International Journal for Parasitology 51 (2021) 1121–1132

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International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Invited Review

Macrocyclic lactone resistance in Dirofilaria immitis: risks for prevention

of heartworm disease
Roger K. Prichard
Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte Anne-de-Bellevue, Quebec H9X 3V9, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Heartworm disease, caused by Dirofilaria immitis, can be lethal in dogs and cats. It is transmitted by mos-
Received 13 July 2021 quitoes, and occurs in many parts of the world. Prevention relies on macrocyclic lactones. Macrocyclic
Received in revised form 29 August 2021 lactones used are ivermectin, selamectin, abamectin, eprinomectin, milbemycin oxime and moxidectin,
Accepted 31 August 2021
administered at 30-day intervals during the transmission season. Some moxidectin formulations are
Available online 27 October 2021
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
Keywords:
a decade later. Understanding factors which promote resistance is important to maintain control. Factors
Heartworm disease
Dirofilaria immitis
important for resistance development are discussed. Better, inexpensive tests to confirm resistance are
Macrocyclic lactone preventives needed. Infection in animals under chemoprophylaxis per se does not imply resistance because lack of
Macrocyclic lactone resistance compliance in preventive use could be the reason. In vivo confirmation of resistance is expensive, slow
Ivermectin and ethically questionable. A microfilariae suppression test can be a surrogate test, but requires a high
Moxidectin dose of a macrocyclic lactone and repeated blood microfilaria counts 2–4 weeks later. DNA single nucleo-
Milbemycin oxime tide 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.
Ó 2021 The Author. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Heartworm disease in domesticated dogs and cats, as well as
their wild cousins, is caused by Dirofilaria immitis. The parasite also
has zoonotic potential and thus could be considered of Public
Health concern (Noack et al., 2021). Humans can be infected with
D. immitis, from a bite of the mosquito vector, and infections may
progress and develop to juvenile adults in the pulmonary vascula-
ture, where they will die. However, the dead worms may cause
pulmonary nodules, which are otherwise usually asymptomatic.
The incidence in humans is relatively rare, but probably not fully
understood due to its rather benign consequences.
Dirofilaria immitis has a worldwide distribution but is most
prevalent in warm/humid areas, particularly the tropics and sub-
tropics. Due to global warming, increasing incidents of hurricanes,
E-mail address: roger.prichard@mcgill.ca
tropical storms and other major rain events in warm temperate
and temperate regions, and its transmissibility by a wide range
of mosquito vectors and resultant periodic increases in mosquito
populations, it appears to be spreading or increasing in temperate
regions such as the northern USA (Bowman et al., 2016), Manitoba
and Quebec in Canada (McGill et al., 2019) and central Europe
(Penezic et al., 2014; Sassnau et al., 2014; Tolnai et al., 2014;
Széll et al., 2020). It is often considered the most important para-
site of dogs in North America.
Dirofilaria immitis belongs to the superfamily Filarioidea (mean-
ing thread-like) and is completely parasitic throughout its life-
cycle. The adult female and male parasites, respectively up to
30 cm and around 18 cm long, but thread-like, live in the pul-
monary artery and its branches in canine hosts. In dogs with high
worm burden, adult D. immitis can also be found in the right ven-
tricle. The adult female worms are viviparous and produce micro-
filariae, which accumulate in the circulation, and can survive up to

https://doi.org/10.1016/j.ijpara.2021.08.006
0020-7519/Ó 2021 The Author. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
R.K. Prichard
2½ years after the adult worms are removed (Bowman and Atkins,
2009). During a blood meal on an infected dog, microfilariae can be
ingested by a mosquito. In the vector they develop in sequence into
the L1, L2 and L3 stages. When the L3s migrate to the mouth parts
of the mosquito host they are infective (iL3) and can be transmitted
during another blood meal to a mammalian host. Within the mam-
malian host they migrate and develop rapidly into L4s and finally
to juvenile (L5) adult parasites which become fecund and fully
mature approximately 6–8 months after the mosquito deposits
the iL3. Much of the prophylaxis of heartworm disease relies on
macrocyclic lactone heartworm preventive killing the extremely
sensitive L3/L4 developing stages in the mammalian host (Fig. 1).
This prevents establishment of adult parasites in the arteries which
are responsible for heartworm disease.
2. Heartworm disease
Heartworm infection in dogs may remain subclinical but often
progresses to clinical disease. The presence of the worms in the
arteries leads to pulmonary hypertension caused by the struc-
tural al-terations of the arteries. Initial symptoms are often a
persistent mild cough and reluctance to exercise. In dogs, young
adult and adult heartworms cause a vascular disease that can
progress to impaired blood flow, and affect the vascular and pul-
monary system. Exercise intolerance and inappetence can be
Fig. 1. Life cycle of Dirofilaria immitis, with some of the life-cycle stages (not to same scale), and points of prevention or therapy.
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International Journal for Parasitology 51 (2021) 1121–1132
observed. In severe cases, usually related to the adult parasite
burden and conversely, the size of the host, the right heart
chambers may be implicated. Pulmonary hypertension may lead
to right-side heart enlargement and progress to right heart fail-
ure. Occasionally, blood flow through the lungs can be
obstructed due to large numbers of adults in the pulmonary
arteries. Thereafter, worms may migrate to the right atrium, ven-
tricle and on to the vena cava. This blockage is known as caval
syndrome and may result in heart failure and mortality
(Bowman and Atkins, 2009). The incidence of heartworm infec-
tion in the USA can be seen on the American Heartworm Society
website (https://www.heartwormsociety.org/veterinary-
resources/incidence-maps, accessed August 19, 2021).
Cats are not considered a natural host for D. immitis and the
incidence of heartworm infection is lower than found in dogs. Feli-
nes usually do not host many parasites, and the parasites often do
not develop into reproducing mature worms (Lee and Atkins,
2010). Similar to dogs, chronic coughing and laboured breathing
may appear in cats. However, it may progress to vomiting and sud-
den death as cats are more immunologically reactive to heartworm
infections than dogs, and show a different range of clinical signs. In
cats, death of immature D. immitis in the pulmonary arteries, as
soon as 70–90 days after infection, can cause severe, pulmonary
symptoms known as heartworm associated respiratory disease
(HARD) (Atkins and Litster, 2006; Dillon et al., 2017).
R.K. Prichard
3. Prophylaxis is important
Because it is the adult stage which causes morbidity, and infec-
tion with a reasonable burden of adult worms can be lethal, the
main thrust of maintaining animal health and welfare is to prevent
the establishment of an adult infection. While mosquito abatement
and trying to screen companion dogs and cats from mosquito
access, or use of insect repellants, may help to reduce exposure
to mosquito bites and parasite transmission, it is difficult to keep
cats and dogs from being bitten, especially in regions of high mos-
quito reproduction. As a result, the main pillar of prevention of
heartworm disease has been the use of the macrocyclic lactone
(ML) class of anthelmintics to truncate infection at the L3/L4 stage
in the mammalian host. These anthelmintics have a range of prop-
erties that make them exquisitely effective against ML-susceptible
populations of L3/L4 D. immitis in dogs and cats. A combination of
the time it takes for L3s to develop to L4s, and the extreme sensi-
tivity of early L4s to MLs, plus the relatively long half-lives of the
MLs used for heartworm prophylaxis, allows treatment at 30-day
intervals, throughout the year or extending over the mosquito
transmission period and a little beyond. This can effectively pre-
vent the larval stages of D. immitis from developing to the patho-
genic adults. Most heartworm preventives are formulated for
administration at 30-day intervals. However, moxidectin (MOX),
which has the longest half-life and appears to be the most potent
of the commercially developed MLs (Prichard and Geary, 2019),
has also been formulated for depot injections to be given at either
6-monthly or 12-monthly intervals.
