Veterinary Parasitology 273(2019)11-16

20190928 入江

Efficacy of imidacloprid 10%/moxidectin 2.5% spot on (Advocate®, Advantage Multi®) anddoxycycline for the treatment of natural Dirofilaria immitis infections in dogs
Marco Genchi, Alice Vismarra, Chiara Lucchetti, Antonio Biglietti, Serena Grosara, Giacomo Gnudi,
Cecilia Quintavalla, Roland Schaper, Laura Kramer
Introduction▶フィラリア症は蚊によって媒介され無治療だと慢性的な肺高血圧によりうっ血性心不全を引き起こす。
▶メラルソミン(MEL)が唯一の成虫に対する治療薬であるが、コスト、有効性、副作用の点からもこれに変わる治療法が研究されている。
▶いくつかの研究では、大環状ラクトン類(MLs)とドキシサイクリン(DOXY)の効果が示されている。
▶目的：モキシデクチン(MOXI)とDOXYを併用した際の成虫への効果を評価し、MELの効果と比較する。
Material and Method▶動物：イタリアで自然感染した犬20頭(組み入れ基準：Table1a)▶2つの群で比較(Fig1)
・MOXI/DOXY群 ：Advocate®(10%イミダクロプリド、2.5% MOXI含有)とDOXYの併用
・MEL群：Immiticide®を3 step プロトコルで使用▶調査項目
・寄生虫学的評価(血中ミクロフィラリア(mf)濃度、抗原検査、抗原濃度)
・呼吸・循環器の評価(胸部レントゲン、心エコー検査):
Advocate®
正常(スコア0)〜重度(スコア3)で評価(Table2)(Mavropoulou et al., 2014)
Results▶MOXI/DOXY群はn=14、MEL群はn=6であった(Table1b)▶寄生虫学的評価：
・血中mf濃度(Fig2)-MOXI/DOXY群の13/14頭が治療開始時にmf(+)→うち10頭は1ヶ月後にmf(-)、4頭は4/mlであった。
-MEL群の2/6頭で治療開始時にmf(+)→1頭は3ヶ月後にmf(-)、もう1頭は9ヶ月でmf(-)となった。
・血中抗原(Fig3)-MOXI/DOXY群の13頭は治療開始後9ヶ月で抗原(-)っとなった。
-MEL群の1頭は治療開始後3ヶ月、3頭は4ヶ月、残り2頭はそれぞれ5, 6ヶ月で抗原(-)となった。
-抗原濃度はMOXI/DOXY群で緩徐に減少、MEL群で始めは上昇し、その後急激に減少した。
▶呼吸・循環器の評価：両パラメーター共に治療開始時には0または1のみで、最終的には全頭で0となり(0→1→0の場合もあり)、MOXI/DOXY群はMEL群より早く改善が認められた。
Discussion▶血中mf濃度：mfに対する効果はMELだけでは弱く、MOXI/DOXY併用での効果が大きかった。
▶血中抗原濃度：MOXI/DOXY群は、成虫の子宮内容の緩徐な減少と、死滅する成虫の抗原の放出が緩徐であるため→抗原濃度の減少が緩徐。MEL群は、成虫の死滅が急激であるため抗原の放出と濃度低下が早い。
▶呼吸・循環器の評価：MOXI/DOXY群で炎症性変化の改善が早かったのは、DOXYによるボルバキアへの効
果や、炎症前駆抗原の減少によるものと考えられる。
▶イタリアでの調査：全体の35%の施設がMELを使用しており、他の29%がMLs/DOXY併用をしている(Genchi et al., 2019)。→MELの代替プロトコルとなりつつある。
批評
・症状のグレードや治療中の運動制限などの表記がなく、細かな部分が不明。
→症状のグレードでは、無症候or軽度では運動制限をすれば問題ないが、中程度or重度or合併症ありならば治療は困難。今回の研究では、画像検査上では、無症候or軽度であると考えられる。
・本研究では、MELのみの投与であったため、AHSのガイドラインに載っている方法との比較も必要。
・コストや投薬の簡便さ、有効性から、フィラリア症の治療としてAdvocate®の使用はオーナーに薦めやすい。
・当院では、治療に対して統一がないため、治療方針の確立の一助になるのではないか。

MOXI/DOXY (tot. 14)
MEL (tot. 6)

