from the black snake genus) and the taipan ( Oxyuranus scutellatus ) and nonvenomous brown-colored snakes like pythons. Brown snake envenomation is characterized by a severe lower motor neuron paralysis with hypocoagulation [ 23 ]. Animals suffer an initial haemodynamic collapse with severe systemic hypotension and thrombocytopenia [ 23 , 24 ]. In an experimental model using anaesthetized dogs hemodynamic effects of brown snake ( Pseudonaja spp.) venom included hypotension with reduced cardiac output and stroke volume and a rise in peripheral vascular resistance and a transient increase and then decrease in heart rate [ 25 ]. Hematological effects consistent with significant derangement of coagulation included marked thrombocytopenia, depletion of serum fibrinogen, prolonged prothrombin, and activated partial thromboplastin time [ 24 ]. The group C prothrombin activators in brown snake venom closely resemble mammalian prothrombinase (Xa:Va) which converts prothrombin into thrombin thus the venom activates coagulation resulting in a consumptive coagulopathy termed venominduced consumptive coagulopathy [ 26 ].

Pseudonaja venom also contains several neurotoxins: a potent presynaptic neurotoxin (textilotoxin) and two postsynaptic peptidic neurotoxins (pseudonajatoxin) [ 27 ]. The clinical signs resulting from these toxins appear to be highly variable amongst envenomated species. Humans rarely demonstrate neurotoxicity of clinical significance (“the brown snake paradox”) [ 27 ], whilst ascending flaccid paralysis and respiratory muscle failure are a much more common finding in dogs and cats [ 15 ].

2.1.2. Tiger Snake ( Notechis scutatus , Elapidae)

Tiger snake venom contains a number of neurotoxins, procoagulant factors, and a weak haemolysin, resulting in a primarily neurological, myolytic and coagulopathic clinical syndrome [ 18 , 28 ]. The complex presentation of tiger snake envenomation has been classified into three categories of clinical signs: (1) a preparalytic phase (acute collapse, vomiting, hypersalivation, defecation, trembling, and tachypnea), a paralytic and lethal phase (skeletal muscle paralysis, coagulopathy, and oliguria, with or without myoglobinuria or haemoglobinuria), and a sublethal or delayed phase (mydriasis, reduced pupillary light reflex, stiffness, ataxia, inability to close the jaw, and/or renal failure) [ 28 ]. During the preparalytic stage collapse, vomiting, salivation, defecation, trembling, and tachypnea are observed. Skeletal muscle paralysis, coagulopathy, and oliguria (which may include either myoglobinuria or haemoglobinuria) are noted in the paralytic stage and dilated pupils with absent pupillary light reflex, stiffness, and ataxia, inability to close the jaws, and renal failure are noted in the sublethal phase.

The principle neurotoxin, notexin, is a toxic phospholipase A2 that depletes acetylcholine [ 18 ]. Notexin is also a potent myotoxin and can cause extensive skeletal muscle degeneration, though with rapid death insufficient time may elapse for significant skeletal muscle changes to occur [ 18 , 29 ]. A procoagulant with factor Xa-like activity is present and histopathological studies on a dog and cat which died for tiger snake envenomation demonstrated extensive thrombus formation [ 18 , 29 ].

Clinical features of a horse diagnosed with tiger snake envenomation by sandwich ELISA included muscle fasciculation, reluctance to move, profuse sweating, tachycardia, tachypnea, and localized hot painful swelling on the muzzle presumed to be the bite site though punctures were not visible [ 18 ]. Significant hematologic abnormalities in this horse included mild neutrophilia with a left shift but no toxic changes and mild elevations in fibrinogen. For clinical chemistry, the horse exhibited a range of hematologic abnormalities with the most notable being increased creatinine kinase and aspartate aminotransferase likely due to muscle damage, and the animal had a significant myoglobinuria.

2.1.3. Red-Bellied Black Snake ( Pseudechis porphyriacus , Elapidae)

Red-bellied black snake venom is reported to be strongly haemolytic and weakly neurotoxic however few reports of envenomation by Pc. porphyriacus in domestic animals are present in the literature [ 23 ]. Envenomation by Pc. porphyriacus has been reported to cause intravascular hemolytic anemia, rhabdomyolysis, and anuric renal failure secondary to myohemoglobinuric pigmenturia in a dog [ 30 ]. In humans, Pc. porphyriacus envenomation causes necrosis around the bite site, pigmenturia, increased serum creatinine kinase, and systemic signs like sweating, nausea, and headache [ 31 ].

2.2. Poisoning by Cane Toads ( Bufo marinus , Anura: Bufonidae)

Cane toads have been an invasive pest in Australia for nearly 80 years and in that time have decimated native animal populations and destroyed pristine habitat [ 4 , 32 ]. The Australian Government Department of the Environment has identified 15 biodiversity hotspots in Australia (Figure 2(a) ) the cane toad is already in five of those locations and has the potential to invade at least three more. The 15th biodiversity hotspot is in Tasmania, where no cane toads have been recorded. To give a clearer picture of the danger cane toads pose to native Australian fauna, the level of species richness has been overlaid with the cane toad population map (Figure 2(b) ).

A map of the current cane toad distribution (6,349 total records marked with red dots (a)) and 14 of the 15 biodiversity hotspots (×, (a)). The cane toad population data is overlaid with species richness data blue indicates higher and yellow lower levels of species diversity, respectively (b). Maps created from [ 33 ].

Cane toad poison induces neurological and cardiovascular effects and exposure to cane toad poison can be lethal to both dogs and cats [ 5 , 8 ]. The poisonous skin of cane toads ( Rhinella = Bufo marinus , Anura: Bufonidae) contains high concentrations of orally active compounds and is the main reason their toxicity in predatory animals is so high. Contaminated drinking water and food is a particularly insidious exposure route and good hygiene can go a long way towards reducing that risk for pet and livestock caretakers. Cane toad poison consists in large part of bufadienolides, a steroid that is a type of cardiac glycoside. Interestingly, compared to other life stages, cane toad eggs contain both the highest number of individual bufadienolides and the highest concentration of those compounds compared to later-stage juveniles [ 7 ]. These compounds act by inhibiting the sodium-potassium pump and increasing the force of contraction by the heart, thus increasing cardiac output.

Cane toad poisoning is not just an Australian problem. In the United States, dogs and cats in Florida, Colorado, Arizona, Texas, and Hawaii have reported intoxication from contact with Bufo toads: B. marinus , the cane toad, and B. alvarius , the Colorado river toad [ 34 ]. Dogs are more commonly poisoned than cats and terriers are disproportionately represented in the demographics [ 8 , 34 ].

Exposure to cane toad poison produces some or all of the following signs: in America, neurological abnormalities, hyperemic mucous membranes, ptyalism, recumbency or collapse, tachypnea, and vomiting [ 34 ] in Australia, ptyalism, hyperemic mucous membranes, and seizures [ 8 ]. Electrocardiographic findings were most commonly sinus arrhythmia, sinus tachycardia, and normal sinus rhythm [ 34 ]. The treatment for animals exposed to cane toad poison is lavage of the mouth and affected areas with tap water and the survival rate for the studies in both America and Australia discussed above was 㺐%.

2.3. Arthropods: Stings, Bites, and Poisoning 2.3.1. Hymenoptera

The insect order Hymenoptera includes the Apoidea (bees), Formicidae (ants), Vespoidea (wasps, hornets, and yellow jackets), and Symphyta (sawflies). Bees lose their stinger after stinging and die, but vespids can sting multiple times and also bite. Ants bite and some secrete venom that travels through the wound created at the bite site. Venoms from the Apoidea and Vespoidea are primarily made of proteins, but formicid venoms are 95% alkaloids [ 35 ]. Although anaphylaxis due to rapid hypersensitivity is the primary concern with Hymenoptera venom [ 10 ], ant bites and stings have long been known to cause severe pain and irritation [ 36 ]. The estimated lethal dose is 20 stings/kg in most mammals, though anaphylactic reactions are not dose-dependent [ 35 ]. No antivenom is available for bites and stings by Hymenoptera in most cases, management of clinical signs (including anaphylaxis) is the only recourse. This can generally be achieved through administration of fluids, corticosteroids, and supportive care [ 37 ].

