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Defining in vitro parasiticide screening and test methods
Chapter 1
Defining in vitro parasiticide screening and test methods Alan A. Marchiondo, MS, PhD1, Larry R. Cruthers, MS, PhD2 and Josephus J. Fourie, PhD3 1
Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States, 2LCruthers Consulting, Chesapeake, VA, United States, 3Clinvet International, Bloemfontein, South Africa
In vitro comes from the Latin root “vitreus” or “vitrum” which means “glassy, glass, transparent” (origin 1890 95). A term which is cognate to this root is “in vitro” which means “in glass.”1 In vitro, often not italicized in English because it has become part of the standard English usage, refers to techniques, experiments, or studies conducted by a given procedure in a controlled environment outside of a living organism or outside their normal biological context.2,3 Colloquially called “test tube experiments,” these studies in biology, medicine, and its subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc. Rearing a parasite adult or immature stage(s) in the laboratory without or on/in a mammalian host to yield large numbers of organisms for testing purposes, including in vivo tests, and to cultivate (maintain/propagate) the parasite in the laboratory will be considered an in vitro test for the purpose of this text. On animal bioassays, where the animal is treated PO or parenterally and the target organism/stage is allowed to imbibe/feed on blood, flesh, or host secretions or infest/infect the treated host in order to determine the efficacy of the parasiticide will be considered an in vivo test. In vitro studies are conducted with whole living organisms, while ex vivo (Latin 5 “out of the living, outside the normal living organism”) uses components of an organism that have been isolated from their usual biological surroundings. In science, ex vivo means that something is experimented or measured in or on cells/tissues from an organism in an external environment with minimal alteration of its natural in vivo conditions. Electrophysiology experiments on Ascaris suum muscle strips are an example. Ex vivo conditions allow experimentation on an organism’s cells or tissue under more controlled conditions than is possible in in vivo experiments (in the intact organism), at
Parasiticide Screening, Vol 1. DOI: https://doi.org/10.1016/B978-0-12-813890-8.00001-8 © 2019 Elsevier Inc. All rights reserved.
1
2
Parasiticide Screening, Vol 1
the expense of altering the “natural” environment. The term ex vivo means that the samples to be tested have been extracted from the organism. The term in vitro (within or in glass) means the parasites to be tested are obtained from a repository, culture, or colony, such as nematodes from the gastrointestinal tract of sheep and cattle or fleas from natural or artificial rearing. These two terms are not synonymous even though the testing in both cases is “within the glass.” One factor during in vitro testing, often overlooked but emphasized by Redi in 1684,4 is that a parasite removed from its host is being tested under abnormal conditions and is actually a dying organism. In vitro results should be interpreted carefully and cautiously as changes resulting from molecular, cellular, physiological, biochemical, and environmental conditions are always open to discussion. An excellent example of an in vitro false lead is the activity of triclabendazole against Hymenolepis in vitro, but later found to be inactive in vivo using a rodent model.5 Advantages of in vitro tests include (1) species-specific results (using the target parasite and stage without the influences of the hosts humeral and cellular responses), (2) enormous level of simplification of the system, (3) simpler and more convenient procedural methods, (4) faster screening and more rapid result often predictive of the likelihood of advancing to in vivo testing, (5) reduced use of test animals, (6) relatively inexpensive, (7) reduced use of the amount of test compound, (8) adaptable to allowing for miniaturization and automation yielding high-throughput screening methods to cope with increasing demands for new parasiticides, and (9) more detailed physiological, biochemical, and morphological analysis than can be done with the whole organism. For confirmation of mortality/lethality in the target parasite species, however, there is no substitute for in vivo parasite screens. Disadvantages of in vitro tests are that they fail to replicate the precise microphysiological and microenvironmental conditions of a parasite in/on a host which makes results very challenging to extrapolate to the target host. Because of this, in vitro studies are complex methods with difficult quality control and may lead to results that do not correspond to the circumstances occurring around a living parasite within the definitive host. The results must never be overinterpreted leading to erroneous conclusions about the parasite and its biological systems. Typically, many candidate drugs that are effective in vitro prove to be ineffective in vivo. This can be due to (1) issues with drug delivery and uptake to the parasite and its microenvironment; (2) absorption, distribution, metabolism, and excretion of the drug and its metabolites in the target host as well as the parasite; (3) parasite species and surrogate differences not applicable to the target host; (4) false positives and reproducibility of the assay; (5) false negatives; (6) target host toxicity; (7) lack of biotransformation; and (8) absence of biokinetics.