Group/No. Animals

4/F 10/M
6 /M

#/sex

2-12 (mean 6.5 years)
3-8 (mean 5.5 years)

Age (mean years)

M. Genchi, et al.

of 0. At 6vs.
monthsfrom the beginning of treatment, one dog with a% vs. 100.00% andscore100.00%100.00%).
Savadelis et al. (2018) reported that positive results ofin adult
worm antigen concentration fromscore of 0 at enrollment had a score of 1.gradualOf the four reduction
dogs with a scorenchi, et al.
9HWHULQDU\3DUDVLWRORJ\ 
² D. immitis-experimentally infected dogs were n
fromof 1 at enrollment, 3 improved (score ofmonths0) while one
remained
1. At theTable1
Fig1 治療プロトコルa: 組み入れ基準、b:組み入れた犬の性別・年齢
onwardfrom
beginningwithdogs
necropsy.
Vencoofet treatment,al. (2017)
showedthatallheat
treat
months 12, 18 and 24, all dogs treated with moxidectin/doxycycline
組み入れ基準antigen test
resultsof samples
from a wideva
one, becoming negative atsitive
9 months.
This
gradualdeclinein circ
had a score of 0.
a
犬種・性別は関係なし
andparasitic infections, including mono-infections w
All dogs in the melarsomine group hada score ofis0similar
at enrollment
antigens
to thatpens,reportedby Savadelisal. (2017).
Tthe aetiologicalagent of et
subcutaneous
dirofil
体重１kg以上
and at 6 months.
dogs showed
1 at 12 months. At months
ctin/doxycycline group
had a Two
cardiacul- a scorebeof due
in many EuropeanCiucă dec
et al
to the gradual losswidespreadof 5ヶ月齢以上
intrauterine
contentcountries.
and a slow
18 and 24 months, all dogs from the melarsomine group had a score ofthat heating of serum samples from dogs with D. rep
ment, while all others
score of 0. At
0 (seehad
Tablea3).
antigen release while worms
are dying, as compared to the
2ヶ月以内にMLやDOXYの使用歴がない
positive results and concluded that where D. repens is®を4週ごとに9ヶ月間投薬
2.5mg/kg投薬し、1ヶ月後に同量を24時間間隔をあけてFor thoracic
radiographs
at 12, 18MELand
24 months non-inferiority of thedogs from Advocate
this grouphad
a scoreof 0. Two
Table 2
D
抗原検査、mfが片方または両方(+)でD.
immitis 感染とみなす
likely consequent
to melarsomine-induced
death, 4. wof samples
not currently
recommended.
2回投薬 release
Enrolled dogs is(moxi/doxy:moxidectin + doxycycline;
mel: melarsomine diDOXY 10mg/kg BIDを30日間投薬
moxidectin/doxycycline
group
vs.
the
melarsomine
group
could
be
provenhydrochloride).
mfカウントと抗原検査
In
up had a scoreof 1 (100.00%
atenrollment,
while
all
the present study,wasmoxidectin/doxycyclinetrea
mfカウントと抗原検査
suddenand vs.
rapid
declinebinInantigenemia
observed at 3–4
mcomb
vs. 66.67%, 100.00% vs. 100.00%
and 100.00%
100.00%).
心エコー検査とレントゲン検査
心エコー検査とレントゲン検査
gradual reduction in adult worm antigen concentra
withmonths, the
two
dogs
with
1
at
enrollment
oresp
In naturally infected dogs,
we onward
cannotfrom
excludethatofimmaturep
1. a. Treatment regimen for moxidectin/doxycycline; b. Treatment regimen for melarsomine.
monthsthe beginning
treatment, withMela
mf: microfilariae).
ultrasoundeenrollment;
anothernormal 3.4.
dogCardiac
at enrollmentwasshow
negativeat 9 months.
This gradualthat are migrating duringone,thebecoming
9-monthtreatment
regimemaydeclal
Table
1
Table2
間質性肺病変・肺高血圧の評価の基準
treat
is similar to that reported by Savadelis et al.
At months 12, 18 and 24, all dogs from thearrived as adults and thenantigens
subsequently
died. The present study
ore of 0 (as shown in Table 3).
also indicate that the combination of moxidectin/doxycycline
12, 18 and 24 months, non-inferiority of
perior adulticide eﬃcacy compared to ivermectin/doxycyclingroup vs. the melarsomine group could be
previous study in naturally-infected dogs treated with doxycyc%).

