diagnostic and clinical aspects of canine anaplasmosis and ehrlichiosis october 2007

8/16/2019 Diagnostic and Clinical Aspects of Canine Anaplasmosis and Ehrlichiosis October 2007

1/12

Diagnostic and Clinical Aspects of Canine Anaplasmosis and Ehrlichiosis October 2007 (Vol 29, No 0!COMPENDIUM ON CONTINUING EDUCATION

Source: Tierärztl Prax 2007; 35 (K):129-136. Tra!late" #$ a" re%rite" &it' %eri!!io ot'e %u#li!'er.

Summary

Objective: Prospective study on the occurrence of *'rlic'ia cai! and +a%la!a

%'a,oc$to%'ilu infections in dogs in Germany and Northern !it"er#and$ Materia# and

methods% The study covered cases of dogs in Germany and !it"er#and &north of the A#ps' that

!ere either sero#ogica##y positive &detection of *. cai! or +. %'a,oc$to%'ilu anti(odies' or

po#ymerase chain reaction &PC)' positive for *'rlic'ia spp DNA$ Anamnestic and c#inica# data!ere compi#ed (y the treating veterinarians on uniform documentation sheets$ )esu#ts% The

study covered *+* cases &anamnestic and c#inica# data (eing avai#a(#e for ,- cases'$ ./ of

these dogs !ere sero#ogica##y and PC) positive$ In contrast0 *1 dogs !ere so#e#y PC) positive

and -/ !ere so#e#y sero#ogica##y positive$ In the second part of the study0 a *12 preva#ence

of +. %'a,oc$to%'ilu anti(odies !as found !ithin -3. random#y chosen dog sera$ In contrast0

the preva#ence !as 4-2 !ithin -5* dogs origina##y re6uested for orrelia 7estern (#ot

ana#ysis$ Conc#usion and c#inica# re#evance% The ris8 for autochthon canine *. cai! infections

seems to (e #o! in Germany and Northern !it"er#and0 and main#y trave##ing or imported dogs

are affected$ In contrast0 canine anap#asmosis must (e c#assified as endemic and consideredas a diagnostic differentia# in cases of tic8 e9posure and c#inica# patho#ogy &e$g$0 arthritic

pro(#ems0 #ameness or neuro#ogic a(norma#ities'$ :or diagnostic investigation0 PC) ana#ysis is

very he#pfu# to detect fresh or reactivated infections and for therapy contro#$

Introduction

T!o forms of ehr#ichiosis are of re#evance in veterinary practices

in Germany and !it"er#andtoday% first0 infections !ith *'rlic'ia cai! &*. cai!'0 the causative

agent of canine monocytic ehr#ichiosis &CME'; second0 infections !ith +a%la!a %'a,oc$to%'ilu & +. %'a,oc$to%'ilu'0 !hich cause granu#ocytic ehr#ichiosis &CGE0 here

termed aa%la!o!i! for (etter differentiation' in the dog$


2/12

(een 8no!n as a c#assic trave#=associated infection$ >o!ever0 increasing tourism !ith dogs

and imports of dogs from Mediterranean countries increasing#y #ed to the introduction of

infected tic8s and autochthon infection in our #atitude &,0 *-'$ A#though the idea# temperature for

the deve#opment of 'i%ice%'alu! !a,uieu! #ies (et!een -+ to 4+ degrees Ce#sius &/, to ,/

degrees :ahrenheit' at high humidity0 tic8s a#so find suita(#e conditions for reproduction inhouses and 8enne#s &.0 **0 *-'$

The vector of +. %'a,oc$to%'ilu0 /xo"e! riciu! &castor (ean tic8'0 is very common

in Germanyand !it"er#and and a#so transmits (orre#iosis and tic8=(orne meningoencepha#itis$

7hi#e infections !ith +. %'a,oc$to%'ilu have on#y recent#y (een noticed in Germany &*0 -0

-+'0 cases of anap#asmoses have (een descri(ed in !it"er#and for some years in dogs and

horses as !e## as in humans &*,0 -5?-10 4-'$ This #ed to the term S&i!! e'rlic'io!i! &--'$

In Germany0 a preva#ence of *?3$*2 !as found for +. %'a,oc$to%'ilu in /xo"e! riciu! &40