Recently, the first account of survival after bumblebee-sting induced anaphylaxis in a dog was reported: “Over the following 48 hours, the dog developed azotemia, severely elevated liver enzyme levels, hypertension, hematochezia, hematemesis, and disseminated intravascular coagulation. The dog’s neurologic status improved slowly, but significant behavioral abnormalities remained. The dog was discharged after seven days with ongoing polyuria, polydipsia, and behavioral changes. The polydipsia and polyuria resolved within a few days, but the behavioral changes continued for six weeks” [ 38 ]. In another case, a dog presented for respiratory stress and shock after being stung by 𾄀 bees acute lung injury/acute respiratory syndrome was diagnosed and after eight days of treatment with oxygen, steroids, antibiotics, and bronchodilators, the dog recovered [ 39 ].

Although not currently present in Australia, Africanized bee stings present a significant threat of veterinary concern should they colonize. A retrospective study of dogs envenomated by Africanized bees in Brazil demonstrated dark-colored kidneys, dark red urine, dark red lungs, and splenomegaly as the major gross changes [ 40 ]. Secondary to massive Africanized bee envenomation (stings by 𾌀 Africanized bees) in a dog, immune-mediated thrombocytopenia was identified [ 41 ]. After a red blood cell transfusion, immunosuppressive dexamethasone, and gastroprotectant therapy, the dog stabilized and platelet count returned to normal within a week. In another case of bee sting envenomation, immune-mediated hemolytic anemia developed in two dogs one dog died and the hemolysis in the other was resolved following prolonged administration of corticosteroids [ 42 ].

Sawfly poisoning in Australia is largely due to Lophotoma spp. and the major toxin that causes poisoning is lophyrotomin, an octapeptide that acts principally on the liver [ 43 ]. The intraperitoneal LD50 in mice for lophyrotomin is 2 mg/kg [ 44 ]. Livestock, particularly sheep and cattle, are exposed to sawfly poisoning when leaves on the ground have sawflies on them and are ingested [ 37 ]. After removing animals from the sawfly source, the recommended management of poisoning consists of administration of silymarin and penicillin and glucose to prevent toxicosis and significant changes to liver enzymes [ 37 ].

In addition to the Hymenoptera, caterpillars of many Lepidoptera (butterflies and moths) contain urticating hairs and spines. In the early 2000s in the United States, eastern tent caterpillars ( Malacosoma americanum , Lepidoptera: Lasiocampidae) were found to be responsible for mare reproductive loss syndrome (MRLS). The combined losses from 2001 to 2002 for the thoroughbred industry due to MRLS were estimated at $ 500 million and more than 4500 equine pregnancies (3,500 of those, or 17%, were from thoroughbreds) were lost [ 45 ]. In Australia, similar incidences of MRLS were reported in the mid-2000s, with Ochrogaster lunifer (Lepidoptera: Thaumetopoeidae) found responsible [ 46 ]. After experimental gavage caterpillar setal fragments were found in multiple organs including the liver and gastrointestinal and reproductive tract and caused serositis, ulceration, and inflammation and it was theorized that the setae could vector bacteria resulting in secondary bacterial abortion [ 46 ].

Australian spiders are notorious for being venomous and deadly. The Australian funnel-web spider is one of a handful of spiders worldwide that are lethal to humans and a bite from the redback spider causes latrodectism (hallmarks of which include pain, muscle rigidity, vomiting, and sweating) [ 47 ]. In addition to having dangerous or lethal effects in humans, animals also experience severe, and sometimes fatal, effects of envenomation.

The distribution of dangerous Australian spiders varies. Australian funnel-web spiders are found primarily on the east coast, which is where the bulk of the human population has settled (Figures 3(a) and 3(b) ). The redback spider, on the other hand, is widely distributed around the coastal areas and throughout the center of the country (Figure 3(c) ). Unlike snakes, all three spiders have been found on the island of Tasmania.

Density and distribution of the three most dangerous spiders in Australia. The Australian funnel-web spiders in the genera Atrax (1,526 records (a)) and Hadronyche (2,108 records (b)) are localized primarily on the east coast and the redback spider ( Latrodectus hasselti ) is more widely distributed (1,297 records, (c)). Maps from [ 48 – 50 ].

The Australian funnel-web spider is classified into 35 species found in three genera, Hadronyche, Illawarra , and Atrax (Araneae: Hexathelidae) [ 53 ]. The lethal toxin in funnel-web spider venom, δ -HXTX-Ar1a, is a 4.8 kDa peptide with three disulfide bonds that was first described in 1985 [ 54 ]. Although the toxin is found in both males and females, only males seem to produce enough toxin to cause lethal effects after an envenomation [ 55 ]. The venom of the funnel-web spider has a wide phylogenetic range: rats, rabbits, and cats seem to be unaffected by a bite from a female spider, whereas 20% of mice and guinea pigs died after a bite from a female and most died after a bite from a male [ 37 ]. Male funnel-web spider bites have also been shown to have transient effects in dogs and cats [ 37 ]. Antivenom was introduced in 1984, after which no human fatalities from A. robustus or related spiders have been reported [ 56 ]. The LD50 of δ -HXTX-Ar1a has been reported as 0.16 mg/kg (33 pmol/g) in mice.

The redback spider, Latrodectus hasselti (Araneae: Theridiidae), is an Australian widow spider in the same genus as the North American black widow ( L. mactans ) and the New Zealand katipo ( L. katipo ). The major toxicity in animals is caused by α -latrotoxin-Lh1a, a 130 kDa presynaptic neurotoxin that causes the exhaustive release of neurotransmitters from presynaptic nerve terminals [ 57 ]. The reported LD50 value for L. tredecimguttatus (the European black widow) crude venom in guinea pigs is 0.0075 mg/kg in guinea pigs and 0.9 mg/kg in mice [ 37 ]. Although not naturally aggressive spiders, redbacks are widely distributed and accidental contact with humans and domestic animals can occur. Cats are particularly sensitive to Latrodectus venom studies have reported an average survival time of 115 h and that 20 of 22 cats died after widow spider bites [ 37 ]. In humans, a bite of the Australian redback spider Latrodectus hasselti (Araneae: Theridiidae) causes latrodectism involving incapacitation through severe local, regional, or systemic pain and autonomic effects such as muscle rigidity and fasciculation, vomiting, dyspnoea, tachycardia, hypertension, weakness, and sweating [ 13 ]. Antivenom is available, but treatment is largely focused on symptom management [ 37 ].

Australian tarantulas or whistling spiders (Araneae: Theraphosidae) are extraordinarily lethal to companion animals. Dogs have been reported to be especially sensitive to tarantula envenomation and death is reported to occur in 30� min for most dogs [ 12 , 58 ]. Phlogiellus and Selenocosmia genus spider bites were reported to kill 7 dogs often within 2 hours of envenomation with apnea and cardiac arrhythmia as clinical features [ 12 ]. Australian tarantulas belong to four genera: Selenotholus , Selenotypus, Coremiocnemis, and Phlogius . Tarantulas are widely distributed throughout the Australian continent and North Queensland has a high concentration of tarantulas and people, which is why many cases of dog death are reported from that region (Figure 4 ).

A distribution map of tarantulas (spiders in the family Theraphosidae), showing 463 occurrence records each marked with a blue dot. Map from [ 51 ].

Tarantula venom contains a variety of peptides with different mechanisms of action and venoms can be expected to contain neurotoxins, as well as possibly cytotoxic and hemolytic toxins. Following a tarantula bite, patients may experience muscle spasms, edema, hemoglobinuria, jaundice, and circulatory shock [ 37 ].

The venom of one species of Australian tarantula, Selenotypus plumipes , has recently been the source of the most potent orally active insecticidal peptide reported from spider venom [ 59 ]. Tarantulas are large, heavy-bodied spiders that live for 5� years in laboratory environments and Australian tarantula venoms contain a particularly large concentration of peptides in the 3𠄶 kDa range, within the size range of many active toxins and pharmaceutical leads [ 60 ].