M. Genchi, et al.

Scoresystem applied
for thoracic
radiographs
andsearchcardiac forultrasoundevaluation(Mavropoulou
et al., 2014).
prod
ability andside-eﬀects
of treatment
have
led to theal2.3. Treatment
protocols
Two dogs in the moxidectin/doxycycline group had a cardiac ul1992
be due to the gradual loss of intrauterine content andaative therapeutic protocols (Kramer et al.,2018).
Pulmonaryinterstitial disease (thoracic
radiography)
間質性肺病変(レントゲン検査)
prop
trasound score of 1 at enrollment, while all others had a score of 0. At
everal studies in both experimentallyand
naturallyinfected
dogs
Treatment
protocolsaresummarized
in
Fig.
1a
and
b.
Briefly,
toantigenrelease
while
worms
are
dying,
ascompared
low
Score
0 正常
1 軽度
2 中程度
3 重度
reported the adulticide eﬀect ofmonths
a combination
macrocyclicof10%
imidacloprid+
2.5%
moxidectin(Adabsen6, 12,of18
and 24, all picaldogsadministrationfrom this grouphad
a score of0.
Tworeleaselikelyconsequent to melarsomine-induced
9HWHULQDU\3DUDVLWRORJ\

²
befor
nes and doxycycline
against D. immitis,Normalthat theseMild
protovocate®)
administrated
14 dogs
once aandmonthfor
9 consecutive
Clinical evaluation
unstructured
interstitialpatternMore diﬀuse
uniform
unstructured
Diﬀuse pulmonary densities, signs compatible withdogs showing
in the melarsomine
group
hadwasalung
score
of 1 attoenrollment,
while
all
cent
sudden and rapid
decline in antigenemia was observe
the caudal lobe
interstitial lung pattern
thrombo-embolism/pneumonia
are safe andeﬀective(Bazzocchi et al., 2008; Bendas et al.,in2017;
monthstogetherwithdoxycycline
(10
mg/kg
BW,
BID)
for
the
first
30
M. Genchi,
et al.
9HWHULQDU\3DUDVLWRORJ\
 ²Euroothershad a score0. At 6 days
months,thewith 1 at enrollment
di et al., 2010; Mavropoulou et al.,2014; Savadelis
et al.,of
2017).
1a). two
For dogs
the melarsominegroup, melarsomineIndihy4. (Fig.
Discussion
naturally infected dogs, we cannot exclude thatowne
imPulmonary 肺高血圧(心エコー検査)
hypertension (echocardiography)ycyclinetargetsmoxidectin
the endosymbiont
Wolbachia,
whose
reduction
drochloride
was administeredat the wasmoment of enrollwere
still
at 1, diwhile
another(Immiticide®)
normaldog
at enrollment(moxi/doxy:+ doxycycline;
mel:scoredmelarsomine(201
that are migrating during the 9-month treatment regim
Table 2
4. Discussions).to worm infertility
and death(Kramer et al., 2007). This activity, 1 軽度
ment, 2.5 mg/kg BW, followed one month
later by two injections of the
incre
Score
0 正常
中程度
重度
scoredwithalterations.
At months
18 and24, weall 2dogs
from thethe adulticide eﬃcacy3 of
In dithe 12,
present
study
evaluatedaEnrolled
dogs (moxi/doxy:moxidectin+ milddoxycycline;
mel: melarsomine
arrivedasadultsandthensubsequently
died.
The
pre
R
bined with the known detrimental eﬀects of macrocyclic lactones,
same dose 24 h apart to six dogs (Fig. 1b).
Fig. 2. Number of mf/ml (average value for positive dogs) expressed ad log10abhydrochloride).
combinationofmoxidectin
anddoxycycline
indogs
naturally
infectedIn
thepresent
study
we
evaluatedtheadulticideeﬃcacyofevaluation
Normal
>
64
ms;
AT/ET
ratio
Slightalterations(AT/ET=
0.42–0.26
ms)
Moderate
alterations
(AT
≤
45
ms;
Markedalterations
pulmonarymelarsomine
group
had
a
score
of
0
(as
shown
in
Table
3).
idect
imals
#/sex
Age
(mean
years)
after
treatment withmoxidectin/doxycycline(moxi/doxy)melarsominenates adultClinical
worms
and
lessensthe(AT
inflammatory
reactionagainstalso
indicatethatthea (moderate/severe
combinationof vs.moxidectin/doxc> 0.42 ms) TRV < 2.8 m/s
TRV < 2.8 m/s