Heartworm preventives have been registered as 100% effective
against ML-sensitive isolates of D. immitis and as preventing heart-
worm infections in required limited field studies. ML heartworm
preventives have been remarkably successful in preventing heart-
worm disease. Because trying to predict the mosquito activity per-
iod for every area and in each year is impractical for the USA, and a
recommendation of a 30 days preventive administration promotes
better owner compliance, it is recommended in the USA that all
dogs should be on a rigorous preventive schedule for 12 months
of the year (American Heartworm Society (AHS) https://
d3ft8sckhnqim2.cloudfront.net/images/pdf/2020_AHS_Canine_
Guidelines_Summary_11_12.pdf?1605556516), accessed June 30,
2021; Companion Animal Parasite Council (CAPC) Guidelines,
2020, https://capcvet.org/guidelines/heartworm/, accessed 29
June, 2021). In other parts of the world, it is recommended that
dogs should receive heartworm preventive throughout the mos-
quito transmission season and extend for 1–2 months beyond to
prevent disease (e.g., Canada, Europe). However, over the last
20 years evidence of ML resistance has begun to appear.
4. Macrocyclic lactones used against filarial infections
The discovery that ivermectin (IVM) was larvicidal and microfi-
laricidal against many filarial parasites represented a significant
advance in the treatment of filarial infections. Initial studies using
IVM in Onchocerca volvulus-infected people were conducted in
1982 (Aziz et al., 1982a,b), and it was found to be safe and effective
in removing the microfilariae, and subsequently found to sterilize
the adult worms for 3–6 months (Greene et al., 1989; Brown,
2002). In 1985, IVM was approved for use in humans at a dose rate
of 150 lg/kg for onchocerciasis (Lindley, 1987), as MectizanÒ for
oral administration. The registration of IVM, as Heartgard-30Ò, as
the first ML oral heartworm preventive in dogs, occurred in 1987
in the USA. It targets the developing L3/L4 stages of D. immitis, with
the low dose rate of 6–12 mg/kg, (USA, Food and Drug Administra-
tion (FDA) NADA 138–412, April 7, 1987). Consistent with its use in
onchocerciasis against the microfilariae, it is of interest that in dogs
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International Journal for Parasitology 51 (2021) 1121–1132
with a patent D. immitis infection, IVM at 50 mg/kg was markedly
microfilaricidal (Blair and Campbell, 1979). However, it was not
registered at this higher dose rate for this indication in dogs over
concerns about possible neurotoxicity in a small subset of dogs
such as the Collie breed.
Abamectin, which is closely related to IVM, and has been used
for many years as an endectocide in livestock, is used for heart-
worm prevention in Australia while eprinomectin, another aver-
mectin used in livestock, is used as a topical preparation, in
combination with praziquantel, in cats.
Milbemycin oxime (MO), at 0.5 mg/kg orally, was approved in
1990 for use as an anthelmintic in dogs, including heartworm pre-
vention (FDA NADA 140-915 InterceptorÒ, June 14, 1990). In some
of the studies for registration, it was observed that in dogs already
infected with adult D. immitis, this MO dose rate was markedly
microfilaricidal. However, it was not registered as a microfilaricide
and was recommended to be used as a preventive in dogs not
already infected with adult heartworms.
Selamectin (SEL) was approved as a topical treatment, at 6 mg/
kg, for heartworm prevention, and treatment of fleas and some
other parasites in dogs and cats in 2000 (FDA NADA 141-152 Revo-
lutionÒ, June 13, 2000).
MOX was originally approved in 1997 for heartworm preven-
tion in dogs from 2 months of age, as ProHeartÒ at an oral dose rate
of 3 mg/kg (FDA NADA 141-051, ProHeartÒ, May 27, 1997), but as
with other heartworm preventives available at the time, treatment
was recommended every 30 days. Subsequently, MOX was devel-
oped as injectable heartworm preventive formulations, at 6-
monthly intervals (FDA NADA 141-189, June 13, 2002, ProHeartÒ
6), at 0.17 mg/kg, and 12-monthly intervals (ProHeartÒ SR-12), in
2001 in Australia; and ProHeartÒ 12 at 0.5 mg/kg, in the USA
(FDA NADA 141-519, July 2, 2019). MOX was also developed as a
topical formulation to be used each 30 days (FDA NADA 141-251,
Advantage MultiÒ (AdvocateÒ), December 20, 2006), for heart-
worm prevention, as a combination product with the ectoparasiti-
cide, imidacloprid. Additional D. immitis microfilaricidal activity
was approved for Advantage MultiÒ (FDA NADA 141-251, October
24, 2013). Another topical formulation of MOX, CoraxisÒ (FDA
NADA 141-147) was registered for heartworm prevention (without
combination with an ectoparasiticide and without microfilaricide
registration). The MLs used for heartworm prevention are shown
in Fig. 2.
It was reported that MOX showed greater potency against D.
immitis compared with other MLs (McCall, J.W., McTier, T.L.,
Holmes, R.A., Greene, T., Strickland, J., Aguilar, R., 1992. Prevention
of naturally acquired heartworm infections in heartworm-naïve
beagles by oral administration of moxidectin at an interval of
either one or two months. In: Soll, M.D. (Ed.), Proc. Heartworm
Symp. ’92. American Heartworm Society, Batavia, IL, USA, pp.
169–178; McTier, T.L., McCall, J.W., Dzimianski, M.T., Aguilar, R.,
Wood, I., 1992. Prevention of experimental heartworm infection
in dogs with single oral doses of moxidectin. In: Soll, M.D. (Ed.),
Proc. Heartworm Symp. ’92. American Heartworm Society, Batavia,
IL,USA, pp. 165–168; Blagburn et al., 2011), and evidence suggests
that this may be generally true for other filariids. MOX was found
to have high potency against microfilariae (Tagboto and Townson,
1996), and anti-reproductive effects on adult Onchocerca spp.
(Trees et al., 2000). MOX was registered for use in humans for
the treatment of onchocerciasis as an 8 mg oral dose by the FDA
on June 13, 2018 (https://www.Prnewswire.com/news-releases/
us-fda-approves-moxidectin-for-thetreatment-of-river-blindness-
300666114.html). It suppressed the microfilaraemia for more than
18 months after a single treatment (Opoku et al., 2018), indicating
not only a strong microfilaricidal effect, but also a profound effect
on the adult parasites, inhibiting their ability to produce new
microfilariae for more than 12 months.
R.K. Prichard
Fig. 2. Structures of commercially available macrocyclic lactone heartworm preventives. Ivermectin (A), abamectin (B), selamectin (C), eprinomectin (D), all avermectins; and
milbemycin oxime (E) and moxidectin (F), which are milbemycins.
5. Macrocyclic lactone resistance in D. immitis
ML resistance in D. immitis in the USA is now well accepted.
