The Bayer Experience 2011

Heartworms and resistance: Truth or fiction?

Byron L. Blagburn, MS, PhD
Distinguished University Professor
College of Veterinary Medicine
Auburn University
Auburn, Ala.

Heartworms Dirofilariasis (heartworm disease) can be a serious and potentially fatal disease of dogs. Heartworm continues to spread throughout the United States, and it is now present in each of the contiguous 48 states and Hawaii. Heartworm is most common in warmer regions, such as the southeastern United States and Hawaii. It is transmitted by many species of mosquitoes, including those in the genera Aedes, Culex, Anopheles, and Psorophora. At least 24 species are documented vectors, based on the presence of Dirofilaria immitis developmental stages in wild-caught mosquitoes.

Historically, heartworm disease was easily prevented by monthly or semi-annual use of available preventives. However, recent research indicates that certain field isolates and laboratory strains of D. immitis may not be fully susceptible to preventive medications.1,2,3 The number of heartworm positive dogs on seemingly compliant prevention has increased demonstrably in certain areas of the central United States. Other variables, such as changes in testing strategies and increases in numbers of capable mosquito vectors, have been ruled out as potential explanations. An emerging concern is that a small sub-population of heartworms in a few limited areas has developed resistance to the heartworm preventive medications. Research conducted at Auburn University's College of Veterinary Medicine and in collaboration with others suggests that both biological and genetic changes have occurred in heartworms present in at least some infected dogs. Recent research also indicates that a laboratory strain of D. immitis that was commonly used in heartworm studies also differs in its susceptibility to heartworm preventives.3

Resistance: Truth or fiction?

A microfilariae-based laboratory bioassay was developed to evaluate the susceptibility of microfilariae of heartworms from dogs from the central United States that were infected while on prevention (see Appendix 1). These same microfilariae were also subjected to genetic analysis to determine whether changes in genes known to be involved in resistance to other worm parasites could be detected. The results indicated that microfilariae of heartworm isolates from dogs in the central United States were more difficult to kill than those from a susceptible isolate.

For example, in the assay, ivermectin at a concentration that should kill approximately 95% of the microfilariae killed only 8%, 55%, and 78% of microfilariae of D. immitis isolates 1, 2, and 3, respectively. Interestingly, when the sequence changes in a gene encoding for P-glycoprotein (a membrane transport protein) was compared for our susceptible heartworm isolate and the three test isolates, there was a direct correlation between reduced susceptibility to ivermectin and the percentage of microfilariae in the population that carried the sequence change.2 These results are strongly supportive of heartworm resistance to the macrocyclic lactone (ML) preventives for the isolates that were tested.

Although most of the reports of heartworm preventive failures are restricted to the central United States, until we have examined more D. immitis samples from other geographic locations, we cannot speculate on the true prevalence and geographic distribution of these isolates.

Not only are there field isolates with reduced susceptibility to heartworm preventives, there are laboratory strains that appear to behave similarly. One such laboratory strain was identified and documented in a recently published article.3 The MP3 laboratory strain survived exposure to both ivermectin (one of 14 dogs) and milbemycin oxime (one of 14 dogs) in the published laboratory efficacy study. Similar results were obtained in an efficacy study to evaluate a new combination preventive product.4 Because of the unique pharmacologic properties of moxidectin in the Advantage Multi® for Dogs formulation, it is speculated that this formulation might be effective against the MP3 laboratory strain of D. immitis. To test this hypothesis, a laboratory study was designed and conducted to compare efficacies of four commercially available heartworm preventives against the MP3 laboratory strain (see Appendix 2). No adult heartworms were recovered from the eight dogs treated with Advantage Multi® for Dogs (100% efficacy). One or more adult heartworm were recovered from seven dogs treated with Heartgard® Plus, Interceptor® Flavor Tabs, and Revolution® (12.5% efficacy). We presume that the difference in efficacy of Advantage Multi® for Dogs and the comparators was due to the potency and extended plasma levels of moxidectin following a single administration. To what extent the data obtained in this laboratory study might be applied to field isolates of D. immitis remains to be determined.