Defining in vitro parasiticide screening and test methods Chapter | 1
3
In vitro laboratory studies include screening studies of the active ingredient, used to establish an innate toxic effect on the test parasite species, dose response tests, or simple laboratory studies, including surface contact tests, etc. These tests should provide an indication of the intrinsic/inherent parasiticidal activity and/or the range of concentrations (dose response curve) over which such activity would be expected. Tests are usually conducted using technical material and performed against well-characterized susceptible and/or resistant strains of laboratory-reared parasite stages under standardized and controlled testing conditions. Negative (no compound) and positive controls (reference compounds) should be included to assess validation of the assay and potential cross-resistance with commonly used parasiticides. Efficacy against a specific parasite depends on the delivery form, application/exposure method, and on the dose administered. Bioassays for in vitro testing of parasiticides against parasites in general should contain a protocol or test method that may specify but is not limited to a study accession number; title; background information, rationale, and/or justification for the study; clearly stated objective(s) with measurable variables; parasite origin, stage, sex, date isolated, and period of time in laboratory cultivation; parasite chemical susceptibility profile; test article chemical characteristics and purity; negative and/or positive controls; handling, disposal, and accountability of test articles; materials specifications and suppliers as appropriate; weight scale verification and validation; inclusion/ exclusion criteria (stage, age, sex, weight, etc., and other viability characteristics of the test parasite); parasite randomization to treatments; study design; treatment method, dose, and regimen; evaluation criteria and variable measurements/observations; human accidental exposure; standard operating procedures for the facility and laboratory; biometrics; and data capture forms as appropriate. Good laboratory practice guidelines should be followed to promote the quality, validity, and reliability of nonclinical test data, common understanding of, and harmonization approach. Internationally accepted in vitro standard methods for toxicological evaluations by the OECD (http://www.oecd.org/) have been developed and the ZEBET (http://www.bfr.bund.de/en/zebet_alternative_methods_to_animal_experiments-53868.html) has collected all available methods for the pharmacological and toxicological testing and assessing the potential effects of chemicals on human health and the environment. Numerous organizations, such as the FAO of the United Nations (http://www.fao.org), IRAC (http:// www.irac-online.org/), and WHOPES (http://www.who.int/whopes/en/), have developed validated in vitro screening methods for the determination of insecticide resistance. These standard screening methods have served as a basis to researchers, who have used or modified these standard methods to determine the inherent parasiticide activity of various compounds and have adapted the methods to various parasitic organisms. This is exemplified by the numerous methods for the determination of the lethal concentration of
4
Parasiticide Screening, Vol 1
parasiticides provided in Chapters 3 5 in Volume 1 and Chapters 1 3 in Volume 2. However, neither standardized nor regulated in vitro guidelines exist for parasiticides, as opposed to the in vivo guidelines by the WAAVP. Typical end points of in vitro tests include morbidity, mortality, and repellency. Morbidity is the quality or state or rate of being morbid, which is defined as a state or symptom of objective aberration in function (sensation, integration, and response) and behavior. Symptoms of peculiar behavior are lack of feeding, inhibited development and phenotype characteristics, and unusual positioning or motor activity (motility and migration). A micromotility meter and motility index (inhibitor concentration) have been developed and adapted to high-throughput screening paradigms for parasiticide discovery and resistance.6 8 Mortality (Latin 5 mortalitas 1300 50) is defined as susceptible to death, the state, or condition of death. Neurotoxicity of parasiticides is exhibited by the two general effects of excitation and inhibition caused by interactions between the target site and parasiticide. These physiological and biochemical interactions can occur in receptor agonism or antagonism, ion channel modulation, and enzyme inhibition, all of which can result in and be expressed as mortality. The concept of selective