AT/ET ≤ 0.25;
≥
2.8
m/s)
TRV
>
3.57
m/s)
et al
combinationof moxidectin
andTRV
doxycyclinein (mel).
dogs naturally
infectedwith
D. andimmitisandcompared
microfilariemia,
andhypertension:
cardiGroup/No.
Animalset al., 2011).
#/sex
Age (mean
years)and dying worms(KramerThe
American ultrasoundHeartworm
ParasitologicalevaluationFor
cardiacat2.4.
12,18
24
months,non-inferiorityof antigenemia
perioradulticideeﬃcacycomparedtoivermectin/d(201
a(tot. 14)
4/Ftime.
10/M
2-12 (mean 6.5 years)
with D. immitis and compared microfilariemia,
antigenemia
and cardiAcceleration
ety
(AHS)
and
the
EuropeanSociety
Dirofilariosis
andAngiosorespiratory
parametersin
dogs
treatedwithmelarsomine.
※AT:
加速時間、ET:
駆出時間、TRV:
三尖弁逆流速度
adult
bMOXI/DOXY
theofmoxidectin/doxycycline
group
vs. theorespiratory
melarsomine
group
could
be treated
4/F3-8
10/M
2-12 (mean
6.5 years)
Ejection time.
3.3. withThoracic
radiographs
6 (tot.
/M 14)(mean 5.5 years)
previousstudy
in naturally-infected dogs treated withparameters
in dogs
melarsomine.
gylosis (ESDA)
currently
includein
theirguidelines
that
in
cases
Blood
were
collected
once
a
month
for
nine
monthsandthe sMelarsomine
administeredwiththethree-step
protocol.
It
has
been
cMEL (tot. 6)
6 /M
3-8 (mean 5.5
years) samples was
Tricuspide valve regurgitation velocity.
provenvs.
100.00%).
administeredwiththethree-step
protocol.
It
has
been
e treatment with melarsomine is notpossible(100.00%
or is contraindicated,
cons
then
at 12, that18, 24Melarsominemonthsfromwasallin thestudy,
apartforFour adulticide
dogs in the moxidectin/doxycycline group had an interstitial
shown
this
protocolis dogs
saferenrolled
and moreeﬀectivequesthat this groupprotocoland moreeﬀectiveadulticide
onthly macrocyclic
lactone along with doxycycline for a 4-week
from
three
dogs inshown
the melarsominethat isdidsafer
not present
at onelungor patternfor
score
of 1 at enrollment, while the remaining 10 had a
Fig2 mfのカウント
Fig3
抗原濃度
treatment
indogs
comparedtothetwo-step
protocolreported
on
the
seroc
onfromthethe beginning of treatment, one dog with aod might be considered (AHS guidelines, 2018; ESDA guidelines,
score of 0.reported
At 6 months
two control visits.treatment in dogs compared to the two-step protocol
Sava
product label, independent of the dog’s clinical statusscore(Keister
et al., had a score of 1. Of the four dogs with a scoreof 0 at enrollment
7).
productlabel,
independent
clinicalstatus(Keisteretal.,Microfilarial concentration
wasdetermined ofin the1 mldog’sof bloodwith
MOXI/DOXYfromMOXI/DOXYof 1 at enrollment, 3 improved (score of 0) while one remained 1. At
Vezzonietal.,1992).
Even
thoughthestep
washe aim of the present study was to evaluate theadulticide
eﬀect
of
1992;
Vezzonietal.,1992).
Even
though
the
three
step
protocolwasthe1992;
modified
Knott’s
test(BalboandPanichi,1968).
Thethreenumberof protocol
necro
months 12, 18 and 24, all dogs treated with moxidectin/doxycycline
MEL
MEL
mbination of moxidectin and doxycycline in dogs
naturally-infected
sitiveproposed,ownerconsent
waslimitedformelarsomine,
explaining
proposed,ownerconsent
waslimitedformelarsomine,
microfilariae(mf)wasexpressed
as
n°
of
mf
/1
ml
blood.
Serumsamhada score explainingof 0. the theandD. immitis from two endemic areas of Italy and to compare eﬃcacy
pleslow
weretested for
antigens
using
PetChek®
HTWM
lownumberofdogs
in thistreatment
It dogs
is thatlikelythat the group had a score of 0 at enrollment
Allin thethemelarsomine
number(6)circulatingof dogs(6)in
thistreatmentgroup.