Ticks affect animal and human health globally and cause significant economic losses directly via feeding, indirectly through the transmission of tick-borne diseases, and through toxicosis, a toxic reaction due to a toxic component present in the saliva. Ticks in the genus Ixodes are well known for their ability to induce paralysis during and after feeding [ 61 ]. The toxicity of ticks, which are hematophagic ectoparasites, comes from antigens in their saliva that modulate the host’s immune response in order to facilitate blood feeding. Tick salivary anticoagulants are reported to act through either the inhibition of thrombin or inhibition of factor X activation [ 62 ]. Ixodes tick paralysis is a toxin-mediated type of acute flaccid paralysis caused by the presynaptic neurotoxin holocyclotoxin, which acts to inhibit acetylcholine release at the neuromuscular junction [ 63 – 65 ]. Death is commonly the result of respiratory failure from a combination of neuromuscular paralysis causing hypoventilation as well as pulmonary parenchymal disease, though unexpected or “sudden” death is also reported [ 65 , 66 ]. Dogs with tick paralysis may exhibit pulmonary congestion and oedema in uncomplicated cases but frequently also show moderate to severe bronchopneumonia with or without evidence of aspiration [ 65 ]. Laryngeal and oesophageal dysfunction, often accompanied by vomiting, is common in tick paralysis and may predispose affected dogs to aspiration pneumonia [ 65 ]. Further, analysis of crude toxin in rats indicates that Ixodes toxins have direct cardiovascular effects suggestive of potassium channel blockade [ 67 ]. Necropsy findings in other tissues are nonspecific and include severe vascular congestion in the liver, kidneys, and myocardium [ 68 ]. The first example of immunization against the paralyzing effects of holocyclotoxin was in dogs, using salivary gland extracts from I. holocyclus after immunization, dogs were able to withstand four times the ED50 [ 69 ]. Australian paralysis ticks have a reported ED50 of 0.48 mg salivary gland protein/kg bodyweight to cause hind limb paralysis in dogs [ 69 ]. Despite the potency of salivary gland extracts, the amount of crude starting material extracted from ticks is extraordinarily small, which complicates discovery-stage work. Research suggests there may be different modes of action for toxins in the saliva of North American and Australian tick species [ 70 ].

The Australian paralysis tick is found primarily on the east coast (Figure 5 ).

A distribution map of Ixodes holocyclus , the Australian paralysis tick. Each occurrence record (174 total) is marked with a blue dot. Map from [ 52 ].

Native hosts of I. holocyclus include the three species of bandicoot, although the tick has been found on a wide variety of native animals and livestock [ 71 ]. Although cats, dogs, and horses present most frequently with signs of tick infestation, paralysis ticks also affect native animals. The spectacled flying fox ( Pteropus conspicillatus , Chiroptera: Pteropodidae) has shown affected electrical cardiac function when infested with I. holocyclus [ 72 ].

3. Potential for Novel Therapeutics

Recent technological advances have provided the gateway to exploring venomics (or, in the case of ticks, sialomics) as a novel source of therapeutics. First, the ability of proteomics and genomics to identify all the venom components, even those expressed in low quantities in the venom, allows more potent toxins to be identified. Second, the advent of high-throughput assays and target-based drug design have led to an explosion of interest in venom toxins, which can act as highly specific pharmacological probes for a single molecular target. Third, the bulk of vertebrate and invertebrate venom toxins hit ion channels, which are critical for nervous system function and an area of particular interest for pharmaceutical companies. Spider venom toxins, for example, represent one-third of known NaV channel modulators [ 73 ].

Animal models of human disease are a critical component of drug discovery, although they can differ significantly from human biology and pathobiology [ 74 ]. Dogs are often a close match for human disorders, particularly for cardiovascular disease [ 75 ]. Thus, an understanding of the clinical effects and signs of envenomation in animals can yield pharmaceutical leads for veterinary use, as well as potential leads for human therapeutics, too.

3.1. Potency and Mechanism of Animal Venoms and Poisons

One of the advantages of using venom and poison toxins for pharmaceutical leads is the potency and highly targeted nature of the individual toxins. Two specific sources of potent potential veterinary leads are discussed further: snake venoms and cane toad poisons.

A common thread between Australian elapid snake venoms is the presence of variations on potent α -neurotoxins [ 76 ]. The “ α -” prefix is used to indicate toxins with postsynaptic activity α -neurotoxins are neurotoxic peptides between 60 and 75 residues in length, which are linked by 4-5 disulfide bridges [ 77 ]. Short- and long-type toxins have similar 3D structures, but different dissociation kinetics with the receptor [ 77 ]. They act as competitive and irreversible antagonists of postsynaptic nicotinic acetylcholine receptors [ 78 ].

A variety of new human pharmaceuticals have been discovered from snake venoms, including several which are in clinical trials. Snake venoms have proved to be a particularly rich source of cardiovascular drugs [ 79 ]. Despite the potency of Australian snake venoms, their pharmaceutical use remains undetermined to date, no human pharmaceuticals have been isolated from Australian snake venoms. Cenderitide, a toxin from the Eastern green mamba ( Dendroaspis angusticeps , Squamata: Elapidae), is indicated in the treatment of congestive heart failure a chemically modified version of a short-chain α -cobrotoxin, a cobra venom toxin (isolated from Naja spp. venom), is indicated in the treatment of HIV and a chemically modified version of a long-chain α -cobrotoxin is indicated in the treatment of multiple sclerosis and perioperative bleeding [ 14 ]. Based on these examples of human pharmaceuticals, there is evidence to suggest novel therapeutics could be developed for veterinary use as well.

The chemistry of snake venoms has been fairly well characterized, primarily due to their importance in human medicine [ 80 ]. Snake venoms are produced in specialized venom glands and snake venom from an individual or within a species can vary widely [ 81 ], making treatment more of a challenge. Snake venom toxins are of particular interest for cardiovascular disease [ 79 ] and as natriuretic peptides, which modulate body fluid volume [ 82 ]. An overview of snake venom toxins is provided, with an emphasis on clinical effects (Table 1 ). Since these classes have major pathophysiological effects in snakebite victims, they are well suited to rational drug design.

An overview of the major toxin classes with clinical effects in snake venom and their indications. Note myotoxins are necrotic and often lead to death via diaphragmatic paralysis.

Several species of toad in the genus Bufo (Anura: Bufonidae) have been reported to have hallucinogenic or psychedelic effects when they are licked by humans. The bulk of these effects are due to the presence of an alkaloid, bufotenine, which is structurally related to the neurotransmitter serotonin, in skin secretions of the toad [ 83 ]. The hallucinogenic effects of licking cane toads have been reported in humans but not so comprehensively studied in dogs. Nonetheless, owners and veterinarians report poisoned dogs as appearing “high.” Not surprisingly, this Australian story has caught the public’s interest at home [ 84 ] and overseas [ 85 – 87 ]. The behavior of these dogs belies some critical clues for the use of the poison extracts as potential therapeutics: (i) the poison components are orally active in dogs (ii) the poison contains active compounds with neurological, cardiac, and potentially psychoactive effects specific to dogs and (iii) after treatment, within 24 h of initial exposure the dog experiences complete recovery with no known long-term effects.

Cane toad poison consists in large part of bufadienolides, a steroid that is a type of cardiac glycoside. Interestingly, compared to other life stages, cane toad eggs contain both the highest number of individual bufadienolides and the highest concentration of those compounds compared to later-stage juveniles [ 7 ]. These compounds act by inhibiting the sodium-potassium pump and increasing the force of contraction by the heart, thus increasing cardiac output.

Despite the toxicity of cane toad poison to other vertebrates, including reptiles and mammals, cane toads and chickens are to be immune to the poison in fact, one chicken was reported to eat 45 cane toads over a two-day experimental period with no ill effects [ 88 ]. The same study showed chickens had no adverse reaction when drinking water cane toads which had been sitting in overnight, suggesting perhaps chickens are nonresponsive to the cardiac glycosides in the cane toad poison. Chickens and cane toads have slightly, but not completely, different gene sequences for the sodium-potassium pump (Figures 6(a) and 6(b) ). The gene sequences for cats and dogs are most similar to each other, and most different from chickens and cane toads. These gene-level differences may explain why companion animals (cats and dogs) are so susceptible to cane toad poisoning and livestock (rabbits, pigs, sheep, and cows) are protected from the severe cardiac effects of cane toad poisoning.