First indications that ML preventives may be failing were reported
in 2005 (Hampshire, 2005), in an analysis of significant numbers of
reports to the FDA of ineffectiveness of heartworm preventives,
described as ‘‘adverse experiences of approved animal products”
(commonly termed ‘LOE’ for loss of effectiveness when compliance
with heartworm prevention guidelines appear to have been strictly
followed). This paper reported that heartworm product complaints
relating to LOEs were first submitted to the FDA Center for Veteri-
nary Medicine (CVM) in 1998, and occurred rarely until 2000. The
numbers increased dramatically between 2000 and 2003, and
occurred mainly in the southern USA. Subsequently, most of these
LOEs were believed to be due to failure of compliance. Heartworm
preventives were registered as being 100% effective based on initial
registration trials, and companies offered reimbursement for the
costs of their products if pet owners claimed lack of efficacy and
some evidence of likely compliance, such as purchase records over
the relevant months.
The microfilaricidal attributes of MLs used as heartworm pre-
ventives was not an original claim when they were registered as
heartworm preventives. However, it had been shown that IVM
and MO could be microfilaricidal (Blair and Campbell, 1979;
Sasaki and Kitagawa, 1993; FDA NADA 140-915, 1990). A clear case
of a lack of expected microfilaricidal efficacy of IVM and MO in vivo
was reported by Bourguinat et al. (2011). These MLs were used
repeatedly, including at relatively very high dose rates, and should
have been microfilaricidal. This study provided the first well docu-
mented evidence for ML resistance in D. immitis.
Around the same time, two reports appeared of a lack of 100%
heartworm preventive effectiveness in dogs experimentally
infected with an isolate called Miss Piggy 3 (MP3). Although the
efficacy based on worm counts was high, respectively 99.7%
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International Journal for Parasitology 51 (2021) 1121–1132
(Snyder et al., 2011), and 94.5, 95.3, 95.3 and 100%, depending
on which commercial heartworm preventive was used (Blagburn
et al., 2011), based on arithmetric means. These data raised the
possibility of low level ML resistance.
Clear examples of unequivocal resistance in two D. immitis iso-
lates, Td2008-2 and Jd2009-2 from the USA, were obtained in three
clinical studies in which efficacies, based on arithmetric means of
worm counts, were respectively 31%, 71.4%, both using IVM at its
recommended dose rate, and 6.5%, using MOX in the ProHeart 6
formulation, with treatment 180 days prior to infection. Besides
the failure to achieve 100% efficacy, there was also evidence of
genetic differences between these isolates and susceptible isolates,
and similar genetic profiles to LOE samples (Bourguinat et al.,
2015). Genetic evidence of resistance, together with the pheno-
typic data, clearly established that it was true drug resistance that
was responsible for the lack of effectiveness and not, for example, a
problem of treatment of the dogs. This conclusion became
unequivocal with the demonstration that the resistance is inher-
ited between generations (Pulaski et al., 2014). Subsequently, other
studies (e.g., McTier et al., 2017a, 2017b) have confirmed ML resis-
tance to heartworm preventive treatment.
Microfilaricidal activity was approved in 2013 for Advantage
MultiÒ, which contains MOX for topical use at a recommended
dose rate of 2.2 mg/kg (NADA 141-251, October 24, 2013) and is
reported in the subsequent publication (Bowman et al., 2015).
Based on the substantial body of work showing that MLs are micro-
filaricidal, an in vivo microfilaraemia suppression test was pro-
posed as a means of detecting heartworm infections containing
ML-resistant D. immitis (Geary et al., 2011). This method was used
in dogs attending clinics in the USA with heartworm infections
(Ballesteros et al., 2018) and at the same time the genetic profiles
of the isolated microfilariae were evaluated using a panel of previ-
ously reported ML resistance genetic markers (Bourguinat et al.,
2015, 2017a). These markers are predictive for resistance. How-
R.K. Prichard
ever, the specific loci for resistance have not yet been determined.
Using these predictive markers and the microfilariae reduction
test, the Ballesteros et al. (2018) study indicated a number of very
resistant infections coming from Louisiana, Tennessee, Arkansas
and Missouri, and other dogs with suspected resistant infections
coming from these states plus Mississippi and Texas. ML resistance
in heartworm preventives has reached serious proportions in parts
of the USA, notably in states surrounding the lower half of the Mis-
sissippi River.
6. Macrocyclic lactone action – implications for resistance in
filarial parasites
The MLs are the most important pharmaceuticals used to con-
trol filarial parasites in animals and humans. In considering how
resistance to these drugs may evolve, it is important to consider
how they work and what stages of the filarial life cycle they target.
The most sensitive targets for the MLs in nematodes, at nM con-
centrations, are glutamate-gated chloride channels (GluCls). The
MLs have a pseudo-irreversible effect of opening these channels
to the influx of Cl-, which results in flaccid paralysis of muscle
(Prichard et al., 2012; Kotze and Prichard, 2016; Wolstenholme,
2012). However, it is also important to note that MLs bind to,
and activate, a wide range of other ligand-gated anion channels
(e.g., GABA- and dopamine-, serotonin- and tyramine-gated chlo-
ride channels) (Prichard et al., 2012), but effects on these ion chan-
nels require higher concentrations of the ML. In Clade 5 nematodes,
such as Caenorhabditis elegans, Haemonchus contortus, hookworms,
etc., the pharynx may be the most sensitive muscle paralyzed by
MLs, resulting in the nematode being unable to take up nutrients.
MLs also cause paralytic effects on body muscle, which in gastroin-
testinal nematode parasites may result in the parasites being
unable to maintain their site of predilection in the gastrointestinal
tract, leading to their physical removal. A more extensive discus-
sion of possible mechanisms of ML resistance in nematodes, and
of outstanding questions that remain about our understanding of
ML resistance mechanisms, is provided by Prichard and Geary
(2019). The effects of MLs seen in gastrointestinal nematodes
may, however, be less important in filarial parasites as filariae
are thought to be able to absorb nutrients through their cuticle
and strong pharyngeal contraction is not required for feeding. Fur-
thermore, some filariae, at some stages of their life-cycle, such as
adult O. volvulus in cutaneous nodules, and adult lymphatic filariae
in lymph glands, may be able to survive a temporary period with
body muscles paralyzed without being physically removed from
their sites of residence.
Although it is not conclusively proven, it has been hypothesized
that GluCl expressed in the excretory pore cell may be paralyzed
following ML exposure. This may result in filariae, particularly
developing larval stages and microfilariae in the blood or tissues,
being unable to adequately maintain the secretion of immunomod-
ulatory chemicals. They may thus become much more susceptible
to immune killing by the host (Moreno et al., 2010).
In adult filariae, MLs can suppress reproduction for prolonged
periods of time. In adult female Brugia malayi, strong GluCl expres-
sion signals were detected in the ovary, developing embryos and
lateral hypodermal chords, with moderate expression in the uterus
wall adjacent to stretched microfilariae (Li et al., 2014). GluCl
genes were also highly expressed in adult male worms in sper-
matogonia, in the wall of the vas deferens, and in the lateral chords
(but not in mature spermatozoa). In addition, the GluCl avr-14 was
highly expressed in somatic muscles adjacent to the terminal end
of the vas deferens. These results suggest that paralysis of muscles
in these sites by MLs may reduce gamete production and embryo-
genesis in filarial worms, and explain the observed suppression of
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International Journal for Parasitology 51 (2021) 1121–1132
microfilaria production following ML treatment. A single ML treat-
ment does not kill a significant proportion of the adult filariae, but
repeated exposure to MLs may reduce the lifespan of the adult
worms (Walker et al., 2018).