activity of anthelmintics and other parasiticides is based on biochemical and physiological differences between the parasite and the host.9,10 Mortality as a measure of the number of dead can be expressed as a lethal dose (LD) at a specific concentration of parasiticide, for example, LD50 or LD90, wherein the amount of substance required to kill 50% or 90% of the parasites (population) under test, respectively. The use of death as a target allows for a quantal test (all or nothing) comparisons between chemicals that kill or do not kill. An effective dose is the dose that measures the reasonable expectancy of the desired or therapeutic effect. Mortality can be calculated according to the Busvine formula.11 The dose effect relationship should be calculated statistically by using an appropriate linear regression method: mo mc mcorr 5 3 100 100 mc where mcorr is the corrected mortality at each concentration tested (in percent), mo is the mean observed mortality in the treated groups (in percent), and mc is the mean observed mortality in the control groups (in percent). Mortality can also be expressed as percent efficacy using Abbott’s formula,12 wherein the percent or number of live organisms in the control group minus the percent or mean number of dead or killed organisms in the treated group equals the percent or mean number dead or killed by the treatment divided by the percent or mean number of live organisms in the control
Defining in vitro parasiticide screening and test methods Chapter | 1
5
group times 100 equals the percent controlled (efficacy), expressed by the equation: Mean number of organisms in control group Percent efficacy 5
2 mean number of organisms in treated group Mean number of organisms in control group
3 100
Mortality bioassays of parasiticides utilize contact exposure and systemic activity via feeding on treated blood or a suitable substance. Contact exposure by direct application on the parasite or immersions and exposing the parasite to a treated substrate (filter paper, glass surfaces, test tubes, Petri dish lids, vials, watch glasses, etc.) have led to unique in vitro tests specific for parasite adult species and stages. Feeding bioassays utilize artificial feeding membranes through which parasites imbibe parasiticide-treated blood or simply allowing parasite stages to feed and crawl through treated rearing medium allow for the assessment of systemic active drugs. In general, a repellent effect will cause the parasite to avoid contact with a treated substrate or animal completely and/or to leave/move away/ fall off a host soon after contact with the treated host surface. Classically, this definition has been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from the chemically treated source. This definition needs to be used with care for crawling arthropod-like fleas and ticks that require physical contact for exposure and may cause the parasite to avoid or leave the host without attaching, biting or feeding, or prevention and disruption of attachment.13,14 In vitro assays to evaluate repellency will be covered in more detail by parasite species. In vitro testing methods are of no value unless one has an ample supply of test organisms. Therefore in vitro cultivation of parasites is invaluable, providing information on the biology and development of the parasite as well as new approaches to the treatment and control of parasites. It is important in diagnosis, research, epidemiology, and teaching.15 In vitro cultivation methods include (1) xenic cultures, that is, cultures of parasites grown in association with an unknown or undefined microbial population, such as the cultivation of Entamoeba histolytica16; (2) monoxenic cultures, that is, parasites grown in association with a single known additional species, such as Acanthamoeba, cultured from a corneal biopsy with Escherichia coli17; (3) axenic (Greek a 5 free from; xenos 5 a stranger) cultures, that is, pure culture without contaminating bacterial association and without the presence of the host, Leishmania species culture18 and Brugia pahangi19 as examples. Parasitic helminths and arthropods are perhaps more challenging to cultivate than many other organisms because of their complex life cycles (free-living to parasitic stages) that require different sets of nutritional and physicochemical conditions.20 22
6
Parasiticide Screening, Vol 1
Chapters 3 5 of Volume 1 and Chapters 1 3 of Volume 2 of this book focus on in vitro tests developed for parasiticide discovery screening and their related rearing/cultivation methods, as applicable and known to the authors, some of which have led to the discovery of novel parasiticides used in veterinary medicine.