An alignment (a) and cladogram (b) of the ATP1A1 gene, which produces the sodium/potassium-transporting ATPase subunit alpha-1. A Jukes-Cantor genetic distance model of the ATP1A1 gene, using a neighbour-joining tree building method with zebrafish as the outgroup (b). Bootstrapping was used as a resampling method with 100 replicates and the support threshold was 50%. An asterisk ( * ) in (a) and (b) indicates a partial sequence from UniProt. Chemical structures in (c) are from ChemSpider ( http://www.chemspider.com/ ) the average mass is reported.

Cardiac glycosides are commonly prescribed to treat congestive heart failure and arrhythmia and several successful drugs have been developed from natural products (Figure 6(c) ).

Not surprisingly, these naturally derived cardiac glycosides are considered lethal when encountered in nature however, therapeutic doses can usually be achieved. Ouabain is an exception, as it is so potent that it is largely only used experimentally. As with the commercially available drugs, the reaction of dogs to cane toad poisoning is delayed. Currently, no specific antidote for cane toad poisoning exists and clinical management relies on lavage of the mouth and exposed areas to decrease toxin exposure, followed by symptomatic management. Bufadienolide is the smallest of the naturally derived cardiac glycosides and its synthesis was first demonstrated in the literature in the 1970s [ 89 ]. Bufadienolides also have demonstrated antitumor activity derivatives of one alkaloid, bufalin, have been shown to have antiproliferation activity against carcinoma and leukemia cells [ 90 ].

Australia has a variety of venomous and poisonous animals that are dangerous to humans, pets, and livestock. Costs of treating a single envenomation event can run into several thousand dollars and, despite extensive medical treatment, many animals die. A greater understanding of individual toxins will enhance our ability to diagnose and treat envenomation and poisoning and to monitor for secondary toxic effects. Although pathophysiology and treatment can be extrapolated from human studies, species differences occur and those venoms that have different effects in animals may prove to be a rich source of novel, specific veterinary therapeutics. Elucidation of the pathophysiology and mechanism of action of venoms and toxins will allow the development of novel human and veterinary therapeutics through rational drug design.


Rabies Patient

Rabies symptoms include malaise, violent movements, terror, mania, and delirium.

Rabies causes about 55,000 human deaths annually worldwide, with 95% of human deaths occurring in Asia and Africa. Roughly 97% of human rabies cases result from dog bites . In the U.S., animal control and vaccination programs have effectively eliminated domestic dogs as reservoirs of rabies. In several countries, including Australia and Japan, rabies carried by terrestrial animals has been eliminated entirely. While rabies was once eradicated in the United Kingdom, infected bats have recently been found in Scotland. In the U.S., the widespread vaccination of domestic dogs and cats and the development of effective human vaccines and immunoglobulin treatments has dropped the number of recorded human deaths from 100 or more annually in the early 20 th century, to one to two per year (mostly caused by bat bites). Modern cell-based vaccines are similar to flu shots in terms of pain and side effects. The old nerve-tissue-based vaccinations that require multiple painful injections into the abdomen with a large needle are cheap, but are being phased out and replaced by affordable World Health Organization intradermal vaccination regimens.


Contents

There is overwhelming scientific consensus that vaccines are a very safe and effective way to fight and eradicate infectious diseases. [15] [16] [17] [18] The immune system recognizes vaccine agents as foreign, destroys them, and "remembers" them. When the virulent version of an agent is encountered, the body recognizes the protein coat on the virus, and thus is prepared to respond, by first neutralizing the target agent before it can enter cells, and secondly by recognizing and destroying infected cells before that agent can multiply to vast numbers. [ citation needed ]

Limitations to their effectiveness, nevertheless, exist. [19] Sometimes, protection fails because of vaccine-related failure such as failures in vaccine attenuation, vaccination regimes or administration or host-related failure due to host's immune system simply does not respond adequately or at all. Lack of response commonly results from genetics, immune status, age, health or nutritional status. [20] It also might fail for genetic reasons if the host's immune system includes no strains of B cells that can generate antibodies suited to reacting effectively and binding to the antigens associated with the pathogen. [ citation needed ]

Even if the host does develop antibodies, protection might not be adequate immunity might develop too slowly to be effective in time, the antibodies might not disable the pathogen completely, or there might be multiple strains of the pathogen, not all of which are equally susceptible to the immune reaction. However, even a partial, late, or weak immunity, such as a one resulting from cross-immunity to a strain other than the target strain, may mitigate an infection, resulting in a lower mortality rate, lower morbidity, and faster recovery. [ citation needed ]

Adjuvants commonly are used to boost immune response, particularly for older people whose immune response to a simple vaccine may have weakened. [21]

The efficacy or performance of the vaccine is dependent on several factors:

  • the disease itself (for some diseases vaccination performs better than for others)
  • the strain of vaccine (some vaccines are specific to, or at least most effective against, particular strains of the disease) [22]
  • whether the vaccination schedule has been properly observed.
  • idiosyncratic response to vaccination some individuals are "non-responders" to certain vaccines, meaning that they do not generate antibodies even after being vaccinated correctly.
  • assorted factors such as ethnicity, age, or genetic predisposition.

If a vaccinated individual does develop the disease vaccinated against (breakthrough infection), the disease is likely to be less virulent than in unvaccinated victims. [23]

Important considerations in an effective vaccination program: [24]

  1. careful modeling to anticipate the effect that an immunization campaign will have on the epidemiology of the disease in the medium to long term
  2. ongoing surveillance for the relevant disease following introduction of a new vaccine
  3. maintenance of high immunization rates, even when a disease has become rare

In 1958, there were 763,094 cases of measles in the United States 552 deaths resulted. [25] [26] After the introduction of new vaccines, the number of cases dropped to fewer than 150 per year (median of 56). [26] In early 2008, there were 64 suspected cases of measles. Fifty-four of those infections were associated with importation from another country, although only thirteen percent were actually acquired outside the United States 63 of the 64 individuals either had never been vaccinated against measles or were uncertain whether they had been vaccinated. [26]

Vaccines led to the eradication of smallpox, one of the most contagious and deadly diseases in humans. [27] Other diseases such as rubella, polio, measles, mumps, chickenpox, and typhoid are nowhere near as common as they were a hundred years ago thanks to widespread vaccination programs. As long as the vast majority of people are vaccinated, it is much more difficult for an outbreak of disease to occur, let alone spread. This effect is called herd immunity. Polio, which is transmitted only among humans, is targeted by an extensive eradication campaign that has seen endemic polio restricted to only parts of three countries (Afghanistan, Nigeria, and Pakistan). [28] However, the difficulty of reaching all children as well as cultural misunderstandings have caused the anticipated eradication date to be missed several times. [ citation needed ]

Vaccines also help prevent the development of antibiotic resistance. For example, by greatly reducing the incidence of pneumonia caused by Streptococcus pneumoniae, vaccine programs have greatly reduced the prevalence of infections resistant to penicillin or other first-line antibiotics. [29]

The measles vaccine is estimated to prevent a million deaths every year. [30]

Adverse effects

Vaccinations given to children, adolescents, or adults are generally safe. [31] [32] Adverse effects, if any, are generally mild. [33] The rate of side effects depends on the vaccine in question. [33] Some common side effects include fever, pain around the injection site, and muscle aches. [33] Additionally, some individuals may be allergic to ingredients in the vaccine. [34] MMR vaccine is rarely associated with febrile seizures. [32]

Host-("vaccinee")-related determinants that render a person susceptible to infection, such as genetics, health status (underlying disease, nutrition, pregnancy, sensitivities or allergies), immune competence, age, and economic impact or cultural environment can be primary or secondary factors affecting the severity of infection and response to a vaccine. [35] Elderly (above age 60), allergen-hypersensitive, and obese people have susceptibility to compromised immunogenicity, which prevents or inhibits vaccine effectiveness, possibly requiring separate vaccine technologies for these specific populations or repetitive booster vaccinations to limit virus transmission. [35]

Severe side effects are extremely rare. [32] Varicella vaccine is rarely associated with complications in immunodeficient individuals, and rotavirus vaccines are moderately associated with intussusception. [32]

At least 19 countries have no-fault compensation programs to provide compensation for those suffering severe adverse effects of vaccination. [36] The United States’ program is known as the National Childhood Vaccine Injury Act, and the United Kingdom employs the Vaccine Damage Payment.