Appendix 1. Characterization of heartworm prevention failures in the central United States1

Background

Veterinarians in the mid-south and coastal mid-south regions of the central United States have reported increased numbers of heartworm positive-dogs that could not be explained by reduced compliance. We developed a laboratory test to determine the susceptibility of microfilariae from these dogs to macrocyclic lactones (the active ingredients in available preventives).

Methods

Microfilariae from heartworm positive dogs with a history of compliant prevention were purified and placed in test wells with two concentrations of macrocyclic lactone (ML; i.e., ivermectin). The percentage of microfilariae that were killed after 24 hours was determined at a concentration that normally kills 95% (LD95) of microfilariae of our laboratory (susceptible) isolate. We also assessed efficacy at two times the concentration that kills 95% of susceptible microfilaria (2X LD95), to determine if an increase in concentration resulted in an increase in mortality.

Results and conclusions

Three heartworm isolates from the central United States that met the criteria for suspected resistance were examined using the laboratory test. Results were as follows (the ML was ivermectin):

D. immitis isolate	Percent kill at LD95	Percent kill at 2X LD95
1	8%	25.5%
2	55.4%	99.2%
3	78.6%	100%

Results indicate that microfilariae of the three heartworm isolates from the central United States were less susceptible to MLs than our laboratory isolate in the laboratory test. An increase to 2X the LD95 concentration resulted in efficacies of greater than or equal to 99% for isolates 2 and 3, but not for isolate 1. We concluded that microfilariae of selected heartworm isolates in the central United States appear less susceptible to MLs than our laboratory strain. However, these results were obtained with a concentrated ML in the laboratory. Results with marketed formulations of preventive products may differ because of differences in formulations and pharmacologic properties of the different products.

Appendix 2. Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis5

Methods

Study dogs/experimental infection: Forty Beagle dogs were assigned based on weight to five treatment groups of eight dogs each. All dogs were infected with 100 laboratory-reared infective larvae of the MP3 strain of D. immitis on study day -30. The MP3 strain of D. immitis was chosen because macrocyclic lactone preventives were not 100% effective against it in previous laboratory studies.4

Treatment

Dogs in each group were treated once as follows on study day 0: Group 1: Heartgard® Plus; Group 2: Interceptor® Flavor Tabs; Group 3: Revolution®; Group 4: Advantage Multi® for Dogs. All products were administered as directed on each product label. Group 5 dogs served as non-treated controls. All dogs were examined for adult heartworms approximately 150 days after infection.

Results and conclusions

Results

A range of 34 to 70 adult D. immitis per dog were recovered from each of the eight dogs in the non-treated control group (geometric mean [GM] = 51.6 worms per dog). No adult heartworms were recovered from the eight dogs treated with Advantage Multi® for Dogs (100% efficacy). One or more adult heartworm were recovered from seven dogs treated with Heartgard® Plus, Interceptor® Flavor Tabs, and Revolution® (12.5% efficacy); more than six heartworms were recovered from one dog in each of these three groups. The GM worm counts were 2.3, 2.4, and 2.3 worms per dog, resulting in efficacies compared to controls of 95.6%, 95.4%, and 95.5%, respectively. In this laboratory study, Advantage Multi® for Dogs was the only product tested that was 100% effective against the MP3 isolate of D. immitis.

References

1. Blagburn B, Dillon R, Prichard R, et al. Characterisation of heartworm prevention failures in the central United States, in Proceedings. 13th Triennial Heartworm Symp 2010;27.

2. Bourguinat C, Keller K, Schenker R, et al. Investigation of genetic changes in Dirofilaria immitis after the use of macrocyclic lactone heartworm preventives, in Proceedings. 13th Triennial Heartworm Symp 2010;28.

3. Snyder DE, Wiseman S, Cruthers LR, et al. Ivermectin and Milbemycin Oxime in experimental adult heartworm (Dirofilaria immitis) infection of dogs. J Vet Intern Med [serial online] 2010. Available at: http://www.acvim.org.

4. Bayer HealthCare Animal Health, Study on File.

5. Blagburn BL, Dillon AR, Arther R, et al. Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis. Vet Parasitol 2011;175:in press.