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Dantas-Torres F, Figueredo LA, Sales KGDS, de Luna RLN, de Sousa-Paula LC, da Silva LG, Bonifácio LLN, Otranto D. Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial. Parasitol Res 2024; 123:94. [PMID: 38212547 DOI: 10.1007/s00436-023-08112-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2023] [Accepted: 12/29/2023] [Indexed: 01/13/2024]
Abstract
The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adult antigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.
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Affiliation(s)
- Filipe Dantas-Torres
- Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz), Recife, Brazil.
| | - Luciana Aguiar Figueredo
- Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz), Recife, Brazil
| | | | | | - Lucas Christian de Sousa-Paula
- Laboratory of Bacteriology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA
| | - Lidiane Gomes da Silva
- Laboratory of Bacteriology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA
| | | | - Domenico Otranto
- Department of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy
- Department of Veterinary Clinical Sciences, City University of Hong Kong, Hong Kong SAR, China
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Successful Removal of Angiostrongylus cantonensis Larvae from the Central Nervous System of Rats 7- and 14-Days Post-Infection Using a Product Containing Moxidectin, Sarolaner and Pyrantel Embonate (Simparica Trio™) in Experimental Infections. Pathogens 2023; 12:pathogens12020305. [PMID: 36839577 PMCID: PMC9959906 DOI: 10.3390/pathogens12020305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Revised: 02/01/2023] [Accepted: 02/09/2023] [Indexed: 02/16/2023] Open
Abstract
Angiostrongylus cantonensis is a nematode with an indirect lifecycle, using molluscs as intermediate hosts. Rats are the definitive host. By administering a suitable anthelmintic, at an appropriate interval, the risk of clinical neuroangiostrongyliasis occurring in paratenic hosts (e.g., dogs, man) can be eliminated. We wanted to determine if infective larvae (L3) of A. cantonensis can be safely killed during their migration through the central nervous system (CNS) by oral administration of an anthelmintic combination containing moxidectin (480 µg/kg, Simparica Trio™; M-S-P), thereby preventing patent infections in rats. Eighteen rats were used: ten received oral M-S-P every four weeks; eight rats were used as controls. Rats were initially given M-S-P as a chew to eat, but an acquired food aversion meant that subsequent doses were given by orogastric lavage. All 18 rats were challenged once or twice with approximately 30 L3 A. cantonensis larvae via orogastric lavage. Infection status was determined by faecal analysis using the Baermann technique and necropsy examination of the heart, pulmonary arteries and lungs. Eight out of ten rats dosed with M-S-P had zero lungworms at necropsy; a single female worm was detected in each of the remaining two rats. No treated rats had L1 larvae in faeces. In contrast, all eight controls were infected with patent infections, with a median of 14.5 worms per rat detected at necropsy. The difference in infection rates was significant (two tailed Fishers Exact; p = 0.0011). Moxidectin given orally once every month killed migrating larvae before they reached the pulmonary arteries in 80% of treated rats, while in 20%, only a single female worm was present. Considering the short half-life of moxidectin in the rat, it is likely that the effectiveness of moxidectin is due to larvicidal action on migrating L3, L4 and L5 larvae in the brain parenchyma or subarachnoid space, either 7 days (L3/L4 in cerebrum and spinal cord) or 14 days (L4/L5 in cerebrum and subarachnoid space) after inoculation. This study is a prelude for future research to determine if monthly moxidectin administration orally as M-S-P could prevent symptomatic neuroangiostrongyliasis in dogs.
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Power RI, Šlapeta J. Exploration of the sensitivity to macrocyclic lactones in the canine heartworm (Dirofilaria immitis) in Australia using phenotypic and genotypic approaches. Int J Parasitol Drugs Drug Resist 2022; 20:145-158. [PMID: 36417831 PMCID: PMC9772245 DOI: 10.1016/j.ijpddr.2022.11.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2022] [Revised: 11/08/2022] [Accepted: 11/08/2022] [Indexed: 11/18/2022]
Abstract
Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic approaches to examine the sensitivity to ML drugs in D. immitis in Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knott's test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotype observed by utilising sequential quantification of microfilariae for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving 'rigorous' heartworm prevention for 12 months prior to the study, according to the owners' assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knott's test can be substituted by qPCR targeting D. immitis or associated Wolbachia endosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed.