Vaccines typically contain dead or inactivated organisms or purified products derived from them.

There are several types of vaccines in use. [37] These represent different strategies used to try to reduce the risk of illness while retaining the ability to induce a beneficial immune response.

Attenuated

Some vaccines contain live, attenuated microorganisms. Many of these are active viruses that have been cultivated under conditions that disable their virulent properties, or that use closely related but less dangerous organisms to produce a broad immune response. Although most attenuated vaccines are viral, some are bacterial in nature. Examples include the viral diseases yellow fever, measles, mumps, and rubella, and the bacterial disease typhoid. The live Mycobacterium tuberculosis vaccine developed by Calmette and Guérin is not made of a contagious strain but contains a virulently modified strain called "BCG" used to elicit an immune response to the vaccine. The live attenuated vaccine containing strain Yersinia pestis EV is used for plague immunization. Attenuated vaccines have some advantages and disadvantages. Attenuated, or live, weakened, vaccines typically provoke more durable immunological responses. But they may not be safe for use in immunocompromised individuals, and on rare occasions mutate to a virulent form and cause disease. [38]

Inactivated

Some vaccines contain inactivated, but previously virulent, micro-organisms that have been destroyed with chemicals, heat, or radiation [39] – "ghosts", with intact but empty bacterial cell envelopes. They are considered an intermediate phase between the inactivated and attenuated vaccines. [40] Examples include IPV (polio vaccine), hepatitis A vaccine, rabies vaccine and most influenza vaccines. [41]

Toxoid

Toxoid vaccines are made from inactivated toxic compounds that cause illness rather than the micro-organism. [42] Examples of toxoid-based vaccines include tetanus and diphtheria. [41] Not all toxoids are for micro-organisms for example, Crotalus atrox toxoid is used to vaccinate dogs against rattlesnake bites. [43]

Subunit

Rather than introducing an inactivated or attenuated micro-organism to an immune system (which would constitute a "whole-agent" vaccine), a subunit vaccine uses a fragment of it to create an immune response. One example is the subunit vaccine against hepatitis B, which is composed of only the surface proteins of the virus (previously extracted from the blood serum of chronically infected patients but now produced by recombination of the viral genes into yeast). [44] Another example is edible algae vaccines, such as the virus-like particle (VLP) vaccine against human papillomavirus (HPV), which is composed of the viral major capsid protein. [45] Another example is the hemagglutinin and neuraminidase subunits of the influenza virus. [41] A subunit vaccine is being used for plague immunization. [46]

Conjugate

Certain bacteria have a polysaccharide outer coat that is poorly immunogenic. By linking these outer coats to proteins (e.g., toxins), the immune system can be led to recognize the polysaccharide as if it were a protein antigen. This approach is used in the Haemophilus influenzae type B vaccine. [47]

Outer membrane vesicle

Outer membrane vesicles (OMVs) are naturally immunogenic and can be manipulated to produce potent vaccines. The best known OMV vaccines are those developed for serotype B meningococcal disease. [48] [49]

Heterotypic

Heterologous vaccines also known as "Jennerian vaccines", are vaccines that are pathogens of other animals that either do not cause disease or cause mild disease in the organism being treated. The classic example is Jenner's use of cowpox to protect against smallpox. A current example is the use of BCG vaccine made from Mycobacterium bovis to protect against tuberculosis. [50]

Viral vector

Viral vector vaccines use a safe virus to insert pathogen genes in the body to produce specific antigens, such as surface proteins, to stimulate an immune response. [51] [52]

An mRNA vaccine (or RNA vaccine) is a novel type of vaccine which is composed of the nucleic acid RNA, packaged within a vector such as lipid nanoparticles. [53] Among the COVID-19 vaccines are a number of RNA vaccines under development to combat the COVID-19 pandemic and some have received emergency use authorization in some countries. For example, the Pfizer-BioNTech and Moderna mRNA vaccines have emergency use authorization in the US. [54] [55] [56]

Experimental

Many innovative vaccines are also in development and use.

  • Dendritic cell vaccines combine dendritic cells with antigens to present the antigens to the body's white blood cells, thus stimulating an immune reaction. These vaccines have shown some positive preliminary results for treating brain tumors [57] and are also tested in malignant melanoma. [58] – The proposed mechanism is the insertion and expression of viral or bacterial DNA in human or animal cells (enhanced by the use of electroporation), triggering immune system recognition. Some cells of the immune system that recognize the proteins expressed will mount an attack against these proteins and cells expressing them. Because these cells live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they will be attacked instantly by the immune system. One potential advantage of DNA vaccines is that they are very easy to produce and store. vector – by combining the physiology of one micro-organism and the DNA of another, immunity can be created against diseases that have complex infection processes. An example is the RVSV-ZEBOV vaccine licensed to Merck that is being used in 2018 to combat ebola in Congo. [59] peptide vaccines are under development for several diseases using models of Valley Fever, stomatitis, and atopic dermatitis. These peptides have been shown to modulate cytokine production and improve cell-mediated immunity.
  • Targeting of identified bacterial proteins that are involved in complement inhibition would neutralize the key bacterial virulence mechanism. [60]
  • The use of plasmids has been validated in preclinical studies as a protective vaccine strategy for cancer and infectious diseases. However, in human studies, this approach has failed to provide clinically relevant benefit. The overall efficacy of plasmid DNA immunization depends on increasing the plasmid's immunogenicity while also correcting for factors involved in the specific activation of immune effector cells. [61] – Similar in principle to viral vector vaccines, but using bacteria instead. [48][48]

While most vaccines are created using inactivated or attenuated compounds from micro-organisms, synthetic vaccines are composed mainly or wholly of synthetic peptides, carbohydrates, or antigens.

Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism. [62] A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. [63] The valency of a multivalent vaccine may be denoted with a Greek or Latin prefix (e.g., tetravalent or quadrivalent). In certain cases, a monovalent vaccine may be preferable for rapidly developing a strong immune response. [64]

When two or more vaccines are mixed in the same formulation, the two vaccines can interfere. This most frequently occurs with live attenuated vaccines, where one of the vaccine components is more robust than the others and suppresses the growth and immune response to the other components. This phenomenon was first noted in the trivalent Sabin polio vaccine, where the amount of serotype 2 virus in the vaccine had to be reduced to stop it from interfering with the "take" of the serotype 1 and 3 viruses in the vaccine. [65] This phenomenon has also been found to be a problem with the dengue vaccines currently being researched, [ when? ] where the DEN-3 serotype was found to predominate and suppress the response to DEN-1, −2 and −4 serotypes. [66]

Adjuvants

Vaccines typically contain one or more adjuvants, used to boost the immune response. Tetanus toxoid, for instance, is usually adsorbed onto alum. This presents the antigen in such a way as to produce a greater action than the simple aqueous tetanus toxoid. People who have an adverse reaction to adsorbed tetanus toxoid may be given the simple vaccine when the time comes for a booster. [67]

In the preparation for the 1990 Persian Gulf campaign, the whole cell pertussis vaccine was used as an adjuvant for anthrax vaccine. This produces a more rapid immune response than giving only the anthrax vaccine, which is of some benefit if exposure might be imminent. [68]