*+0 *30 *.0 4/'$ In!it"er#and0 the infection rate !as (et!een +$, and *$32 &-*0 4+0 4*'$

C#inica# signs reported in (oth infections are very varia(#e and depend on the stage of i##ness

&/0 4.'$ CME usua##y cannot (e differentiated from anap#asmosis in the acute phase$ >o!ever0

even no!0 it remains a common perception that infections !ith *. cai! usua##y are more

severe than anap#asmoses &3'$ The most common #a(oratory findings of (oth infections are

throm(ocytopenia0 anemia0 and #eu8openia$ In addition0 one may often see hyperproteinemia0

hypoa#(uminemia0 and sometimes0 e#evated #iver en"ymes$ Usua##y0 the diagnosis is made (y

sero#ogic detection of specific anti(odies via immunof#uorescence anti(ody testing &I:AT' &-40

43'$

7hi#e an endemic infection is often not considered as a diagnostic differentia#0 #a(oratoryprofi#es for detection of trave#=associated infections usua##y on#y inc#ude an anti(ody test for *.

cai! (ut not for +. %'a,oc$to%'ilu$ The aim of the present study !as to ans!er the

fo##o!ing 6uestions%

• hou#d !e pay more attention to infections !ith +. %'a,oc$to%'ilu in the dog@

• 7hat ro#e does the direct0 mo#ecu#ar genetic detection of +. %'a,oc$to%'ilu *. cai!0 and +.

%lat$!(y PC) p#ay for diagnosis of fresh or reactivated infections and contro# of therapy@

Material and Methods

Study design

Part * of the study !as conducted from Apri# -++. to May -++/ in the #a(oratory AOMED

&)ado#f"e##0 Germany'$ The study inc#uded dogs either that !ere sero#ogica##y positive &i$e$0 had

anti(odies against *. cai! or +. %'a,oc$to%'ilu'0 or in !hich the agentBs DNA !as detected


3/12

in EDTA (#ood via PC)$ The (#ood samp#es su(mitted for ana#ysis !ere from dogs #iving

in Germanyand !it"er#and &north of the A#ps' that had (een presented to different

veterinarians$

The cases came from three groups%

*$ uspected cases from practices &n *1'% specific re6uest (y the treating veterinarians

to confirmru#e out the c#inica##y suspected anap#asmosisehr#ichiosis (y #a(oratory

tests$

-$ Trave#=associated infection profi#e &n 33'% re6uest (y the treating veterinarians to test

the samp#e from a trave##ing or imported dog for Mediterranean infectious diseases$

4$ uspected cases AOMED &n 4,'% PC) testing of patients !ith suspicious #a(oratory

va#ues or c#inica# signs$

The treating veterinarians fi##ed out a form documenting patient history and c#inica# data$ Do9y=cyc#ine &*+?-+ mg8g (ody !eightday divided into t!o doses per day' for three !ee8s &in case

of chronic infection for 3=/ !ee8s' !as recommended as the treatment of choice$

In part - of the study0 the seropreva#ence of anti(odies against +. %'a,oc$to%'ilu !as tested

in t!o co##ectives$ On the one hand0 -3. random#y chosen canine sera samp#es sent to the

AOMED #a(oratory in May -++/ !ere tested$ The second co##ective consisted of -5* canine

sera samp#es that had (een su(mitted (et!een :e(ruary and August -++/ (y German and

!iss veterinarians for detection of orrelia=specific anti(odies via 7estern (#ot ana#ysis

&recom*E Het$ Med$ &)IC>IA canisJ and :UOANAPAMA ph$J0 MegaCor0

>Kr(ran"0 Austria'$ The test !as performed according to the manufacturerBs instructions0 and a

titer of *%,+ or higher !as regarded as positive$

Mo#ecu#ar (io#ogic tests !ere performed as descri(ed ear#ier &*/'$ :or DNA purification for

mo#ecu#ar genetic testing of *'rlic'ia0 the Nuc#eopin=


4/12

!as e9tracted0 and the DNA !as disso#ved in .+ L# of e#ution (uffer at the end of purification$

:ive L# of purified DNA !as amp#ified in a 6uantitative rea#=time PC) via ightCyc#erJ &)oche

Diagnostics0 Pen"(erg0 Germany'$ In addition to the po#ymerase0 the reagent contained a##

necessary additives &Mi9 :ast start DNA Master )=)ev &.=TAG CAC TCA TCG TTT ACA GC=4'

&-.'$ :irst0 the po#ymerase !as activated for *+ minutes at 1.C$ This !as fo##o!ed (y 3.

cyc#es of denaturation * second each at 1.C0 . seconds annea#ing at ..C0 and *. seconds

e#ongation at 5-C$ u(se6uent#y0 me#ting curve ana#ysis !as performed0 !hich a##o!ed

ana#ysis of fragment #ength and specificity$ To test the sensitivity of the used rea#=time PC)

assays0 defined num(ers of copies of the */ r)NA genes of *. cai! and +.