Itgroup.
is PF
likelyCut-oﬀ
pens,
elarsomine.
and at 6 months. Two dogs showed a score of 1 at 12 months. At months(IDEXX), following the manufacturer’s instructions. Antigen conabsenceofpre-treatment
withdoxycycline
andamacrocyclic
lactoneabsenceofpre-treatment
withdoxycycline
andamacrocyclic
lactonewide
18 and 24 months, all dogs from the melarsomine group had a score ofcentration was also evaluated reading the plates with a spectrobefore
melarsomine
administration,asrecommended
by
the
most
rethatbefore melarsomine
administration,asrecommended
most0by(seetheTable3). rephotometer at 650cent
nm. guidelines
The cut-oﬀ of
wasdefinedas theaverage value
of
posit
the
AmericanHeartworm
Society(2018)radiographs
and of the
For thoracic
guidelines of the American Heartworm Society (2018)
and of the at 12, 18 and 24 months non-inferiority of theMaterials and methodsofsa
all cent
the negative
controls
provided
by
the
the
kits
used
EuropeanSocietyfor manufacturer
Dirofilariosis ofandAngiostrongylosis(2017),group
ledvs. the melarsomine group could be provenmoxidectin/doxycycline
In
EuropeanSocietyfor
Dirofilariosis
andAngiostrongylosis(2017),
led100.00%
duringin each antigenic
3SD. In
particular,
testing(100.00%
66.67%,
ownerstest
to session
refuse +enrollment.
Indeed, nine
as reported
byvs.Genchi
et al. vs. 100.00% and 100.00% vs. 100.00%).
grad
Study design
sessions were done and all the samples with OD values higher than theownerstorefuseenrollment.
Indeed,asreportedbyGenchietal.
(2019),
combination
protocolswithdoxycycline
and
MLs
are
becomingmont
cut-oﬀ were considered as positive. The mean OD value of all the dogs
3.4.
Cardiacultrasound(2019),
combination
protocolswithdoxycycline
and
MLs
are
becoming
one,
increasingly
popularin
Italy.
he present study evaluated the eﬃcacy of 10% w/v imidaclopridfor
each
point was considered
in order to compare
the decrease in
antig
Fig.
3.timeCirculating
antigens
evaluation
after
moxidectin/doxycycline
andmelarsomine treatments.
increasingly
popular
inofItaly.
Results
thepresent study
confirm
the adulticide
eﬀects ofmox2. Number(Advocate®,of mf/ml (averagevalue Multi®,
for positivedogs)
ad log10
Two dogs in the moxidectin/doxycycline group had a cardiac ul2.5% w/vFig.
moxidectin
Advantage
Bayer)
inexpressedbe duantigenconcentration between the two treatment groups.


3.2.2. Antigenemia
All dogs treatedtigen negative bymelarsomine becam(p.e), three at four
reports the average
doxycycline and mconsidered positive
melarsomine showe
duction in antigen
was more gradual i
For antigenemi
moxidectin/doxycy
proven (100.00% v
100.00%)3.2.1. Microfilariae
Thirteen of the f
had circulating mic
negative after 1 moof blood at day 30.
consistent reduction
month (Fig. 2). On
melarsomine had ci
three months fromquired 9 months t
expected as melaso
For microfilarae
moxidectin/doxycy
proven (100.00% v

3.2. Parasitological

Fourteen dogs
imidacloprid and 2.
with melarsomine dage and sex of dogs
an average age of 7
The group treatedaverage age of 5.5
possible to enroll
however this had n

3.1. Animals and tr

3. Results

analysis were trans
SOPs by means of t
The analysis w
Version 6.7 and Tes
software, hardware
evaluation steps we
Continuous varimean, median, stanCategorical variabl
quency and percent
The study has btistical analysis wason Bauer and Köhnof eight patients inwere now only six
Non parametricences or equality odefined for a non-i

Fig. 3. Circulating antigens evaluation after moxidectin/doxycycline and melarsomine treatments.