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Affiliation(s)
- Rosemonde Isabella Power
- Sydney School of Veterinary Science, Faculty of Science, University of Sydney, New South Wales, 2006, Australia
| | - Jan Šlapeta
- Sydney School of Veterinary Science, Faculty of Science, University of Sydney, New South Wales, 2006, Australia,The University of Sydney Institute for Infectious Diseases, New South Wales, 2006, Australia,Corresponding author. Sydney School of Veterinary Science, Faculty of Science, University of Sydney, New South Wales, 2006, Australia.
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Savadelis MD, McTier TL, Kryda K, Maeder SJ, Woods DJ. Moxidectin: heartworm disease prevention in dogs in the face of emerging macrocyclic lactone resistance. Parasit Vectors 2022; 15:82. [PMID: 35277180 PMCID: PMC8915515 DOI: 10.1186/s13071-021-05104-7] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Accepted: 11/18/2021] [Indexed: 12/04/2022] Open
Abstract
Heartworm (Dirofilaria immitis) disease continues to increase and spread, remaining one of the most important and pathogenic parasitic diseases of dogs, despite the regular use of macrocyclic lactones (MLs) in preventive products. Dogs harboring strains of D. immitis resistant to MLs, the only drug class available for heartworm prevention in the United States, have been documented and proven. As no new products are available utilizing a novel drug class for the prevention of this disease, the only options for combating ML resistance include increasing the dose and/or changing the dosage regime of current MLs, or by optimizing the formulations of MLs currently available. Moxidectin provides a unique opportunity for optimization of the dose and formulation, which may provide improved efficacy against ML-resistant strains. Currently there are oral, topical, and injectable moxidectin products approved for heartworm prevention in the USA. Two new products (ProHeart® 12 and Simparica Trio®), available in many countries around the world including the USA, take advantage of the unique attributes of moxidectin for providing robust heartworm prevention against the strains of heartworm to which most dogs in the USA will likely be exposed. Both products have demonstrated 100% preventive efficacy in laboratory studies against recently collected field strains of heartworm, and also in large field studies, where the majority of dogs were living in the southern USA in areas where ML resistance has been confirmed to occur, therefore under elevated heartworm challenge. Based on the data summarized here, these products offer important advances in heartworm prevention and provide additional options for veterinarians and pet owners to protect their dogs from developing heartworm disease.
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Macrocyclic lactone resistance in Dirofilaria immitis: risks for prevention of heartworm disease. Int J Parasitol 2021; 51:1121-1132. [PMID: 34717929 DOI: 10.1016/j.ijpara.2021.08.006] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2021] [Revised: 08/29/2021] [Accepted: 08/31/2021] [Indexed: 11/21/2022]
Abstract
Heartworm disease, caused by Dirofilaria immitis, can be lethal in dogs and cats. It is transmitted by mosquitoes, and occurs in many parts of the world. Prevention relies on macrocyclic lactones. Macrocyclic lactones used are ivermectin, selamectin, abamectin, eprinomectin, milbemycin oxime and moxidectin, administered at 30-day intervals during the transmission season. Some moxidectin formulations are long-acting injectables. In the USA, preventives are recommended throughout the year. Loss of efficacy of macrocyclic lactone preventives was reported in 2005 and proof of resistance in the USA was published a decade later. Understanding factors which promote resistance is important to maintain control. Factors important for resistance development are discussed. Better, inexpensive tests to confirm resistance are needed. Infection in animals under chemoprophylaxis per se does not imply resistance because lack of compliance in preventive use could be the reason. In vivo confirmation of resistance is expensive, slow and ethically questionable. A microfilariae suppression test can be a surrogate test, but requires a high dose of a macrocyclic lactone and repeated blood microfilaria counts 2-4 weeks later. DNA single nucleotide polymorphism markers have been successfully used. However, the specific genetic changes which cause resistance are unknown. Surveys to map and follow the extent of resistance are needed. Long acting mosquito repellants and insecticides can play a useful role. High dose rate formulations of moxidectin, coupled with mosquito biting mitigation may reduce transmission of resistant genotypes. Doxycycline, daily for 28 days, as anti-Wolbachia treatment, can reduce transmission and remove adult parasites. However, new classes of heartworm preventives are needed. While any preventive strategy must be highly effective, registration requirements for 100% efficacy may hinder development of useful new classes of preventives. Continued reliance on macrocyclic lactone preventives, when they do not work against resistant genotypes, will spread resistance, and allow for more disease.