References 1. American Heritage Publishing Company. American Heritages Dictionary of the English language. 5th ed. Houghton Mifflin Harcourt Publishing Company; 2015. p. 2112. 2. Gregoline B. Non-English words, phrases, and accent marks. In: Iverson C, editor. AMA manual of style: a guideline for authors and editors. 10th ed. Oxford: Oxford University Press; 2007. 3. Coghill AM, Garson LR. ACS style guide. American Chemical Society; 2006. p. 388. 4. de Carneri I, Vita G. In: Cavier R, Hawkins F, editors. Chemotherapy of helminthiasis, vol. 1. Oxford: Pergamon Press; 1973. p. 145 213. 5. Coles GC. Anthelmintic activity of triclabendazole. J Helminthol 1986;60:210 12. 6. Folz SD, Pax RA, Thomas EM, Bennett JL, Lee BL, Conder GA. Detecting in vitro anthelmintic effects with a micromotility meter. Vet Parasitol 1987;24(2 3):241 50. 7. Smout MJ, Kotze AC, McCarthy JS, Loukas A. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility. PLoS Negl Trop Dis 2010. Available from: https://doi.org/10.1371/journal. pntd.0000885. 8. Paveley RA, Mansour NR, Hallyburton I, Bleicher LS, Benn AE, Mikic I, et al. Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases. PLoS Negl Trop Dis 2012. Available from: https://doi.org/10.1371/journal. pntd.0001762. 9. Saz HJ, Bueding E. Relationships between anthelmintic effects and biochemical and physiological mechanisms. Pharmacol Rev 1966;18(1):871 94. 10. K¨ohler P. The biochemical basis of anthelmintic action and resistance. Int J Parasitol 2001;31(4):335 45. 11. Busvine JR. Toxicological statistics. A critical review of the techniques for testing insecticides. 2nd ed. Slough, England: Commonwealth Agricultural Bureaux; 1971. p. 263 88. 12. Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol 1925;18:265 7. 13. Halos L, Banet G, Beugnet F, Bowman AS, Chomel B, Farkas R, et al. Defining the concept of “tick repellency” in veterinary medicine. Parasitology 2012;139(4):419 23. 14. Prullage JB, Hair JA, Everett WR, Yoon SS, Cramer LG, Franke S, et al. The prevention of attachment and the detachment effect of a novel combination of fipronil, amitraz and (S)-methoprene for Rhipicephalus sanguineus and Dermacentor variabilis on dogs. Vet Parasitol 2011;179(4):311 17. 15. Ahmed NH. Cultivation of parasites. Trop Parasitol 2014;4(2):80 9. 16. Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 2002;15(3):329 41. 17. Garcia LS. Diagnostic medical parasitology. 4th ed. Washington, DC: ASM Press; 2001. p. 850 72. 18. Gupta N, Goyal N, Rastogi AK. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 2001;17(3):150 3.
Defining in vitro parasiticide screening and test methods Chapter | 1
7
19. Wisnewski N, Weinstein PP. Growth and development of Brugia pahangi larvae under various in vitro conditions. J Parasitol 1993;79(3):390 8. 20. Weinstein PP, Jones MF. Development in vitro of some parasitic nematodes of vertebrates. Ann NY Acad Sci 1959;77:137 62. 21. Taylor AER, Baker JR. The cultivation of parasites in vitro. Hoboken, NJ: Blackwell Scientific; 1968. p. 377. 22. Smyth JD. In vitro cultivation of parasitic helminths. Boca Raton, FL: CRC Press; 1990. p. 144.