To summarize, developing L3/L4 stages of filariae such as D.
immitis are exquisitely sensitive to MLs (the basis of heartworm
preventive control). Microfilariae are sensitive to slightly higher
concentrations of MLs for the microfilaricidal approach to reducing
onchocerciasis and lymphatic filariasis transmission and morbidity
(IVM and MOX) and the registered claim of microfilaricidal activity
of Advantage MultiÒ (MOX) against heartworm. Furthermore,
reproduction in adult filariae (the anti-fecundity effects of MLs that
are critically important to reducing transmission of filariae such as
O. volvulus and Wuchereria bancrofti, and contributing to reducing
morbidity in onchocerciasis and lymphatic filariasis) is impaired
by MLs. When ML resistance evolves in filariae, resistant L3s/L4s
can still develop into reproductively active adult parasites
(Bourguinat et al., 2015); resistant microfilariae survive ML treat-
ment (Bourguinat et al., 2011; Ballesteros et al., 2018) and can sub-
sequently be transmitted by the relevant vector; and reproduction
in the adult worms is supressed for a shorter period of time, allow-
ing more transmission (Osei-Atweneboana et al., 2007). If the ML
effect on any of these stages is diminished, the surviving parasites
will be able to reproduce and pass on genes involved in the resis-
tance phenotype.
7. Factors that may play a role and amplify selection for
macrocyclic lactone resistance in D. immitis
ML resistance in heartworm preventives has reached serious
proportions in states along the lower half of the Mississippi River
in the USA, and there is concern that it will spread to other regions
of North America and arise in other parts of the world. Much is still
to be learnt about whether it has occurred independently at differ-
ent locations or whether it is spread by movement of dogs or
migration of infected vectors. What is the general fitness cost of
being resistant, and what can we expect in terms of its spread
and our ability to control heartworm infections and disease in dogs
and cats? What role, if any, will infections in untreated dogs and
wild canines play in providing refugia from drug selection to mit-
igate against a worsening of the problem? Many other questions
need to be answered to provide guidance for limiting the spread
of resistance and maintaining our ability to prevent heartworm
infection. Some of these questions are discussed below.
7.1. Selection for ML resistance in heartworm
If we assume, based on the report of Hampshire (2005), that
resistance first appeared around 1998, ML heartworm preventives
had been used and recommended as the first line means to prevent
heartworm disease for over 10 years (Heartgard-30 was first
approved in April, 1987, see above). Given that heartworm preven-
tives were registered as being 100% effective, we cannot assume
that the genetic change(s) that causes ML resistance only arose
and was selected in that 10–11 year period. Resistant lines of D.
immitis may have been present prior to the use of the MLs for con-
trol, but were very rare. Despite the fact that heartworm preven-
tives were registered as being 100% effective, and studies were
submitted to the FDA showing 100% efficacy, it is very probable
that they were never truly 100% effective. Vidyashankar et al.
(2017) have argued, very cogently, that this may be the case. Their
arguments include the facts that early registration studies were
performed with very few D. immitis isolates, which were main-
tained under experimental conditions, that severe ‘bottlenecking’
occurs during the lifecycle of D. immitis, and that very few dogs
R.K. Prichard
are used in treatment and control groups (typically 8–15 dogs), and
each untreated dog has an infection with very few (breeding) adult
male and female worms (typically 7–18 male or female worms/-
dog). Thus, efficacy studies that are used for registration purposes
do not adequately reflect the likely genetic and phenotypic diver-
sity that occurs in the wider D. immitis population; thousands of
dogs infected with tens of thousands of worms from different iso-
lates would need to be tested to adequately estimate the true vari-
ability in responsiveness of D. immitis to a heartworm preventive.
Clearly, that is not practical and so a requirement for 100% effec-
tiveness by registration authorities is a moot point. Given normal
biological variation in organisms that are not clonal, very few if
any pharmaceuticals are 100% effective in all circumstances. Nev-
ertheless, a high level of efficacy should be expected when a pre-
ventive is registered.
A recent study (McTier et al., 2019a) clearly demonstrates that
against a very ML-resistant strain of D. immitis (JYD-34), the effi-
cacy of MOX, used as a preventive in dogs, is dependent on the
dose rate and the number of monthly treatments. When the dose
rate was low (e.g., at the original registered monthly dose rate of
3 mg/kg) and the challenged dogs are not treated at 4-week inter-
vals, more adult worms will establish and could subsequently
breed and pass on their resistant genes. In contrast, when a high
dose rate (24–60 mg/kg) was strictly repeated, at 4-week intervals,
efficacy against JYD-34 developing larvae was very high and
approached 100%. These data are important for understanding
how ML resistance in D. immitis likely is selected. ML heartworm
preventives that were commercialized for monthly treatment were
registered and used at dose rates that could provide apparent 100%
efficacy in the limited studies that were required for registration,
on the assumption that animal owners would treat their dogs at
the recommended minimum dose rates and at the stipulated 30-
day frequency. Studies of the pharmacokinetics of IVM heartworm
preventive show that peak blood levels for the chewable oral for-
mulation at 6 mg/kg occurs at 8.5 h (3.7 ng/gm equivalent) and
decays rapidly over the first 3 days, and was less than 0.2 ng/gm
equivalent by 10 days after treatment (Daurio et al., 1992). For
the 6 mg/kg topical application of SEL, the peak plasma concentra-
tion, occurring at around 5 days, reported in male dogs as 12.7 ng/
ml and for female dogs was 22.7 ng/ml. For both sexes, the mean
residence time was 12.55 days (Dupuy et al., 2004). Oral adminis-
tration of 0.5 mg/kg of MO reaches peak plasma concentration by
1–4 days, with a t1/2 of only 1.6 days (A3 form) and 3 days (A4
form) (Letendre et al., 2017). The pharmacokinetic studies show
that these ML heartworm preventives reach peak concentrations
not long after administration and then are eliminated from the cir-
culation within a few days. After the peak concentration is reached,
decreasing levels are available to act on developing D. immitis L3s/
L4s until the next dose of preventive. If the next dose is longer than
30 days later, or is missed in some months, developing larvae are
likely to be exposed to sub-therapeutic levels of the ML. Any D.
immitis with a genotype that makes them less susceptible to MLs
will be more likely to survive and subsequently reproduce than
fully susceptible parasites. In this way, with selection pressure
every month, and given that ML heartworm preventives have been
recommended since 1987, ML resistance in D. immitis is not sur-
prising. Prophylaxis for heartworm infection has been less inten-
sive in other countries and regions than in the highly endemic
southern USA, and it is not surprising that ML resistance has not
so far been confirmed in other countries (Diakou, A., Koutinas, C.,
Bourguinat, C., Ballesteros, C., Dimzas, D., Chalkias, V., Batra, M.,
Traversa, D., Prichard, R., 2018. Heartworm infection in military
dogs under preventive treatment: Questions and suggested
answers. Abstract (P14), 6th European Dirofilaria and
Angiostrongylus Days, Belgrade, Serbia; Curry et al., unpublished
data).
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The extent of refugia may not be high in some regions due to
long-standing campaigns for suppressive treatment of every dog
and cat. In addition, we should remember that MLs act on other life
cycle stages, but at normal monthly dose rates that are not lethal,
or are possibly only selectively lethal, to some life-cycle stages.
7.2. Refugia
In contrast to gastrointestinal nematodes where most of the
nematode population will often be free-living stages on pasture
and/or in soil, which are not subject to anthelmintic selection, filar-
ial nematodes do not have any free-living stages; all of the filarial
nematode population will be in a mammalian host (L3, L4, adult,
microfilariae) or an insect vector (L1–L3). The vast majority of
the population (>99.9%) overall will be the microfilariae in the
mammalian host. These microfilariae can survive in a dog for 2½
years and may total billions in a single dog. The proportion in
the insect vector is miniscule. Thus, the proportion of the filarial
population that is in refugia will be the miniscule fraction in vec-
tors and those in infected mammalian hosts not subject to
chemotherapy. In effect, the proportion of the domestic and wild
canid population that is not under chemotherapy will be the main
reservoir for refugia. In North America, and particularly in the
southern USA where heartworm transmission is most intense,
there have been very strong recommendations for many years to
have all domesticated dogs and cats on heartworm preventives
12 months of the year because heartworm disease is very serious.