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Concern for Dirofilaria immitis and Macrocyclic Lactone Loss of Efficacy: Current Situation in the USA and Europe, and Future Scenarios. Pathogens 2021; 10:pathogens10101323. [PMID: 34684273 PMCID: PMC8541013 DOI: 10.3390/pathogens10101323] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 10/11/2021] [Accepted: 10/13/2021] [Indexed: 12/02/2022] Open
Abstract
Dirofilaria immitis infection is one of the most severe parasitic diseases in dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of the ineffectiveness of MLs, reported as “loss of efficacy” (LOE), were generally attributed to owners’ non-compliance, or other reasons associated with inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of (i) the great extent of refugia, (ii) the complexity of resistance development to MLs, and (iii) the possible large number of genes involved in resistance selection. Nevertheless, today, it is unequivocally proven that ML-resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a “decision tree” (algorithm), including compliance and preventatives’ purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for the validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive ML treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiological characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons, and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for the timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.
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Jacobson LS, DiGangi BA. An Accessible Alternative to Melarsomine: "Moxi-Doxy" for Treatment of Adult Heartworm Infection in Dogs. Front Vet Sci 2021; 8:702018. [PMID: 34386540 PMCID: PMC8353148 DOI: 10.3389/fvets.2021.702018] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Accepted: 06/30/2021] [Indexed: 11/29/2022] Open
Abstract
Canine heartworm infection, caused by the filarial parasite Dirofilaria immitis, represents a serious and expanding animal welfare concern that is expected to increase due to the effects of climate change and the COVID-19 pandemic. A body of evidence has emerged to support the use of a non-arsenical adulticide treatment protocol, using moxidectin and doxycycline to kill adult heartworms over a prolonged period. While a three-dose protocol using the arsenical drug melarsomine is currently the safest and most effective treatment for heartworm infection, this drug is not available in some countries and is inaccessible for many owners and animal shelters. Moxidectin-doxycycline (moxi-doxy) provides a viable alternative to no treatment at all, in cases where arsenical treatment is not possible. Based on current evidence, the most effective non-arsenical treatment regimen is doxycycline 10 mg/kg PO q 12 or 24 h for 28 days, combined with topical moxidectin at label dose. Moxidectin is repeated monthly until no antigen detected (NAD) status is confirmed. Sustained release injectable moxidectin, in combination with doxycycline, may provide an alternative in remote regions or in settings where significant compliance or accessibility concerns exist, but more studies are needed. In moxi-doxy protocols, doxycycline should be repeated annually until NAD. This review summarizes the safety and efficacy of moxi-doxy, addresses controversies surrounding this treatment approach, and provides detailed recommendations for treatment regimens and post-treatment testing.
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Affiliation(s)
- Linda S. Jacobson
- Shelter Medicine Advancement, Toronto Humane Society, Toronto, ON, Canada
| | - Brian A. DiGangi
- Shelter and Veterinary Services, American Society for the Prevention of Cruelty to Animals, New York, NY, United States
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Schraven AL, Stannard HJ, Old JM. A systematic review of moxidectin as a treatment for parasitic infections in mammalian species. Parasitol Res 2021; 120:1167-1181. [PMID: 33615411 DOI: 10.1007/s00436-021-07092-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2020] [Accepted: 02/16/2021] [Indexed: 11/30/2022]
Abstract
Moxidectin (MOX) is a macrocyclic lactone approved worldwide for the treatment of both endo- and ecto-parasites in many mammalian species. The aim of this study was to assess the efficacy of MOX as a treatment against parasites in a range of mammalian species. An electronic literature search was performed for publications to the 1st September 2020. A total of 205 papers were retrieved and screened against all required criteria; hence, 35 were papers were reviewed in this study. The level of evidence and methodological quality was analysed, where a total of 13 publications were categorised as a 'randomised control trial', seven were categorised as a 'non-randomised control trial' and 15 as an 'experimental control trial'. The overall methodological quality of the publications was considered low, low to moderate, moderate, moderate to high and high in ten, four, twelve, five and a further four, respectively. We assessed the treatment and possible toxicity of MOX in 13 mammalian species, six investigations reported adverse effects to MOX in a small percentage of individuals. The authors reported observed reactions that were typically mild symptoms that did not require additional therapies, and/or resolved themselves. Further studies are needed to assess the efficacy of MOX treatment in a larger number of species, particularly in wildlife.