Defining in vitro parasiticide screening and test methods Alan A. Marchiondo, MS, PhD1, Larry R. Cruthers, MS, PhD2 and Josephus J. Fourie, PhD3 1
Adobe Veterinary Parasitology Consulting LLC, Santa Fe, NM, United States, 2LCruthers Consulting, Chesapeake, VA, United States, 3Clinvet International, Bloemfontein, South Africa
In vitro comes from the Latin root “vitreus” or “vitrum” which means “glassy, glass, transparent” (origin 1890 95). A term which is cognate to this root is “in vitro” which means “in glass.”1 In vitro, often not italicized in English because it has become part of the standard English usage, refers to techniques, experiments, or studies conducted by a given procedure in a controlled environment outside of a living organism or outside their normal biological context.2,3 Colloquially called “test tube experiments,” these studies in biology, medicine, and its subdisciplines are traditionally done in test tubes, flasks, Petri dishes, etc. Rearing a parasite adult or immature stage(s) in the laboratory without or on/in a mammalian host to yield large numbers of organisms for testing purposes, including in vivo tests, and to cultivate (maintain/propagate) the parasite in the laboratory will be considered an in vitro test for the purpose of this text. On animal bioassays, where the animal is treated PO or parenterally and the target organism/stage is allowed to imbibe/feed on blood, flesh, or host secretions or infest/infect the treated host in order to determine the efficacy of the parasiticide will be considered an in vivo test. In vitro studies are conducted with whole living organisms, while ex vivo (Latin 5 “out of the living, outside the normal living organism”) uses components of an organism that have been isolated from their usual biological surroundings. In science, ex vivo means that something is experimented or measured in or on cells/tissues from an organism in an external environment with minimal alteration of its natural in vivo conditions. Electrophysiology experiments on Ascaris suum muscle strips are an example. Ex vivo conditions allow experimentation on an organism’s cells or tissue under more controlled conditions than is possible in in vivo experiments (in the intact organism), at
Parasiticide Screening, Vol 1. DOI: https://doi.org/10.1016/B978-0-12-813890-8.00001-8 © 2019 Elsevier Inc. All rights reserved.
1
2
Parasiticide Screening, Vol 1
the expense of altering the “natural” environment. The term ex vivo means that the samples to be tested have been extracted from the organism. The term in vitro (within or in glass) means the parasites to be tested are obtained from a repository, culture, or colony, such as nematodes from the gastrointestinal tract of sheep and cattle or fleas from natural or artificial rearing. These two terms are not synonymous even though the testing in both cases is “within the glass.” One factor during in vitro testing, often overlooked but emphasized by Redi in 1684,4 is that a parasite removed from its host is being tested under abnormal conditions and is actually a dying organism. In vitro results should be interpreted carefully and cautiously as changes resulting from molecular, cellular, physiological, biochemical, and environmental conditions are always open to discussion. An excellent example of an in vitro false lead is the activity of triclabendazole against Hymenolepis in vitro, but later found to be inactive in vivo using a rodent model.5 Advantages of in vitro tests include (1) species-specific results (using the target parasite and stage without the influences of the hosts humeral and cellular responses), (2) enormous level of simplification of the system, (3) simpler and more convenient procedural methods, (4) faster screening and more rapid result often predictive of the likelihood of advancing to in vivo testing, (5) reduced use of test animals, (6) relatively inexpensive, (7) reduced use of the amount of test compound, (8) adaptable to allowing for miniaturization and automation yielding high-throughput screening methods to cope with increasing demands for new parasiticides, and (9) more detailed physiological, biochemical, and morphological analysis than can be done with the whole organism. For confirmation of mortality/lethality in the target parasite species, however, there is no substitute for in vivo parasite screens. Disadvantages of in vitro tests are that they fail to replicate the precise microphysiological and microenvironmental conditions of a parasite in/on a host which makes results very challenging to extrapolate to the target host. Because of this, in vitro studies are complex methods with difficult quality control and may lead to results that do not correspond to the circumstances occurring around a living parasite within the definitive host. The results must never be overinterpreted leading to erroneous conclusions about the parasite and its biological systems. Typically, many candidate drugs that are effective in vitro prove to be ineffective in vivo. This can be due to (1) issues with drug delivery and uptake to the parasite and its microenvironment; (2) absorption, distribution, metabolism, and excretion of the drug and its metabolites in the target host as well as the parasite; (3) parasite species and surrogate differences not applicable to the target host; (4) false positives and reproducibility of the assay; (5) false negatives; (6) target host toxicity; (7) lack of biotransformation; and (8) absence of biokinetics.