Preservatives

Vaccines may also contain preservatives to prevent contamination with bacteria or fungi. Until recent years, the preservative thiomersal (A.K.A. Thimerosal in the US and Japan) was used in many vaccines that did not contain live viruses. As of 2005, the only childhood vaccine in the U.S. that contains thiomersal in greater than trace amounts is the influenza vaccine, [69] which is currently recommended only for children with certain risk factors. [70] Single-dose influenza vaccines supplied in the UK do not list thiomersal in the ingredients. Preservatives may be used at various stages of the production of vaccines, and the most sophisticated methods of measurement might detect traces of them in the finished product, as they may in the environment and population as a whole. [71]

Many vaccines need preservatives to prevent serious adverse effects such as Staphylococcus infection, which in one 1928 incident killed 12 of 21 children inoculated with a diphtheria vaccine that lacked a preservative. [72] Several preservatives are available, including thiomersal, phenoxyethanol, and formaldehyde. Thiomersal is more effective against bacteria, has a better shelf-life, and improves vaccine stability, potency, and safety but, in the U.S., the European Union, and a few other affluent countries, it is no longer used as a preservative in childhood vaccines, as a precautionary measure due to its mercury content. [73] Although controversial claims have been made that thiomersal contributes to autism, no convincing scientific evidence supports these claims. [74] Furthermore, a 10–11-year study of 657,461 children found that the MMR vaccine does not cause autism and actually reduced the risk of autism by seven percent. [75] [76]

Excipients

Beside the active vaccine itself, the following excipients and residual manufacturing compounds are present or may be present in vaccine preparations: [77]

    salts or gels are added as adjuvants. Adjuvants are added to promote an earlier, more potent response, and more persistent immune response to the vaccine they allow for a lower vaccine dosage. are added to some vaccines to prevent the growth of bacteria during production and storage of the vaccine.
  • Egg protein is present in the influenza vaccine and yellow fever vaccine as they are prepared using chicken eggs. Other proteins may be present. is used to inactivate bacterial products for toxoid vaccines. Formaldehyde is also used to inactivate unwanted viruses and kill bacteria that might contaminate the vaccine during production. (MSG) and 2-phenoxyethanol are used as stabilizers in a few vaccines to help the vaccine remain unchanged when the vaccine is exposed to heat, light, acidity, or humidity. is a mercury-containing antimicrobial that is added to vials of vaccines that contain more than one dose to prevent contamination and growth of potentially harmful bacteria. Due to the controversy surrounding thiomersal, it has been removed from most vaccines except multi-use influenza, where it was reduced to levels so that a single dose contained less than a microgram of mercury, a level similar to eating ten grams of canned tuna. [78]

Various fairly standardized abbreviations for vaccine names have developed, although the standardization is by no means centralized or global. For example, the vaccine names used in the United States have well-established abbreviations that are also widely known and used elsewhere. An extensive list of them provided in a sortable table and freely accessible is available at a US Centers for Disease Control and Prevention web page. [79] The page explains that "The abbreviations [in] this table (Column 3) were standardized jointly by staff of the Centers for Disease Control and Prevention, ACIP Work Groups, the editor of the Morbidity and Mortality Weekly Report (MMWR), the editor of Epidemiology and Prevention of Vaccine-Preventable Diseases (the Pink Book), ACIP members, and liaison organizations to the ACIP." [79]

Some examples are "DTaP" for diphtheria and tetanus toxoids and acellular pertussis vaccine, "DT" for diphtheria and tetanus toxoids, and "Td" for tetanus and diphtheria toxoids. At its page on tetanus vaccination, [80] the CDC further explains that "Upper-case letters in these abbreviations denote full-strength doses of diphtheria (D) and tetanus (T) toxoids and pertussis (P) vaccine. Lower-case "d" and "p" denote reduced doses of diphtheria and pertussis used in the adolescent/adult-formulations. The 'a' in DTaP and Tdap stands for 'acellular', meaning that the pertussis component contains only a part of the pertussis organism." [80]

Another list of established vaccine abbreviations is at the CDC's page called "Vaccine Acronyms and Abbreviations", with abbreviations used on U.S. immunization records. [81] The United States Adopted Name system has some conventions for the word order of vaccine names, placing head nouns first and adjectives postpositively. This is why the USAN for "OPV" is "poliovirus vaccine live oral" rather than "oral poliovirus vaccine".

A vaccine licensure occurs after the successful conclusion of the development cycle and further the clinical trials and other programs involved through Phases I–III demonstrating safety, immunoactivity, immunogenetic safety at a given specific dose, proven effectiveness in preventing infection for target populations, and enduring preventive effect (time endurance or need for revaccination must be estimated). [82] As part of a multinational licensing of a vaccine, the World Health Organization Expert Committee on Biological Standardization developed guidelines of international standards for manufacturing and quality control of vaccines, a process intended as a platform for national regulatory agencies to apply for their own licensing process. [82] Vaccine manufacturers do not receive licensing until a complete clinical cycle of development and trials proves the vaccine is safe and has long-term effectiveness, following scientific review by a multinational or national regulatory organization, such as the European Medicines Agency (EMA) or the US Food and Drug Administration (FDA). [83] [84]

Upon developing countries adopting WHO guidelines for vaccine development and licensure, each country has its own responsibility to issue a national licensure, and to manage, deploy, and monitor the vaccine throughout its use in each nation. [82] Building trust and acceptance of a licensed vaccine among the public is a task of communication by governments and healthcare personnel to ensure a vaccination campaign proceeds smoothly, saves lives, and enables economic recovery. [85] [86] When a vaccine is licensed, it will initially be in limited supply due to variable manufacturing, distribution, and logistical factors, requiring an allocation plan for the limited supply and which population segments should be prioritized to first receive the vaccine. [85]

World Health Organization

Vaccines developed for multinational distribution via the United Nations Children's Fund (UNICEF) require pre-qualification by the WHO to ensure international standards of quality, safety, immunogenicity, and efficacy for adoption by numerous countries. [82]

The process requires manufacturing consistency at WHO-contracted laboratories following Good Manufacturing Practice (GMP). [82] When UN agencies are involved in vaccine licensure, individual nations collaborate by 1) issuing marketing authorization and a national license for the vaccine, its manufacturers, and distribution partners and 2) conducting postmarketing surveillance, including records for adverse events after the vaccination program. The WHO works with national agencies to monitor inspections of manufacturing facilities and distributors for compliance with GMP and regulatory oversight. [82]

Some countries choose to buy vaccines licensed by reputable national organizations, such as EMA, FDA, or national agencies in other affluent countries, but such purchases typically are more expensive and may not have distribution resources suitable to local conditions in developing countries. [82]

European Union

In the European Union (EU), vaccines for pandemic pathogens, such as seasonal influenza, are licensed EU-wide where all the member states comply ("centralized"), are licensed for only some member states ("decentralized"), or are licensed on an individual national level. [83] Generally, all EU states follow regulatory guidance and clinical programs defined by the European Committee for Medicinal Products for Human Use (CHMP), a scientific panel of the European Medicines Agency (EMA) responsible for vaccine licensure. [83] The CHMP is supported by several expert groups who assess and monitor the progress of a vaccine before and after licensure and distribution. [83]

United States

Under the FDA, the process of establishing evidence for vaccine clinical safety and efficacy is the same as for the approval process for prescription drugs. [87] If successful through the stages of clinical development, the vaccine licensing process is followed by a Biologics License Application which must provide a scientific review team (from diverse disciplines, such as physicians, statisticians, microbiologists, chemists) and comprehensive documentation for the vaccine candidate having efficacy and safety throughout its development. Also during this stage, the proposed manufacturing facility is examined by expert reviewers for GMP compliance, and the label must have a compliant description to enable health care providers' definition of vaccine-specific use, including its possible risks, to communicate and deliver the vaccine to the public. [87] After licensure, monitoring of the vaccine and its production, including periodic inspections for GMP compliance, continue as long as the manufacturer retains its license, which may include additional submissions to the FDA of tests for potency, safety, and purity for each vaccine manufacturing step. [87]

Postmarketing surveillance

Until a vaccine is in use for the general population, all potential adverse events from the vaccine may not be known, requiring manufacturers to conduct Phase IV studies for postmarketing surveillance of the vaccine while it is used widely in the public. [82] [87] The WHO works with UN member states to implement post-licensing surveillance. [82] The FDA relies on a Vaccine Adverse Event Reporting System to monitor safety concerns about a vaccine throughout its use in the American public. [87]