To the extent that such campaigns have been successful, refugia
will have diminished. It is well established in other parasitic infec-
tions that when refugia is low, resistance selection is likely to be
high (Leathwick et al., 2019).
7.3. Intensity of transmission
The study of Leathwick et al. (2019) also provides evidence that
‘years to resistance’ is likely to decrease in environments where
parasite transmission is high. For heartworm infection, the Lower
Mississippi River region is an environment of very high mosquito
vector density, which would be conducive to resistance develop-
ment. The high availability of mosquito vectors, coupled with
attempts to suppress D. immitis infections, would favour parasites
able to survive the effects of MLs, namely resistant genotypes
breeding, and being transmitted, while the ability of susceptible
genotypes to be transmitted would be truncated by constant treat-
ment with ML heartworm preventives. Selection for resistance is
also likely to be greatly amplified by inbreeding in zones of high
transmission.
7.4. Inbreeding
Another factor that is likely to play a role in the development of
ML resistance in D. immitis is inbreeding in D. immitis. A host with
an adult breeding population of heartworm will often have only a
few adult worms and any microfilariae taken up by a biting mos-
quito will be the progeny of those few female and male worms liv-
ing in the mammalian host. Thus, a good proportion of the
microfilariae acquired by the transmitting mosquito are likely to
be siblings or half-siblings. The small number of microfilariae
acquired during a blood meal can then develop to iL3s in a few
days and be transmitted to a new host, when the mosquito takes
another blood meal. Thus, the L3s infecting the recipient mam-
malian host will also likely have a proportion of siblings and
half-siblings similar to the microfilariae that the mosquito initially
acquired. These related L3s will subsequently develop into breed-
ing, genetically related, adult worms, unless the L3/L4 stages are
truncated by effective chemoprophylaxis. Obviously, if the devel-
R.K. Prichard
oping larvae are somewhat tolerant of the chemoprophylaxis, they
may survive the intervention. If resistance is polygenic, subsequent
inbreeding will produce some progeny even more resistant to ML
prophylaxis than their parents were. In this way, a moderately high
propensity to inbreeding in filarial infections is likely to be more
conducive to resistance selection (Churcher et al., 2008) than
would be parasite populations with little inbreeding, such as gas-
trointestinal nematodes that have very large and diverse popula-
tions on pasture or in the soil.
7.5. Effects of ML heartworm preventives on different life-cycle stages
While ML heartworm preventives primarily target the L3/L4
stages in the mammalian host, they do have effects on other life-
cycle stages such as microfilariae and adult worms. Indeed, killing
microfilariae is the primary purpose of treatment of human filarial
infections such as in onchocerciasis (IVM or MOX) and lymphatic
filariasis (IVM). Against D. immitis microfilariae, the high MOX dose
rate formulation, Advantage MultiÒ, has a registered claim as being
microfilaricidal (see Bowman et al., 2015). However, IVM and MO
also have microfilaricidal effects (Blair and Campbell, 1979;
Bowman, D.D., Johnson, R.C., Ulrich, M.E., Neumann, N., Lok, J.B.,
Zhang, Y., Knight, D.H., 1992. Effects of long-term administration
of ivermectin and milbemycin oxime on circulating microfilariae
and parasite antigenemia in dogs with patent heartworm infec-
tions. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Sympo-
sium ’92. Amer. Heartworm Soc., Batavia, IL, USA, pp. 151–158;
FDA NADA 140-915 InterceptorÒ, June 14, 1990). The effectiveness
of MLs as microfilaricides will depend on the anthelmintic dose
rate, frequency of treatment and the degree of resistance of a pop-
ulation of D. immitis. This was illustrated by the observations of
McTier et al. (2017b), who showed that microfilariae of the ZoeMO
isolate, a relative of the highly ML-resistant JYD-34 isolate, were
essentially eliminated over several months in dogs treated with
MOX extended release products, but were incompletely reduced
by a single high oral dose of MOX (0.25 mg/kg). Thus, ML heart-
worm preventives will more likely remove fully susceptible micro-
filariae, while any microfilariae more tolerant to the ML treatment
may survive, enriching the gene pool for resistance in subsequent
generations.
A similar consideration is relevant to the effects of MLs on adult
filariae. While MLs do not kill a significant proportion of adult
worms, they markedly interrupt their reproduction for in excess
of 30 days. This has been well documented for IVM (Duke et al.,
1991) and MOX in O. volvulus (Opoku et al., 2018), in which the
suppression of fecundity by IVM lasts from 3 to 6 months, while
for MOX it extended more than 15 months. Indeed, sub-optimal
response to IVM in onchocerciasis is manifested as a shortening
of the anti-fecundity effect (Osei-Atweneboana et al., 2011). Blair
and Campbell (1979) showed that a 50 mg/kg dose of IVM sup-
pressed production of new D. immitis microfilariae for approxi-
mately 2 months in the dog. A reduction of the anti-fecundity
effect of MLs against D. immitis would manifest in adult heartworm
showing a reduced susceptibility to the ML, as it does on O. volvulus
(Osei-Atweneboana et al., 2007). Thus, for a period after ML heart-
worm preventive treatment, adult heartworms which were less
affected by the treatment would be more likely to continue releas-
ing microfilariae (replacing microfilariae eliminated by the treat-
ment) than would more susceptible adult worms, and so more
resistant adult worms would have a reproductive advantage over
more susceptible worms, leading to selection for ML resistance.
One could speculate that reliance on the ‘slow-kill’ treatment
method (Dixon-Jimenez et al., 2018) to eliminate heartworm infec-
tions in dogs, in which repeated ‘sub-therapeutic’ administration
of ML is used, would facilitate resistance selection acting on the
adult worm fertility, and on the microfilaricidal effects of MLs.
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International Journal for Parasitology 51 (2021) 1121–1132
Thus ML resistance selection in D. immitis may occur at several
points in the life cycle, particularly on L3s/L4s, the primary target
for heartworm preventives, but be assisted by variable efficacy
against microfilariae and any reduction in the anti-fecundity effect
if MLs are used while adult worms are present.
As almost all of the filarial population exists in the mammalian
host, recommendations to try to suppress heartworm infection by
year round monthly (or long acting) treatment, in as many dogs as
possible, are likely to reduce refugia, which is mainly represented
by infections in untreated dogs. The relative potency of different
heartworm preventive formulations against all of the life-cycle
stages on which they can act, is also likely to be a factor in selection
pressure for resistance. Formulations that are more potent will
reduce any selective advantage of D. immitis populations with
resistance genotypes (see e.g., McTier et al., 2017a,b, 2019a).
7.6. Migration of mammalian hosts and vectors, and general fitness
While ML resistance in heartworm is mainly confined to the
Lower Mississippi River region (Ballesteros et al., 2018), the move-
ment of dogs could spread it. Indeed, there is evidence of consider-
able migration of dogs in North America and of this migration
increasing levels of heartworm infections (Drake and Parrish,
2019). Movement of dogs can introduce ML-resistant D. immitis
to areas well away from the Lower Mississippi River region, as
has already been reported (Bourguinat et al., 2011). So far, very lit-
tle survey work has been done to provide information on the pos-
sible spread of ML resistance in heartworm.