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Affiliation(s)
- Andrea L Schraven
- School of Science, Western Sydney University, Hawkesbury, NSW, Australia
| | - Hayley J Stannard
- School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
| | - Julie M Old
- School of Science, Western Sydney University, Hawkesbury, NSW, Australia.
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Paterson T, Fernandez C, Burnett PJ, Lessey L, Hockley T, Hagen R, Coomansingh C, Sharma B, Chandrashekar R, Schaper R. Heartworm control in Grenada, West Indies: Results of a field study using imidacloprid 10% + moxidectin 2.5% and doxycycline for naturally-acquired Dirofilaria immitis infections. Vet Parasitol 2020; 284:109194. [PMID: 32866837 DOI: 10.1016/j.vetpar.2020.109194] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 07/21/2020] [Accepted: 07/21/2020] [Indexed: 11/27/2022]
Abstract
Canine heartworm disease (CHD) results from infection with Dirofilaria immitis and while it is of global concern, it is most prevalent in tropical climates where conditions support the parasite and vector life cycles. Melarsomine dihydrochloride is the sole treatment for CHD recommended by the American Heartworm Society. However, in cases where cost or access to melarsomine precludes treatment of an infected dog, therapeutic alternatives are warranted. This randomized, controlled field study evaluated the adulticidal efficacy of a combination therapeutic protocol using 10 % imidacloprid + 2.5 % moxidectin spot-on and a single 28-day course of doxycycline and compared with that of a 2-dose melarsomine dihydrochloride protocol. Of 37 naturally-infected domestic dogs with class 1, 2 or early class 3 CHD enrolled in the study, 30 were evaluated for a minimum of 12 months. Seven dogs were withdrawn due to canine ehrlichiosis, non-compliance, or wrongful inclusion. Dogs were randomly assigned to a control (CP, n = 15) or investigational (IVP, n = 15) treatment group. CP dogs received two injections of melarsomine dihydrochloride (2.5 mg/kg) 24 -hs apart and maintained on monthly ivermectin/pyrantel. IVP dogs were treated with oral doxycycline (10 mg/kg twice daily for 28 days) and topical 10 % imidacloprid + 2.5 % moxidectin once monthly for 9 months. Dogs were evaluated up to 18 months - monthly for the first 9 months, then every 3 months. Parasiticidal efficacy was based on antigen status using the IDEXX PetChek® 34 Heartworm-PF Antigen test. By month 18, antigen was not detected in any study dog except one from the IVP group. One other IVP dog was persistently antigenemic and treated with melarsomine at month 12 according to the initial study protocol. Mean antigen concentration (based on optical density) decreased more rapidly in the CP group and by month 15 was 0.11 for the IVP and 0.07 for CP groups, with equivalent median concentrations (0.04) in both groups. Conversion following heat-treatment of antigen-negative samples occurred frequently and at similar rates in both treatment groups. Based on the bias of diagnostic tests towards detection of female worms, we conclude that monthly application of 10 % imidacloprid + 2.5 % moxidectin for 9 months combined with a course of doxycycline twice daily for 28 days resulted in effective therapy against female adults in CHD. This therapeutic option may be particularly useful in cases where financial constraint or access to melarsomine precludes treatment of an infected individual. This study was supported by Bayer Animal Health.