In order to provide the best protection, children are recommended to receive vaccinations as soon as their immune systems are sufficiently developed to respond to particular vaccines, with additional "booster" shots often required to achieve "full immunity". This has led to the development of complex vaccination schedules. In the United States, the Advisory Committee on Immunization Practices, which recommends schedule additions for the Centers for Disease Control and Prevention, recommends routine vaccination of children against [88] hepatitis A, hepatitis B, polio, mumps, measles, rubella, diphtheria, pertussis, tetanus, HiB, chickenpox, rotavirus, influenza, meningococcal disease and pneumonia. [89]

The large number of vaccines and boosters recommended (up to 24 injections by age two) has led to problems with achieving full compliance. To combat declining compliance rates, various notification systems have been instituted and many combination injections are now marketed (e.g., Pneumococcal conjugate vaccine and MMRV vaccine), which protect against multiple diseases. [ citation needed ]

Besides recommendations for infant vaccinations and boosters, many specific vaccines are recommended for other ages or for repeated injections throughout life – most commonly for measles, tetanus, influenza, and pneumonia. Pregnant women are often screened for continued resistance to rubella. The human papillomavirus vaccine is recommended in the U.S. (as of 2011) [90] and UK (as of 2009). [91] Vaccine recommendations for the elderly concentrate on pneumonia and influenza, which are more deadly to that group. In 2006, a vaccine was introduced against shingles, a disease caused by the chickenpox virus, which usually affects the elderly. [ citation needed ]

One challenge in vaccine development is economic: Many of the diseases most demanding a vaccine, including HIV, malaria and tuberculosis, exist principally in poor countries. Pharmaceutical firms and biotechnology companies have little incentive to develop vaccines for these diseases because there is little revenue potential. Even in more affluent countries, financial returns are usually minimal and the financial and other risks are great. [92]

Most vaccine development to date has relied on "push" funding by government, universities and non-profit organizations. [93] Many vaccines have been highly cost effective and beneficial for public health. [94] The number of vaccines actually administered has risen dramatically in recent decades. [95] This increase, particularly in the number of different vaccines administered to children before entry into schools may be due to government mandates and support, rather than economic incentive. [96]

Patents

According to the World Health Organization, the biggest barrier to vaccine production in less developed countries has not been patents, but the substantial financial, infrastructure, and workforce requirements needed for market entry. Vaccines are complex mixtures of biological compounds, and unlike the case for prescription drugs, there are no true generic vaccines. The vaccine produced by a new facility must undergo complete clinical testing for safety and efficacy by the manufacturer. For most vaccines, specific processes in technology are patented. These can be circumvented by alternative manufacturing methods, but this required R&D infrastructure and a suitably skilled workforce. In the case of a few relatively new vaccines, such as the human papillomavirus vaccine, the patents may impose an additional barrier. [97]

When increased production of vaccines was urgently needed during the COVID-19 pandemic in 2021, the World Trade Organization and governments around the world evaluated whether to waive intellectual property rights and patents on COVID-19 vaccines, which would "eliminate all potential barriers to the timely access of affordable COVID-19 medical products, including vaccines and medicines, and scale up the manufacturing and supply of essential medical products." [98]

Vaccine production is fundamentally different from other kinds of manufacturing – including regular pharmaceutical manufacturing – in that vaccines are intended to be administered to millions of people of whom the vast majority are perfectly healthy. [99] This fact drives an extraordinarily rigorous production process with strict compliance requirements that go far beyond what is required of other products. [99]

Depending upon the antigen, it can cost anywhere from US$50 to $500 million to build a vaccine production facility, which requires highly specialized equipment, clean rooms, and containment rooms. [100] There is a global scarcity of personnel with the right combination of skills, expertise, knowledge, competence and personality to staff vaccine production lines. [100] With the notable exceptions of Brazil, China, and India, many developing countries' educational systems are unable to provide enough qualified candidates, and vaccine makers based in such countries must hire expatriate personnel to keep production going. [100]

Vaccine production has several stages. First, the antigen itself is generated. Viruses are grown either on primary cells such as chicken eggs (e.g., for influenza) or on continuous cell lines such as cultured human cells (e.g., for hepatitis A). [101] Bacteria are grown in bioreactors (e.g., Haemophilus influenzae type b). Likewise, a recombinant protein derived from the viruses or bacteria can be generated in yeast, bacteria, or cell cultures. [102] [103]

After the antigen is generated, it is isolated from the cells used to generate it. A virus may need to be inactivated, possibly with no further purification required. Recombinant proteins need many operations involving ultrafiltration and column chromatography. Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as needed. The adjuvant enhances the immune response to the antigen, stabilizers increase the storage life, and preservatives allow the use of multidose vials. [102] [103] Combination vaccines are harder to develop and produce, because of potential incompatibilities and interactions among the antigens and other ingredients involved. [104]

The final stage in vaccine manufacture before distribution is fill and finish, which is the process of filling vials with vaccines and packaging them for distribution. Although this is a conceptually simple part of the vaccine manufacture process, it is often a bottleneck in the process of distributing and administering vaccines. [105] [106] [107]

Vaccine production techniques are evolving. Cultured mammalian cells are expected to become increasingly important, compared to conventional options such as chicken eggs, due to greater productivity and low incidence of problems with contamination. Recombination technology that produces genetically detoxified vaccines is expected to grow in popularity for the production of bacterial vaccines that use toxoids. Combination vaccines are expected to reduce the quantities of antigens they contain, and thereby decrease undesirable interactions, by using pathogen-associated molecular patterns. [104]

Vaccine manufacturers

In 2012 the increasing role of Indian and Chinese vaccine manufacturers in meeting the global demand for vaccine doses was noted. [108] The Serum Institute of India was at that point the world's largest manufacturer of vaccines against measles and rubella, as well as combination DTP vaccines. The Serum Institute of India made a name for itself as a developer of vaccines when it brought into production its measles vaccine using a MRC-5 cell culture instead of chicken eggs, allowing for a productivity increase at 10% to 20% compared to Merck Group and GlaxoSmithKline. In 2012 it was estimated that two out of three vaccinated children globally had been immunized using a vaccine manufactured by the Serum Institute of India. In 2012 China ranked as the largest vaccine manufacturing country in the world, with 46 registered vaccine manufacturers focusing on meeting China's domestic need for vaccine doses. 90% of doses for the Chinese National Immunization Program were supplied by the state-owned China National Pharmaceutical Group. [109]

One of the most common methods of delivering vaccines into the human body is injection.

The development of new delivery systems raises the hope of vaccines that are safer and more efficient to deliver and administer. Lines of research include liposomes and ISCOM (immune stimulating complex). [110]

Notable developments in vaccine delivery technologies have included oral vaccines. Early attempts to apply oral vaccines showed varying degrees of promise, beginning early in the 20th century, at a time when the very possibility of an effective oral antibacterial vaccine was controversial. [111] By the 1930s there was increasing interest in the prophylactic value of an oral typhoid fever vaccine for example. [112]

An oral polio vaccine turned out to be effective when vaccinations were administered by volunteer staff without formal training the results also demonstrated increased ease and efficiency of administering the vaccines. Effective oral vaccines have many advantages for example, there is no risk of blood contamination. Vaccines intended for oral administration need not be liquid, and as solids, they commonly are more stable and less prone to damage or spoilage by freezing in transport and storage. [113] Such stability reduces the need for a "cold chain": the resources required to keep vaccines within a restricted temperature range from the manufacturing stage to the point of administration, which, in turn, may decrease costs of vaccines.

A microneedle approach, which is still in stages of development, uses "pointed projections fabricated into arrays that can create vaccine delivery pathways through the skin". [114]

An experimental needle-free [115] vaccine delivery system is undergoing animal testing. [116] [117] A stamp-size patch similar to an adhesive bandage contains about 20,000 microscopic projections per square cm. [118] This dermal administration potentially increases the effectiveness of vaccination, while requiring less vaccine than injection. [119]

Vaccinations of animals are used both to prevent their contracting diseases and to prevent transmission of disease to humans. [120] Both animals kept as pets and animals raised as livestock are routinely vaccinated. In some instances, wild populations may be vaccinated. This is sometimes accomplished with vaccine-laced food spread in a disease-prone area and has been used to attempt to control rabies in raccoons.