The spread of ML resistance could also be facilitated by migra-
tion of mosquitoes. Although mosquitoes tend to remain in their
local environment, mosquitoes could play a role in spreading resis-
tant strains locally, particularly in areas of high transmission.
Another factor that could affect the spread of ML resistance is if
resistance confers some general fitness cost to resistant popula-
tions compared with susceptible populations, i.e., the greater the
resistance fitness cost, the lower the tendency for resistance to
spread. This has yet to be studied. However, a preliminary finding
(McTier et al., 2017a) suggests that there could be some fitness cost
of ML resistance.
8. Diagnosis, detection and monitoring for macrocyclic lactone
resistance
Diagnosis of heartworm infection relies on antigen tests. These
antigens are produced in detectable quantities by adult female D.
immitis and the tests do not detect pre-adult infections (i.e., prior
to about 5 months after infection by a mosquito). There are a num-
ber of commercial antigen test kits that are available to clinics and
laboratories (e.g., SNAP 4Dx PlusTM Test - IDEXX, DiroChekTM - Zoetis,
etc., see Henry et al., 2018). Infection with adult worms is con-
firmed by the presence of microfilariae in the blood (see e.g., CAPC
Guidelines, 2020, https://capcvet.org/guidelines/heartworm/,
accessed 29 June, 2021). Microfilariae usually cannot be detected
before 6 months after initial infection, once the adult worms are
producing microfilariae. In cases where the owner believes that
their companion animal has been on regular heartworm preventive
treatment, these tests may prompt an enquiry whether the infec-
tion was caused by ML-resistant parasites. However, they do not
prove, one way or the other, whether it is a case of resistance,
unless the infections were conducted under very controlled condi-
tions when treatments are strictly monitored (such as clinical
research studies).
Confirmation of a resistance diagnosis in heartworm infections
is often confounded because records of when treatments occurred
may be incomplete, or difficult to confirm. A dog, which has been
R.K. Prichard
on monthly heartworm preventive, may not have received a treat-
ment in one or more months or the interval between monthly
treatments is sometimes greater than 30 days. In these situations,
if a dog becomes infected, it is likely to have been due to non-
compliance with the treatment recommendations (Atkins et al.,
2014). Thus, the existence of a heartworm infection in a dog whose
owner believed that the dog had been on preventive is not de facto
proof of ML resistance.
The ‘gold standard’ for showing that a D. immitis isolate contains
ML-resistant parasites is by challenging a naïve dog which is on
heartworm preventive, under controlled conditions in which the
treatment, timing and infection challenge are all carefully moni-
tored. This has been done in a number of studies (e.g., Pulaski
et al., 2014; Bourguinat et al., 2015; McTier et al., 2017a) showing
unequivocally that ML resistance existed. Furthermore, in these or
associated studies it was shown that resistance was inherited (had
a genetic basis), and that changes at the DNA level were associated
with the resistance trait (Pulaski et al., 2014; Bourguinat et al.,
2015, 2017a,b).
DNA-based tests of single nucleotide polymorphisms (SNPs)
which correlate with ML resistance were developed by
Bourguinat et al. (2011, 2015). These SNP markers were further
refined (Bourguinat et al., 2017a) and it was shown that a two
SNP DNA test was very predictive of ML resistance as measured
in a microfilariae suppression test carried out on field isolates in
many states of the USA (Ballesteros et al., 2018). While these SNP
markers do not tell us the mechanism of ML resistance in heart-
worm, they have been highly predictive in these and other unpub-
lished analyses (R. Prichard, unpublished data). The genotyping
done in these studies was based on either whole genome sequenc-
ing (Illumina) or MiSeq analysis of the SNPs of interest. These tech-
niques are useful for research studies, but are too slow and
expensive for application in a clinic on individual cases of apparent
breakthrough heartworm infection. New techniques for analysis of
the genotypes of individual cases of heartworm infection are under
development (Kumar et al., unpublished data). Most importantly,
further research is required to pinpoint the genetic changes that
cause resistance.
Based on the early evidence that MLs have a microfilaricidal
effect on D. immitis (Blair and Campbell, 1979), Geary et al.
(2011)proposed a microfilariae suppression test as a means of con-
firming a resistance diagnosis in heartworm infections (Geary
et al., 2011). The test involves undertaking a blood microfilaria
count in a dog that is infected with heartworm, treating the dog
with a dose rate of a ML that is usually microfilaricidal (for most
MLs, this will be a higher dose rate than used in heartworm pre-
ventives; e.g., for IVM 50 mg/kg rather than the 6–12 mg/kg used
in HeartgardTM formulations). Then 2–4 weeks later having a second
microfilarial count taken in order to determine the reduction in the
microfilariae count. In infections in which the heartworms are sus-
ceptible, the microfilarial count should be substantially reduced
(>90%), while in cases of ML resistance the microfilarial count will
not decrease significantly. This test was used and refined further
with the microfilaricidal treatment Advantage MultiÒ, which is
registered as a microfilaricidal MOX formulation, and the follow-
up blood count conducted 2–4 weeks after treatment (Ballesteros
et al., 2018). This study, which used both the microfilariae reduc-
tion test and analysis of genetic markers for resistance, also pro-
vided some limited information on the distribution of ML
resistance in USA. However, there is a great need to expand on that
study and to monitor for the presence of ML resistance in D. immitis
more widely and over time to better map the distribution of the
resistance and to see if it is spreading. Such a study would also pos-
sibly provide information on factors that are involved in the spread
of resistance, as well as any control practices which may delay or
enhance the spread of the resistance and would be a service to help
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International Journal for Parasitology 51 (2021) 1121–1132
maintain animal health in heartworm endemic areas. It would also
be prudent to monitor for ML heartworm preventive resistance in
other parts of the world where D. immitis infection is common and
receive preventives. Preliminary genotyping studies of D. immitis
from infected dogs in Greece (Diakou et al., 2018), Italy, Hungary
and the Canary Islands, Spain (Curry et al., unpublished data) sug-
gest that, so far, there is no hard evidence of ML resistance in these
European locales.
9. Current treatment and prevention options in areas of high
transmission and apparent inefficacy of heartworm preventive
formulations
Melarsomine dihydrochloride (ImmiticideTM, DirobanTM) is an
arsenic-containing drug that is approved in a number of jurisdic-
tions to kill adult heartworms in dogs. It is administered by deep
injection into the lumbar muscles to treat dogs with stabilized
stage 1, 2, and 3 disease. Usually, two or three doses of the arseni-
cal are given under supervised conditions in the clinic. The second
dose is usually administered about 4 weeks after the first dose,
while a third dose may be administered 24 h to a few days after
the second dose. The initial treatment kills most of the adult
worms in the blood vessels, but the presence of disintegrating filar-
ial worms and a massive amount of worm antigen can provoke
reactions. Treatment can be potentially toxic, including life-
threatening thromboembolism to the dog’s lungs. The treatment
is expensive because it requires multiple visits to the veterinarian,
blood tests, x-rays, hospitalization, and a series of injections.
Another drug, Advantage MultiTM for dogs (MOX and imidaclo-
prid), is approved to remove circulating microfilariae. Advantage
MultiTM for dogs is a high dose rate of MOX for topical application
to the skin.
Prevention remains by far the preferred way of keeping dogs
and cats free of heartworm disease and the approved ML preven-
tives remain the mainstay of prevention of heartworm disease,
despite evidence that resistance to MLs occurs in the USA.