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Affiliation(s)
- T Paterson
- Small Animal Clinic, School of Veterinary Medicine, St. George's University, PO Box 7, W.I., Grenada; Small Animal Medicine & Surgery Department, School of Veterinary Medicine, St. George's University, PO Box 7, Grenada, W.I., Grenada.
| | - C Fernandez
- Small Animal Clinic, School of Veterinary Medicine, St. George's University, PO Box 7, W.I., Grenada; 1305 Newfound Harbor Drive Merritt Island, FL 32952, USA
| | - P J Burnett
- Small Animal Clinic, School of Veterinary Medicine, St. George's University, PO Box 7, W.I., Grenada; Island Animal Hospital, 105 Mcleod St, Merritt Island, FL 32953, USA
| | - L Lessey
- Small Animal Clinic, School of Veterinary Medicine, St. George's University, PO Box 7, W.I., Grenada; McMaster University, Life Sciences Building, 1280 Main St. W., Hamilton, Ontario L8S 4K1, Canada
| | - T Hockley
- SVM Student, School of Veterinary Medicine, St. George's University, PO Box 7, Grenada, W.I., Grenada
| | - R Hagen
- Small Animal Medicine & Surgery Department, School of Veterinary Medicine, St. George's University, PO Box 7, Grenada, W.I., Grenada
| | - C Coomansingh
- Pathobiology Department, School of Veterinary Medicine, St. George's University, PO Box 7, Grenada, W.I., Grenada
| | - B Sharma
- Pathobiology Department, School of Veterinary Medicine, St. George's University, PO Box 7, Grenada, W.I., Grenada
| | - R Chandrashekar
- IDEXX Laboratories, One IDEXX Drive, Westbrook, ME 04092, USA
| | - R Schaper
- Bayer Animal Health GmbH, 51368 Leverkusen, Germany
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Sanchez J, Dharmarajan G, George MM, Pulaski C, Wolstenholme AJ, Gilleard JS, Kaplan RM. Using population genetics to examine relationships of Dirofilaria immitis based on both macrocyclic lactone-resistance status and geography. Vet Parasitol 2020; 283:109125. [PMID: 32535487 DOI: 10.1016/j.vetpar.2020.109125] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Revised: 03/24/2020] [Accepted: 04/25/2020] [Indexed: 12/18/2022]
Abstract
Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
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Affiliation(s)
- Julie Sanchez
- University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States
| | - Guha Dharmarajan
- Savannah River Ecology Laboratory, University of Georgia, Aiken, SC, United States
| | - Melissa M George
- University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States
| | - Cassan Pulaski
- University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States
| | - Adrian J Wolstenholme
- University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States
| | - John S Gilleard
- Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada
| | - Ray M Kaplan
- University of Georgia College of Veterinary Medicine, Department of Infectious Diseases, Athens, GA, United States.
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Prichard RK, Geary TG. Perspectives on the utility of moxidectin for the control of parasitic nematodes in the face of developing anthelmintic resistance. INTERNATIONAL JOURNAL FOR PARASITOLOGY-DRUGS AND DRUG RESISTANCE 2019; 10:69-83. [PMID: 31229910 PMCID: PMC6593148 DOI: 10.1016/j.ijpddr.2019.06.002] [Citation(s) in RCA: 82] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 06/12/2019] [Accepted: 06/12/2019] [Indexed: 12/22/2022]
Abstract
Macrocyclic lactone (ML) anthelmintics are the most important class of anthelmintics because of our high dependence on them for the control of nematode parasites and some ectoparasites in livestock, companion animals and in humans. However, resistance to MLs is of increasing concern. Resistance is commonplace throughout the world in nematode parasites of small ruminants and is of increasing concern in horses, cattle, dogs and other animals. It is suspected in Onchocerca volvulus in humans. In most animals, resistance first arose to the avermectins, such as ivermectin (IVM), and subsequently to moxidectin (MOX). Usually when parasite populations are ML-resistant, MOX is more effective than avermectins. MOX may have higher intrinsic potency against some parasites, especially filarial nematodes, than the avermectins. However, it clearly has a significantly different pharmacokinetic profile. It is highly distributed to lipid tissues, less likely to be removed by ABC efflux transporters, is poorly metabolized and has a long half-life. This results in effective concentrations persisting for longer in target hosts. It also has a high safety index. Limited data suggest that anthelmintic resistance may be overcome, at least temporarily, if a high concentration can be maintained at the site of the parasites for a prolonged period of time. Because of the properties of MOX, there are reasonable prospects that strains of parasites that are resistant to avermectins at currently recommended doses will be controlled by MOX if it can be administered at sufficiently high doses and in formulations that enhance its persistence in the host. This review examines the properties of MOX that support this contention and compares them with the properties of other MLs. The case for using MOX to better control ML-resistant parasites is summarised and some outstanding research questions are presented.