Defining in vitro parasiticide screening and test methods Chapter | 1
3
In vitro laboratory studies include screening studies of the active ingredient, used to establish an innate toxic effect on the test parasite species, dose response tests, or simple laboratory studies, including surface contact tests, etc. These tests should provide an indication of the intrinsic/inherent parasiticidal activity and/or the range of concentrations (dose response curve) over which such activity would be expected. Tests are usually conducted using technical material and performed against well-characterized susceptible and/or resistant strains of laboratory-reared parasite stages under standardized and controlled testing conditions. Negative (no compound) and positive controls (reference compounds) should be included to assess validation of the assay and potential cross-resistance with commonly used parasiticides. Efficacy against a specific parasite depends on the delivery form, application/exposure method, and on the dose administered. Bioassays for in vitro testing of parasiticides against parasites in general should contain a protocol or test method that may specify but is not limited to a study accession number; title; background information, rationale, and/or justification for the study; clearly stated objective(s) with measurable variables; parasite origin, stage, sex, date isolated, and period of time in laboratory cultivation; parasite chemical susceptibility profile; test article chemical characteristics and purity; negative and/or positive controls; handling, disposal, and accountability of test articles; materials specifications and suppliers as appropriate; weight scale verification and validation; inclusion/ exclusion criteria (stage, age, sex, weight, etc., and other viability characteristics of the test parasite); parasite randomization to treatments; study design; treatment method, dose, and regimen; evaluation criteria and variable measurements/observations; human accidental exposure; standard operating procedures for the facility and laboratory; biometrics; and data capture forms as appropriate. Good laboratory practice guidelines should be followed to promote the quality, validity, and reliability of nonclinical test data, common understanding of, and harmonization approach. Internationally accepted in vitro standard methods for toxicological evaluations by the OECD (http://www.oecd.org/) have been developed and the ZEBET (http://www.bfr.bund.de/en/zebet_alternative_methods_to_animal_experiments-53868.html) has collected all available methods for the pharmacological and toxicological testing and assessing the potential effects of chemicals on human health and the environment. Numerous organizations, such as the FAO of the United Nations (http://www.fao.org), IRAC (http:// www.irac-online.org/), and WHOPES (http://www.who.int/whopes/en/), have developed validated in vitro screening methods for the determination of insecticide resistance. These standard screening methods have served as a basis to researchers, who have used or modified these standard methods to determine the inherent parasiticide activity of various compounds and have adapted the methods to various parasitic organisms. This is exemplified by the numerous methods for the determination of the lethal concentration of
4
Parasiticide Screening, Vol 1
parasiticides provided in Chapters 3 5 in Volume 1 and Chapters 1 3 in Volume 2. However, neither standardized nor regulated in vitro guidelines exist for parasiticides, as opposed to the in vivo guidelines by the WAAVP. Typical end points of in vitro tests include morbidity, mortality, and repellency. Morbidity is the quality or state or rate of being morbid, which is defined as a state or symptom of objective aberration in function (sensation, integration, and response) and behavior. Symptoms of peculiar behavior are lack of feeding, inhibited development and phenotype characteristics, and unusual positioning or motor activity (motility and migration). A micromotility meter and motility index (inhibitor concentration) have been developed and adapted to high-throughput screening paradigms for parasiticide discovery and resistance.6 8 Mortality (Latin 5 mortalitas 1300 50) is defined as susceptible to death, the state, or condition of death. Neurotoxicity of parasiticides is exhibited by the two general effects of excitation and inhibition caused by interactions between the target site and parasiticide. These physiological and biochemical interactions can occur in receptor agonism or antagonism, ion channel modulation, and enzyme inhibition, all of which can result in and be expressed as mortality. The concept of selective