In areas with evidence of resistance to some ML preventives,
some clinicians are recommending a combination of a monthly
heartworm preventive and periodic doxycycline (10 mg/kg twice
per day for 28 days) treatment. Dirofilaria immitis harbour a symbi-
otic, intracellular, gram-negative bacteria, Wolbachia pipientis. This
doxycycline regimen will eliminate these commensal bacteria and
without these bacteria, the nematode parasite is unable to produce
viable progeny and will, over time (e.g., about a year for adult
worms), die. Doxycycline, given over a month-long regimen, as
indicated above, also prevents microfilariae from developing into
infective larvae if taken up by a mosquito (McCall et al, 2014;
CAPC, 2020, https://capcvet.org/guidelines/heartworm/, accessed
29 June, 2021), and this treatment may reduce the transmission
of ML-resistant D. immitis. There is evidence that treatment of dogs
with doxycycline, to eliminate Wolbachia prior to adulticide arseni-
cal therapy, may improve adulticidal and microfilaricidal efficacy,
and reduce the gross pulmonary pathology by reducing the
immunological reaction caused by the release of the bacteria when
the worms die.
High dose rate MOX formulations such as Advantage MultiTM,
Proheart 12TMand Symperica TrioTM, although not registered for
use against resistant D. immitis, appear to be more efficacious in
preventing heartworm infections, including against isolates that
are resistant to other heartworm preventives (Blagburn et al.,
2016; McTier et al., 2017a, 2017b, 2019a, 2019b; Kryda et al.,
2019). Some of the underlying reasons why MOX can be more
effective than other MLs was previously reviewed (Prichard et al.,
2012; Prichard and Geary, 2019). These formulations may be par-
ticularly useful in areas where ML resistance to monthly heart-
R.K. Prichard
worm preventives is of concern. In addition, a veterinarian-
administered long-acting formulation such as Proheart 12TM has
the added advantage of virtually eliminating any concerns about
compliance with heartworm prevention, compared with owner
administered monthly treatments.
In addition to the use of heartworm preventives, reducing mos-
quito bites to pets can help in preventing heartworm infection and
reducing transmission, including ML-resistant genotypes. How-
ever, pets are very attractive to female mosquitoes seeking a blood
meal in order to reproduce. Keeping pets indoors and/or in
mosquito-screened premises can reduce mosquito bites, but is dif-
ficult in most instances. Mosquito repellants/insecticides were
found to help prevent heartworm infection (AHS, 2015, https://
d3ft8sckhnqim2.cloudfront.net/images/bulletin/AHS-1511-Nov-
15-Winter-Bulletin.pdf?1535052705, accessed June 30, 2021).
The current situation for prevention, treatment, and occurrence
of ML resistance is summarized in Table 1.
10. What is the likely progression of macrocyclic lactone
resistance under existing options, policies and practices?
In the USA, both the Companion Animal Parasite Council (CAPC)
(https://capcvet.org/guidelines/heartworm/ accessed June 30,
2021) and the American Heartworm Society (AHS) (https://
d3ft8sckhnqim2.cloudfront.net/images/pdf/2020_AHS_Canine_
Guidelines_Summary_11_12.pdf?1605556516 accessed June 30,
2021) recommend that all dogs in the USA be protected from
heartworm infection by using (ML) preventives year round. The
European Society of Dirofilariosis and Angiostrongylosis (ESDA)
states that infection of dogs with D. immitis has been diagnosed
in many European countries and cases are spreading. The ESDA
provides prevalence maps and recommends prevention of heart-
worm disease in all endemic regions by use of heartworm preven-
tives year round; even in winter, as urban heat islands allow
mosquitoes to survive (https://www.esda.vet/wp-content/
uploads/2017/11/GUIDELINES-FOR-CLINICAL-MANAGEMENT-OF-
CANINE-HEARTWORM-DISEASE.pdf accessed June 30, 2021). The
European Scientific Counsel Companion Animal Parasites (ESCCAP)
guidelines state that in most parts of Europe where infection is
endemic, the transmission season of heartworm lasts from April
to October/November (depending on the climate). Recently, topical
Table 1
Summary of the current situation, preventions or treatments, and threats to sustained
prevention and control of Heartworm disease.
Current situation, treatment or preventive measures or threats to sustained
control
1 Heartworm disease is a serious, potentially fatal, infection in dogs and
cats and prevention is strongly recommended
2 The macrocyclic lactone (ML) class is currently the only class of
antiparasiticide used for heartworm prevention. Therefore, resistance to
MLs poses a serious threat to sustained prevention of heartworm disease
3 ML-resistance in heartworm preventives occurs in the USA. This appears
to be predominantly in the areas around the lower Mississippi River
4 To date, there are no confirmed cases of ML-resistance transmission in
other parts of the world. However, vigilance is required
5 Diethylcarbamazine is an anti-filarial used in human medicine. However,
for heartworm prevention it requires daily administration during the
mosquito transmission season which is much more challenging than
treatment each month, or at longer intervals, with ML heartworm
preventives
6 Mosquito repellents and insecticides can be used to reduce transmission,
particularly of ML-resistant genotypes
7 Arsenical adulticides remain effective against established infections with
ML-resistant genotypes
8 Month-long doxycycline treatment can stop microfilarial development to
iL3 in the mosquito and, in the long-term, remove infections with adult
parasites
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International Journal for Parasitology 51 (2021) 1121–1132
administration of permethrin with dinotefuran has shown repel-
lent efficacy against mosquitoes on dogs for at least 4 weeks
(https://www.esccap.org/uploads/docs/ubs8uae7_0778_ESCCAP_
GL1_v15_1p.pdf accessed July 1, 2021). While the Australian
Heartworm Advisory Panel guidelines state that all domestic dogs
should be on year-round heartworm prevention (MLs) irrespective
of their location in Australia (Australian Heartworm Advisory
Panel, 2014), veterinary bodies in other parts of the world where
D. immitis is endemic also argue for the use of heartworm
preventives. As can be seen from these authoritative bodies, heart-
worm ML preventives are strongly recommended throughout the
mosquito transmission season, and in many parts of the
world, notably in the USA, the recommendation is year round
prevention.
In the absence of an alternative class of heartworm preventives,
this suppressive treatment with MLs will impose significant selec-
tion pressure for genotypes of D. immitis that are able to survive
the preventive chemotherapy to reproduce, leading eventually to
resistance. The various factors that may enhance or delay that
resistance selection are discussed above. However, continuation
of current practices is likely to lead to resistance appearing in
new locations and to its spread and increase in intensity, unless
other measures are taken to reduce dependence on this single class
of heartworm preventive therapies. Furthermore, once a heart-
worm preventive has been approved in the USA, where resistance
exists, the FDA does not withdraw registration, or require that the
failed product only be used to prevent infection by susceptible par-
asites (probably an impractical request). Therefore, continued use
of the product to which there is resistance may continue to select
for higher levels of resistance and facilitate transmission of resis-
tant parasites to other areas. In the absence of new classes of heart-
worm preventives and the limited use of other approaches to
mitigating infections, the situation is likely to get worse and
become an increasing problem for animal health and welfare.
Steps to reduce mosquito biting and transmission should affect
both ML-susceptible and ML-resistant D. immitis similarly, and so
represent an additional useful approach to prevention that does
not enhance resistance to the MLs. The periodic use of anti-Wohl-
bachia chemotherapy (e.g., doxycycline or minocycline used daily
for at least 28 days) may also be useful in helping remove estab-
lished infections and in reducing transmission of ML-resistant
genotypes. However, novel non-ML compounds suitable for pre-
ventive treatment are needed in order to control or delay the
development of ML-resistant D. immitis populations and to expand
the range of options for preventing heartworm disease.