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Affiliation(s)
- Roger K Prichard
- Institute of Parasitology, McGill University, Sainte Anne-de-Bellevue, Quebec, Canada, H9X3V9.
| | - Timothy G Geary
- Institute of Parasitology, McGill University, Sainte Anne-de-Bellevue, Quebec, Canada, H9X3V9.
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Ballesteros C, Pulaski CN, Bourguinat C, Keller K, Prichard RK, Geary TG. Clinical validation of molecular markers of macrocyclic lactone resistance in Dirofilaria immitis. Int J Parasitol Drugs Drug Resist 2018; 8:596-606. [PMID: 30031685 PMCID: PMC6288007 DOI: 10.1016/j.ijpddr.2018.06.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 06/18/2018] [Accepted: 06/22/2018] [Indexed: 12/20/2022]
Abstract
Prophylaxis with macrocyclic lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2-4 weeks post-treatment. Each sample was processed by performing a modified Knott's Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts.
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Affiliation(s)
- Cristina Ballesteros
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne de Bellevue, H9X 3V9, QC, Canada.
| | - Cassan N Pulaski
- School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA, 70803, USA
| | - Catherine Bourguinat
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne de Bellevue, H9X 3V9, QC, Canada
| | - Kathy Keller
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne de Bellevue, H9X 3V9, QC, Canada
| | - Roger K Prichard
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne de Bellevue, H9X 3V9, QC, Canada.
| | - Timothy G Geary
- Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne de Bellevue, H9X 3V9, QC, Canada.
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Grant T, Wiseman S, Snyder DE. Effects of milbemycin oxime, combined with spinosad, when administered orally to microfilaremic dogs infected with adult heartworms (Dirofilaria immitis). J Am Vet Med Assoc 2018; 252:1084-1089. [PMID: 29641338 DOI: 10.2460/javma.252.9.1084] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
OBJECTIVE To evaluate the safety of PO administration of a milbemycin oxime (MBO) and spinosad product to heartworm (Dirofilaria immitis)-positive microfilaremic dogs. DESIGN Randomized, blinded, complete block trial. ANIMALS 32 purebred Beagles with a patent heartworm infection. PROCEDURES Dogs ranked by sex and microfilaria counts (range, 398 to 1,980 microfilaria/mL) were assigned to 4 groups of 8 to receive 3 treatments PO at 28-day intervals beginning on day 0: placebo (control group) or spinosad-MBO tablets containing MBO at the upper end of the label dose range (0.75 to 1 mg/kg [0.34 to 0.45 mg/lb]; 1× group) or 3 (3× group) or 5 (5× group) times that dose. Blood samples were collected at various points for adult heartworm antigen and Knott tests. Necropsies were performed on day 65, and recovered adult heartworms were counted. RESULTS 1 control dog died from heartworm-associated complications. Other adverse events included mild, transient emesis (1 dog in each of the 1× and 5× groups and 3 dogs in the 3× group). Similar adult heartworm counts (range, 13 to 41) were obtained for all 4 groups. Results of blood antigen and microfilaria tests were positive throughout the study, with 1 exception in each of the 3× and 5× groups. Mean microfilaria counts increased with time in the control group, whereas reductions from baseline in treated groups ranged from 61.5% to 96.4%. CONCLUSIONS AND CLINICAL RELEVANCE The evaluated MBO-spinosad formulation caused no severe adverse events when administered PO to microfilaremic dogs. Although microfilaria counts decreased following treatment, repeated monthly MBO treatments were incompletely microfilaricidal, suggesting MBO should not be used as a microfilaricide.