%'a,oc$to%'ilu &each c#oned into a vector' !ere added to -++ L# of EDTA (#ood of a non=

infected dog$ After DNA e9traction0 di#utions of up to one copy of infectious DNA per -++ L# of

(#ood cou#d (e detected via PC)$ e6uence ana#ysis !as performed for further species typing$

The PC) products of a## positive samp#es !ere purified using the uia6uic8 Ge# E9traction=

Fit &iagen0 >i#den0 Germany' and ana#y"ed in a A


5/12

not received the do9ycyc#ine regu#ar#y$ In t!o of the seven successfu##y treated dogs0

symptoms recurred after some time; ho!ever0 no infectious DNA cou#d (e detected in their

(#ood$

The four dogs that tested positive on#y in the PC) had (een imported immediate#y (efore

presentation &4 pain0 * Greece'$ At the time of e9amination0 three of the dogs !ere youngerthan *- months and one dog !as */ months o#d$ T!o dogs sho!ed no c#inica# symptoms

!hatsoever$ The t!o dogs that did !ere free of symptoms three and five days after the start of

do9ycyc#ine therapy0 respective#y$

A#so0 in the third group of the *5 dogs that had on#y tested positive sero#ogica##y0 */ had (een

imported &*+ pain0 4 Greece0 * ici#y0 - Portuga#'$ In one case0 no history !as avai#a(#e$

Eight dogs &.+2' !ere descri(ed as symptomatic0 !hi#e the remaining eight dogs had neither

c#inica# symptoms nor hemato#ogic or c#inico=chemica# #a(oratory changes$ In t!o cases !ith

severe symptoms and a#tered #a(oratory resu#ts0 it !as reported that they had received

prophy#actic do9ycyc#ine therapy prior to (#ood samp#ing$ ymptomatic dogs sho!ed mar8edimprovement after do9ycyc#ine therapy$ In t!o dogs0 DNA of *. cai! !as found in (one

marro!; in one dog0 it !as found in #ymph node aspirate$ A## three dogs had a high *.

cai! titer of Q*%*-,+ (ut !ere free of symptoms$ To chec8 the success of therapy in the

symptomatic dog0 a PC) of the (one marro! !as performed; the resu#t !as negative$

Cases of granulocytic ehrlichiosis (infection/contact ith $"

phagocytophilum#

The #a(oratory resu#ts and c#inica# data of the /+ anap#asmosis cases are presented in Ta(#e4 $ Information regarding history and c#inica# data !ere avai#a(#e for -3 of the 41 dogs that

tested positive sero#ogica##y and in the PC)$ According to the o!ners0 *, of these dogs had

never (een a(road to Mediterranean countries$ Ten of these dogs !ere from !it"er#and and

eight from Germany$ Three dogs had (een imported &- >ungary0 * pain'$ Three dogs had

accompanied their o!ner on vacations to :rance &n -' and >ungary &n *'0 respective#y0

(efore the occurrence of symptoms$ At the time of diagnosis0 *, dogs &5.2' disp#ayed

symptoms and si9 dogs !ere unremar8a(#e$ */ patients had (oth neuro#ogic and arthritic

symptoms$ In a## documented cases0 symptoms improved 6uic8#y after do9ycyc#ine therapy$

PC) from EDTA (#ood to chec8 the success of therapy in eight dogs gave negative resu#ts$)ecurrence of symptoms !as not reported in any case$

>istory and c#inica# data !ere avai#a(#e for *+ of the *- dogs that !ere on#y positive in the

PC)$ According to these0 nine of the dogs had never (een outside Germany &n .' or