activity of anthelmintics and other parasiticides is based on biochemical and physiological differences between the parasite and the host.9,10 Mortality as a measure of the number of dead can be expressed as a lethal dose (LD) at a specific concentration of parasiticide, for example, LD50 or LD90, wherein the amount of substance required to kill 50% or 90% of the parasites (population) under test, respectively. The use of death as a target allows for a quantal test (all or nothing) comparisons between chemicals that kill or do not kill. An effective dose is the dose that measures the reasonable expectancy of the desired or therapeutic effect. Mortality can be calculated according to the Busvine formula.11 The dose effect relationship should be calculated statistically by using an appropriate linear regression method: mo mc mcorr 5 3 100 100 mc where mcorr is the corrected mortality at each concentration tested (in percent), mo is the mean observed mortality in the treated groups (in percent), and mc is the mean observed mortality in the control groups (in percent). Mortality can also be expressed as percent efficacy using Abbott’s formula,12 wherein the percent or number of live organisms in the control group minus the percent or mean number of dead or killed organisms in the treated group equals the percent or mean number dead or killed by the treatment divided by the percent or mean number of live organisms in the control
Defining in vitro parasiticide screening and test methods Chapter | 1
5
group times 100 equals the percent controlled (efficacy), expressed by the equation: Mean number of organisms in control group Percent efficacy 5
2 mean number of organisms in treated group Mean number of organisms in control group
3 100
Mortality bioassays of parasiticides utilize contact exposure and systemic activity via feeding on treated blood or a suitable substance. Contact exposure by direct application on the parasite or immersions and exposing the parasite to a treated substrate (filter paper, glass surfaces, test tubes, Petri dish lids, vials, watch glasses, etc.) have led to unique in vitro tests specific for parasite adult species and stages. Feeding bioassays utilize artificial feeding membranes through which parasites imbibe parasiticide-treated blood or simply allowing parasite stages to feed and crawl through treated rearing medium allow for the assessment of systemic active drugs. In general, a repellent effect will cause the parasite to avoid contact with a treated substrate or animal completely and/or to leave/move away/ fall off a host soon after contact with the treated host surface. Classically, this definition has been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from the chemically treated source. This definition needs to be used with care for crawling arthropod-like fleas and ticks that require physical contact for exposure and may cause the parasite to avoid or leave the host without attaching, biting or feeding, or prevention and disruption of attachment.13,14 In vitro assays to evaluate repellency will be covered in more detail by parasite species. In vitro testing methods are of no value unless one has an ample supply of test organisms. Therefore in vitro cultivation of parasites is invaluable, providing information on the biology and development of the parasite as well as new approaches to the treatment and control of parasites. It is important in diagnosis, research, epidemiology, and teaching.15 In vitro cultivation methods include (1) xenic cultures, that is, cultures of parasites grown in association with an unknown or undefined microbial population, such as the cultivation of Entamoeba histolytica16; (2) monoxenic cultures, that is, parasites grown in association with a single known additional species, such as Acanthamoeba, cultured from a corneal biopsy with Escherichia coli17; (3) axenic (Greek a 5 free from; xenos 5 a stranger) cultures, that is, pure culture without contaminating bacterial association and without the presence of the host, Leishmania species culture18 and Brugia pahangi19 as examples. Parasitic helminths and arthropods are perhaps more challenging to cultivate than many other organisms because of their complex life cycles (free-living to parasitic stages) that require different sets of nutritional and physicochemical conditions.20 22
6
Parasiticide Screening, Vol 1
Chapters 3 5 of Volume 1 and Chapters 1 3 of Volume 2 of this book focus on in vitro tests developed for parasiticide discovery screening and their related rearing/cultivation methods, as applicable and known to the authors, some of which have led to the discovery of novel parasiticides used in veterinary medicine.