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Moorhead AR, Evans CC, Kaplan RM. A diagnostic algorithm for evaluating cases of potential macrocyclic lactone-resistant heartworm. Parasit Vectors 2017; 10:479. [PMID: 29143642 PMCID: PMC5688499 DOI: 10.1186/s13071-017-2441-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND The emergence of macrocyclic lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly. METHODS With the aim of identifying likely cases of macrocyclic lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case. RESULTS This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance. CONCLUSION Heartworm isolates identified through this method can be used in research to better understand macrocyclic lactone resistance so prevention strategies can be adapted.
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Affiliation(s)
- Andrew R Moorhead
- Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 D. W. Brooks Drive, Athens, GA, 30605, USA.
| | - Christopher C Evans
- Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 D. W. Brooks Drive, Athens, GA, 30605, USA
| | - Ray M Kaplan
- Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 D. W. Brooks Drive, Athens, GA, 30605, USA
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Frangipane di Regalbono A, Di Cesare A, Traversa D, Simonato G, Poser H, Danesi P, Furnari C, Russi I, Raele DA, Crisi P, Pampurini F, Pietrobelli M. Microfilaricidal efficacy of a single administration of Advocate(®) (Bayer Animal Health) in dogs naturally infected with Dirofilaria immitis or Dirofilaria repens. Vet Parasitol 2016; 226:30-4. [PMID: 27514879 DOI: 10.1016/j.vetpar.2016.06.024] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2016] [Revised: 06/15/2016] [Accepted: 06/16/2016] [Indexed: 11/19/2022]
Abstract
The present study evaluated the microfilaricidal efficacy of a single application of the spot-on containing imidacloprid 10%/moxidectin 2.5% (Advocate(®), Bayer Animal Health) in dogs naturally infected either by Dirofilaria immitis or Dirofilaria repens. Dogs living in north-eastern and central-southern Italy, endemic for D. immitis and D. repens respectively, were randomly screened. Sixteen animals, eight infected with D. immitis and eight with D. repens, and fulfilling inclusion criteria were enrolled. Dogs infected with D. immitis received an adulticide treatment prior to the study and Advocate(®) 3 weeks after. The animals were divided in blocks of two (1:1, T1:T2) animals each, where Day 0 (D0) had an interval of 15days to compare T2 vs. T1 dogs during the first fortnight of examination (i.e. T2 dogs acted as control animals at each examination). At baseline (Days -15 and 0 for T2 and T1 dogs, respectively) the animals had a range of microfilaraemia of 180-99.700mff/ml (D. immitis) and 60-750 mff/ml (D. repens). All animals received a topical administration of Advocate(®) at D0 and were examined for microfilariae with microscopic and molecular tests at D15, D30, D60 and D90. All animals scored negative for mff at the first control post-treatment and throughout the study, with the exception of two D. immitis- infected animals that had a 2 mff/ml count at D15, and then become negative from Day 30 onwards. No adverse events were observed. The present study demonstrates the safety and the high microfilaricidal efficacy (99.97% and 100% for D. immitis and D. repens, respectively) of a single dose of moxidectin contained in Advocate(®) in naturally infected dogs.
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Affiliation(s)
| | - Angela Di Cesare
- Teaching Veterinary Hospital, Faculty of Veterinary Medicine, University of Teramo, Italy.
| | - Donato Traversa
- Teaching Veterinary Hospital, Faculty of Veterinary Medicine, University of Teramo, Italy
| | - Giulia Simonato
- Department of Animal Medicine, Production and Health, University of Padua, Italy
| | - Helen Poser
- Department of Animal Medicine, Production and Health, University of Padua, Italy
| | - Patrizia Danesi
- Parasitology-Mycology Laboratory, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy
| | | | - Ilaria Russi
- Teaching Veterinary Hospital, Faculty of Veterinary Medicine, University of Teramo, Italy
| | - Donato Antonio Raele
- Canile-Rifugio Ente Nazionale Protezione Animali (ENPA) Località Posta del Fosso, Manfredonia, Italy
| | - Paolo Crisi
- Teaching Veterinary Hospital, Faculty of Veterinary Medicine, University of Teramo, Italy
| | | | - Mario Pietrobelli
- Department of Animal Medicine, Production and Health, University of Padua, Italy
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