!it"er#and &n 3'$ One dog had (een on a vacation trip to Ita#y (efore occurrence of

symptoms$ Nine out of ten dogs had neuro#ogic symptoms; three had additiona# arthritic

symptoms$ A## documented cases sho!ed fast improvement of symptoms after do9ycyc#ine

http://www.vetlearn.com/compendium/diagnostic-and-clinical-aspects-of-canine-anaplasmosis-and-ehrlichiosishttp://www.vetlearn.com/compendium/diagnostic-and-clinical-aspects-of-canine-anaplasmosis-and-ehrlichiosishttp://www.vetlearn.com/compendium/diagnostic-and-clinical-aspects-of-canine-anaplasmosis-and-ehrlichiosishttp://www.vetlearn.com/compendium/diagnostic-and-clinical-aspects-of-canine-anaplasmosis-and-ehrlichiosis


6/12

therapy$ In three cases0 PC) of EDTA (#ood !as performed to chec8 the success of therapy0 a##

!ith negative resu#ts$

A#so0 in the third group of nine on#y sero#ogica##y positive dogs0 seven dogs had never (een

a(road in Mediterranean countries &3 !it"er#and0 4 Germany'$ T!o dogs had accompanied

their o!ners on vacations to >ungary and outhern :rance0 respective#y$ Three dogs had

reported#y (een suffering from recurrent epi#eptic sei"ures for months or years0 respective#y0

!hich disappeared after do9ycyc#ine therapy$ evere Roint disease !as reported in eight dogs;

three of these a#so disp#ayed neuro#ogic symptoms$ i9 dogs a#so in this group sho!ed

improvement after do9ycyc#ine therapy; ho!ever0 in some cases0 success of therapy !as on#y

visi(#e after more than three !ee8s$ :ive of the nine sero#ogica##y positive dogs origina##y had

the presumptive diagnosis of (orre#iosis0 !ith no orrelia=specific anti(odies detecta(#e in

7estern (#ot ana#ysis in four of these five patients$

$nother diagnosed form of ehrlichiosis (infection ith $" platys#

In three of the sero#ogica##y unremar8a(#e dogs0 PC) !as positive$ e6uence ana#ysis of the

amp#ified PC) product diagnosed an infection !ith +. %lat$!$ T!o of these dogs originated from

a(road &Tur8ey and outh America0 respective#y'$ The third dog sho!ed severe symptoms

after a vacation in pain$ A## dogs had anemia and #eu8openia in addition to a mar8ed

throm(ocytopenia$ The reports #isted the c#inica# symptoms as !eight #oss &-'0 #ethargy &4'0

apathy &4'0 anore9ia &-'0 fever &-'0 and neuro#ogic &-' and arthritic symptoms &-'$

Seroprevalence of antibodies against $" phagocytophilum

Part - of this study dea#t !ith the re#evance of autochthon infections !ith +.

%'a,oc$to%'ilu in the dog in Germany and !it"er#and$ To date0 #itt#e data have (een

pu(#ished regarding the seropreva#ence of +. %'a,oc$to%'ilu in the dog &*0 -0 4-'$ Therefore0

!e tested t!o groups of dogs$

In the first group0 -3. random#y se#ected dog sera su(mitted to the #a(oratory in May -++/

!ere tested for anti(odies via I:AT$ The test resu#ts are sho!n in :igure * $ C#inico=chemica#

and hemato#ogic parameters !ere avai#a(#e for -1 of the 35 sero#ogica##y positive dogs$


7/12

are sho!n in:igure - $ 7hi#e no significant concentration of orrelia=specific anti(odies cou#d

(e detected in *,5 of these sera0 5+ &452' of these !ere positive in the +.

%'a,oc$to%'ilu I:AT$ C#inico=chemica# and hemato#ogic parameters !ere avai#a(#e for *, of a

tota# of ,5 sero#ogica##y positive dogs$ :ive of these !ou#d have (een graded as suspicious

(ased on these #a(oratory resu#ts$ In eight dogs0 direct detection via PC) !as re6uestedafter!ard0 and the resu#t !as positive in three cases$ In three dogs from this co##ective0 DNA

of +. %'a,oc$to%'ilu !as detecta(#e in synovia$ A## three dogs had severe arthritic sym=

ptoms$

%iscussion

It !as the aim of the present study to gather information regarding the re#evance of

anap#asmoses and ehr#ichioses in dogs in Germany and the northern cantons of !it"er#and$ A

tota# of *+* cases !ere inc#uded in the study over a period of *4 months$ In addition to /4c#inica##y suspected cases0 at the re6uest of the treating veterinarian for sero#ogic tests or direct

detection of pathogen (y PC)0 PC) !as performed in 4, cases suspected (ecause of

#a(oratory test resu#ts$

On the one hand0 the resu#ts support ear#ier findings that autochthon infections of dogs !ith *.