References 1. American Heritage Publishing Company. American Heritages Dictionary of the English language. 5th ed. Houghton Mifflin Harcourt Publishing Company; 2015. p. 2112. 2. Gregoline B. Non-English words, phrases, and accent marks. In: Iverson C, editor. AMA manual of style: a guideline for authors and editors. 10th ed. Oxford: Oxford University Press; 2007. 3. Coghill AM, Garson LR. ACS style guide. American Chemical Society; 2006. p. 388. 4. de Carneri I, Vita G. In: Cavier R, Hawkins F, editors. Chemotherapy of helminthiasis, vol. 1. Oxford: Pergamon Press; 1973. p. 145 213. 5. Coles GC. Anthelmintic activity of triclabendazole. J Helminthol 1986;60:210 12. 6. Folz SD, Pax RA, Thomas EM, Bennett JL, Lee BL, Conder GA. Detecting in vitro anthelmintic effects with a micromotility meter. Vet Parasitol 1987;24(2 3):241 50. 7. Smout MJ, Kotze AC, McCarthy JS, Loukas A. A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility. PLoS Negl Trop Dis 2010. Available from: https://doi.org/10.1371/journal. pntd.0000885. 8. Paveley RA, Mansour NR, Hallyburton I, Bleicher LS, Benn AE, Mikic I, et al. Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases. PLoS Negl Trop Dis 2012. Available from: https://doi.org/10.1371/journal. pntd.0001762. 9. Saz HJ, Bueding E. Relationships between anthelmintic effects and biochemical and physiological mechanisms. Pharmacol Rev 1966;18(1):871 94. 10. K¨ohler P. The biochemical basis of anthelmintic action and resistance. Int J Parasitol 2001;31(4):335 45. 11. Busvine JR. Toxicological statistics. A critical review of the techniques for testing insecticides. 2nd ed. Slough, England: Commonwealth Agricultural Bureaux; 1971. p. 263 88. 12. Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol 1925;18:265 7. 13. Halos L, Banet G, Beugnet F, Bowman AS, Chomel B, Farkas R, et al. Defining the concept of “tick repellency” in veterinary medicine. Parasitology 2012;139(4):419 23. 14. Prullage JB, Hair JA, Everett WR, Yoon SS, Cramer LG, Franke S, et al. The prevention of attachment and the detachment effect of a novel combination of fipronil, amitraz and (S)-methoprene for Rhipicephalus sanguineus and Dermacentor variabilis on dogs. Vet Parasitol 2011;179(4):311 17. 15. Ahmed NH. Cultivation of parasites. Trop Parasitol 2014;4(2):80 9. 16. Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 2002;15(3):329 41. 17. Garcia LS. Diagnostic medical parasitology. 4th ed. Washington, DC: ASM Press; 2001. p. 850 72. 18. Gupta N, Goyal N, Rastogi AK. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 2001;17(3):150 3.
Defining in vitro parasiticide screening and test methods Chapter | 1
7
19. Wisnewski N, Weinstein PP. Growth and development of Brugia pahangi larvae under various in vitro conditions. J Parasitol 1993;79(3):390 8. 20. Weinstein PP, Jones MF. Development in vitro of some parasitic nematodes of vertebrates. Ann NY Acad Sci 1959;77:137 62. 21. Taylor AER, Baker JR. The cultivation of parasites in vitro. Hoboken, NJ: Blackwell Scientific; 1968. p. 377. 22. Smyth JD. In vitro cultivation of parasitic helminths. Boca Raton, FL: CRC Press; 1990. p. 144.