The requirements of many different jurisdictions for registra-
tion of novel heartworm preventive products were briefly summa-
rized by Noack et al. (2021). The US FDA requirements are
currently: two laboratory dose-confirmation studies, using D.
immitis isolates, not prior-screened for susceptibility to the pro-
posed product, or isolated more than 5 years previously, and one
multisite field safety and effectiveness study, which must demon-
strate heartworm preventive efficacy. The FDA has historically
required 100% efficacy in these studies for registration.
Vidyashankar et al. (2017) have made the case that initial registra-
tion studies were based on just a few strains of parasites, and
therefore were not representative of the full assortment of circulat-
ing biotypes. Furthermore, they were done in years when resis-
tance to MLs in D. immitis was unknown, and was probably very
rare in the whole population of the parasite in the USA. These
authors also make the point that the UGA-TRS strain used for the
majority of product approval studies prior to 2000 (McCall,
2011), would be predicted to be highly bottlenecked and have very
low genetic diversity as a consequence of the repeated passage of
this strain over 30 years. The measured efficacy of an anti-
parasiticide is affected by a large range of biological and pharma-
R.K. Prichard International Journal for Parasitology 51 (2021) 1121–1132

ceutical factors affecting the parasite, the host and the active ingre- Table 2
dient in the formulation. To accurately determine the true efficacy Summary of actions helpful for maintenance of prevention to stop or delay a
worsening of the prevalence of heartworm disease, and relevant actors for these
of a product, both numbers of infected dogs tested and numbers of actions.
worms recovered from non-treated animals need to be very large
(thousands of dogs of various parentage infected with tens of thou- Suggested action Organizations/individuals who
could implement action
sands of worms). However, this is not economically feasible and
would be of concern for animal welfare. Therefore, tests occur in 1 Improve diagnosis of macrocyclic Animal health industry, academic
lactone (ML) resistance to researchers, veterinarians
a small sample of dogs (typically 8–15/group), that are relatively heartworm preventives to the point
uniform with regard to breed, age, weight, etc. For heartworm when tests can become available in
infections, usually 30 to 100 iL3s are used to inoculate each dog. the clinic
Furthermore, in untreated dogs, establishment rates vary but aver- 2 Survey the prevalence of ML Academic researchers, animal
resistance in Dirofilaria immitis health industry, veterinarians
age around 50%. Thus, tests are being conducted on only a small
across the USA, and commence
number of worms (typically only about 250 worms being screened vigilance for detecting ML
by the preventive in a treatment group). Vidyashankar et al. (2017) resistance in other countries and
also explain that the statistical methods of analysis typically used regions
are inadequate, and should be improved. We are left with a conclu- 3 Discourage use of ‘slow-kill’ Animal health and welfare non-
method to remove established profits, academic researchers
sion that claims of 100% efficacy being applied to the general pop-
infections with repeated sub-
ulation of D. immitis are somewhat bogus. Simulations of efficacy therapeutic treatments with MLs
that provide 95% confidence interval would be more meaningful, 4 Recommend the use of mosquito Animal health and welfare non-
rather than accepting a claim of true 100% efficacy. While evidence repellents and insecticides, and profits, academic researchers,
other measures to reduce mosquito animal health industry
of very high efficacy (e.g., >98%) should be expected for a heart-
biting
worm preventive, a 100% standard is in fact not verifiable and is 5 Until new classes of heartworm Veterinarians, animal health
a significant disincentive for new classes of potentially preventive become available, industry, academic researchers
resistance-breaking heartworm preventives being found, regis- encourage the use of high dose rate
tered and potentially commercially viable. This is an animal health moxidectin formulations in areas
where ML resistance is prevalent.
and welfare issue because the current limitations to developing
Long-acting formulations may also
novel heartworm preventives are leading to resistant strains being be helpful for reducing non-
increasingly selected and potentially spreading. In the long term, compliance frequently observed
this is not sustainable. Resistance to the ML class of heartworm with monthly preventives
6 Encourage discovery and Animal health industry, research
preventives should be recognized by both registration authorities
registration of novel classes of funders, academic researchers,
and animal health companies, and the registration of novel preven- heartworm preventives and registration authorities and
tives that are highly active against ML-resistant D. immitis should therapies governments
be encouraged by revision of registration requirements and recog- 7 Relaxation of rules requiring 100% Registration authorities and
nition of the fact of susceptibility and resistance in claims for efficacy for registration of novel governments, animal health and
classes of heartworm preventives, welfare non-profits, animal health
heartworm preventives.
while maintaining a requirement of industry
very high efficacy, but below 100%;
11. The way forward – what is needed especially against ML-resistant
isolates of D. immitis
8 Discovery and development of anti- Research funding organizations,
Faster, less expensive methods for the detection of ML resis-
Wolbachia therapies requiring only academic researchers, animal
tance are needed to help guide clinicians in advice on prevention a short course of treatment health industry
and treatment (see Table 2). Ideally, these should be rapid genetic 9 Testing dogs for heartworm Veterinarians, animal health and
or in vitro tests for resistance that can be performed as part of a infection, and treating if necessary, welfare non-profits
consultation in the clinic. In order to improve the accuracy of tests prior to their transportation to new
regions or countries in order to
for resistance, and perhaps envision ways of surmounting resis- reduce the risk of importing
tance, we need knowledge of the mechanisms and specific genetic resistance
changes that cause resistance. In optimizing prevention of infec-
tion, in the face of the possibility that some parasites may be resis-
tant to ML heartworm preventives, we need to have surveys of In regions where resistance to current preventives occurs, the
countries and regions, where resistance occurs or is suspected; use of more potent, higher dose rate formulations of MOX, rather
and such surveys should be repeated periodically to assess changes than other MLs, may provide a better measure of prevention of
in distribution of resistance. infection with moderately resistant D. immitis isolates. However,
Strong recommendations to pet owners to try to avoid their in the longer term, based on experience in gastrointestinal nema-
companion animals being bitten by mosquitoes would be helpful. todes (see e.g., Prichard and Geary, 2019), continued selection
The routine use of mosquito repellents and insecticides on pets, pressure with MLs is likely to lead to higher levels of resistance
particularly products with persistent effects for 4 weeks or longer, to all MLs, including to higher dose rate MOX formulations. Never-
would be very helpful as an adjunct to the use of heartworm pre- theless, more use of such high dose rate MOX formulations may
ventives. Many ML heartworm preventives for dogs and cats are buy some time until new classes of heartworm preventives can
now combined with ectoparasiticides such as those from the isox- become available.
azoline class (Noack et al, 2020). Depending on the dose rate and New classes of heartworm preventives need to be discovered,
persistence of these ectoparasiticides in the combination they registered and commercialised. Acceptance of the limitations of
may, for a period, kill mosquitoes that take blood meals from trea- current ML heartworm preventives, in the face of ML resistance,
ted dogs. This may reduce the canine-feeding target population, by registration authorities, and a recognition that facilitation of
and kill mosquitoes before they could transmit the heartworms new resistance-breaking classes of preventive, even if they provide
ingested. very high efficacy, but not always a theoretical 100%, would be

1130
R.K. Prichard
beneficial to reduce heartworm disease, transmission of ML-
resistant genotypes and the spread of ML resistance.
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