Where rabies occurs, rabies vaccination of dogs may be required by law. Other canine vaccines include canine distemper, canine parvovirus, infectious canine hepatitis, adenovirus-2, leptospirosis, Bordetella, canine parainfluenza virus, and Lyme disease, among others.

Cases of veterinary vaccines used in humans have been documented, whether intentional or accidental, with some cases of resultant illness, most notably with brucellosis. [121] However, the reporting of such cases is rare and very little has been studied about the safety and results of such practices. With the advent of aerosol vaccination in veterinary clinics, human exposure to pathogens not naturally carried in humans, such as Bordetella bronchiseptica, has likely increased in recent years. [121] In some cases, most notably rabies, the parallel veterinary vaccine against a pathogen may be as much as orders of magnitude more economical than the human one.

DIVA vaccines

DIVA (Differentiation of Infected from Vaccinated Animals), also known as SIVA (Segregation of Infected from Vaccinated Animals) vaccines, make it possible to differentiate between infected and vaccinated animals. DIVA vaccines carry at least one epitope less than the equivalent wild microorganism. An accompanying diagnostic test that detects the antibody against that epitope assists in identifying whether the animal has been vaccinated or not. [ citation needed ]

The first DIVA vaccines (formerly termed marker vaccines and since 1999 coined as DIVA vaccines) and companion diagnostic tests were developed by J. T. van Oirschot and colleagues at the Central Veterinary Institute in Lelystad, The Netherlands. [122] [123] They found that some existing vaccines against pseudorabies (also termed Aujeszky's disease) had deletions in their viral genome (among which was the gE gene). Monoclonal antibodies were produced against that deletion and selected to develop an ELISA that demonstrated antibodies against gE. In addition, novel genetically engineered gE-negative vaccines were constructed. [124] Along the same lines, DIVA vaccines and companion diagnostic tests against bovine herpesvirus 1 infections have been developed. [123] [125]

The DIVA strategy has been applied in various countries to successfully eradicate pseudorabies virus from those countries. Swine populations were intensively vaccinated and monitored by the companion diagnostic test and, subsequently, the infected pigs were removed from the population. Bovine herpesvirus 1 DIVA vaccines are also widely used in practice. [ citation needed ] Considerable efforts are ongoing to apply the DIVA principle to a wide range of infectious diseases, such as classical swine fever, [126] avian influenza, [127] Actinobacillus pleuropneumonia [128] and Salmonella infections in pigs. [129]

Prior to the introduction of vaccination with material from cases of cowpox (heterotypic immunisation), smallpox could be prevented by deliberate variolation with smallpox virus. The earliest hints of the practice of variolation for smallpox in China come during the 10th century. [130] The Chinese also practiced the oldest documented use of variolation, dating back to the fifteenth century. They implemented a method of "nasal insufflation" administered by blowing powdered smallpox material, usually scabs, up the nostrils. Various insufflation techniques have been recorded throughout the sixteenth and seventeenth centuries within China. [131] : 60 Two reports on the Chinese practice of inoculation were received by the Royal Society in London in 1700 one by Martin Lister who received a report by an employee of the East India Company stationed in China and another by Clopton Havers. [132]

Mary Wortley Montagu, who had witnessed variolation in Turkey, had her four-year-old daughter variolated in the presence of physicians of the Royal Court in 1721 upon her return to England. [131] Later on that year Charles Maitland conducted an experimental variolation of six prisoners in Newgate Prison in London. [133] The experiment was a success, and soon variolation was drawing attention from the royal family, who helped promote the procedure. However, several days after Prince Octavius of Great Britain was inoculated he died in 1783. [134] In 1796 the physician Edward Jenner took pus from the hand of a milkmaid with cowpox, scratched it into the arm of an 8-year-old boy, James Phipps, and six weeks later variolated the boy with smallpox, afterwards observing that he did not catch smallpox. [135] [136] Jenner extended his studies and in 1798 reported that his vaccine was safe in children and adults and could be transferred from arm-to-arm reducing reliance on uncertain supplies from infected cows. [134] Since vaccination with cowpox was much safer than smallpox inoculation, [137] the latter, though still widely practiced in England, was banned in 1840. [138]

Following on from Jenner's work, the second generation of vaccines was introduced in the 1880s by Louis Pasteur who developed vaccines for chicken cholera and anthrax, [13] and from the late nineteenth century vaccines were considered a matter of national prestige. National vaccination policies were adopted and compulsory vaccination laws were passed. [135] In 1931 Alice Miles Woodruff and Ernest Goodpasture documented that the fowlpox virus could be grown in embryonated chicken egg. Soon scientists began cultivating other viruses in eggs. Eggs were used for virus propagation in the development of a yellow fever vaccine in 1935 and an influenza vaccine in 1945. In 1959 growth media and cell culture replaced eggs as the standard method of virus propagation for vaccines. [139]

Vaccinology flourished in the twentieth century, which saw the introduction of several successful vaccines, including those against diphtheria, measles, mumps, and rubella. Major achievements included the development of the polio vaccine in the 1950s and the eradication of smallpox during the 1960s and 1970s. Maurice Hilleman was the most prolific of the developers of the vaccines in the twentieth century. As vaccines became more common, many people began taking them for granted. However, vaccines remain elusive for many important diseases, including herpes simplex, malaria, gonorrhea, and HIV. [135] [140]

Generations of vaccines

First generation vaccines are whole-organism vaccines – either live and weakened, or killed forms. [141] Live, attenuated vaccines, such as smallpox and polio vaccines, are able to induce killer T-cell (TC or CTL) responses, helper T-cell (TH) responses and antibody immunity. However, attenuated forms of a pathogen can convert to a dangerous form and may cause disease in immunocompromised vaccine recipients (such as those with AIDS). While killed vaccines do not have this risk, they cannot generate specific killer T cell responses and may not work at all for some diseases. [141]

Second generation vaccines were developed to reduce the risks from live vaccines. These are subunit vaccines, consisting of specific protein antigens (such as tetanus or diphtheria toxoid) or recombinant protein components (such as the hepatitis B surface antigen). They can generate TH and antibody responses, but not killer T cell responses. [ citation needed ]

RNA vaccines and DNA vaccines are examples of third generation vaccines. [141] [142] [143] In 2016 a DNA vaccine for the Zika virus began testing at the National Institutes of Health. Separately, Inovio Pharmaceuticals and GeneOne Life Science began tests of a different DNA vaccine against Zika in Miami. Manufacturing the vaccines in volume was unsolved as of 2016. [144] Clinical trials for DNA vaccines to prevent HIV are underway. [145] mRNA vaccines such as BNT162b2 were developed in the year 2020 with the help of Operation Warp Speed and massively deployed to combat the COVID-19 pandemic.

Since at least 2013, scientists were trying to develop synthetic 3rd-generation vaccines by reconstructing the outside structure of a virus it was hoped that this will help prevent vaccine resistance. [146]

Principles that govern the immune response can now be used in tailor-made vaccines against many noninfectious human diseases, such as cancers and autoimmune disorders. [147] For example, the experimental vaccine CYT006-AngQb has been investigated as a possible treatment for high blood pressure. [148] Factors that affect the trends of vaccine development include progress in translatory medicine, demographics, regulatory science, political, cultural, and social responses. [149]

Plants as bioreactors for vaccine production

The idea of vaccine production via transgenic plants was identified as early as 2003. Plants such as tobacco, potato, tomato, and banana can have genes inserted that cause them to produce vaccines usable for humans. [150] In 2005, bananas were developed that produce a human vaccine against hepatitis B. [151] Another example is the expression of a fusion protein in alfalfa transgenic plants for the selective directioning to antigen presenting cells, therefore increasing vaccine potency against Bovine Viral Diarrhea Virus (BVDV). [152] [153]