cai! p#ay no ro#e in our #atitudes &,0 **0 *-0 */'$ A## 4/ cases that had a 8no!n history !ere

imported dogs from Mediterranean countries or dogs that had (een diagnosed short#y after a

vacation in a Mediterranean country$ On the other hand0 43 of the 34 anap#asmosis cases !ith

a 8no!n history had never #eft Germany or !it"er#and0 according to their o!ners$ In contrast

to the typica# import infection !ith *. cai!0 anap#asmosis seems to (e an indigenous infection$C#inica# and #a(oratory data of these cases as !e## as a seropreva#ence of *12 in random#y

se#ected dogs are a c#ear indication that infections !ith +. %'a,oc$to%'ilu are of significant

re#evance in veterinary practice$ ystematic epidemio#ogic investigations are needed to c#arify

!here and to !hat degree +. %'a,oc$to%'ilu appears autochthon in Germany and

!it"er#and$


8/12

detecta(#e in the (#ood; ho!ever0 in three patients0 it !as found in the synovia$ In tota#0 --

&/*2' of the descri(ed dogs !ith anap#asmosis had (oth neuro#ogic and arthritic symptoms$

:or three reasons0 it is sti## very difficu#t to ma8e a definitive diagnosis of an infection

!ith *'rlic'ia$ :irst0 microscopic eva#uation of a (#ood smear on#y rare#y gives morpho#ogic

c#ues$ econd0 seroconversion can (e much de#ayed or remain a(sent$ Third0 severa#

infectious agents hard#y sho! any cross=reactivity in I:AT$ et sero#ogic testing for *. cai! ?

or +$ %'a,oc$to%'ilu ?specific anti(odies remains the most common#y used diagnostic

method &-3'$ >o!ever0 in many cases0 it seems 6uestiona(#e to assume the presence of an

infectious agent (ased on anti(ody detection and to use this as (asis for therapy$

Direct detection of the infectious agent !ith the high#y sensitive mo#ecu#ar genetic method

descri(ed herein offers the advantage that an acute infection can (e differentiated from a

possi(#e sero#ogic scar &1'$ :urthermore0 DNA of three re#evant *'rlic'ia species is tested in

a sing#e method$ In addition to *. cai! and +. %'a,oc$to%'ilu0 +. %lat$!0 the agent of the

infectious canine cyc#ic throm(ocytopenia0 is a#so detected$ This disease occurs !or#d!ide in

!arm c#imate "ones$ In European countries0 it is thought to occur in Greece &*5'0 :rance0

pain &44'0 and outhern Ita#y &-/'$ Therefore0 the ris8 for infection must (e considered !hen

trave##ing to these countries or importing dogs from them$

There are severa# #imitations to the standard use of PC) in the diagnosis of ehr#ichiosis$ These

are0 in particu#ar0 the ris8 for fa#se=positive resu#ts due to contamination0 high costs0 and time

re6uired as !e## as #imited practica#ity for routine use$ >o!ever0 none of these #imitations

app#ies to the rea#=time PC) method esta(#ished (y us &*/'$ It is high#y specific and very

sensitive and ta8es on#y t!o hours in an emergency to e9tract the DNA from the (#ood andinvestigate via PC)$ The c#osed system of rea#=time PC) reduces the ris8 for contamination to

a minimum$

Conclusions for practice

As a resu#t of the presented data0 the fo##o!ing recommendations can (e made for diagnosis%

PC) detection of *'rlic'ia DNA from EDTA (#ood is the #a(oratory method of choice for

diagnosis of ehr#ichiosis or anap#asmosis in the ear#y or acute phase or in reactivated

infections and to chec8 the success of therapy$

Detection of specific anti(odies against *. cai! or +. %'a,oc$to%'ilu may (e usefu# in

asymptomatic dogs &e$g$0 imported anima#s' to e9c#ude past contact !ith the agent or a #atent

infection$ ince the agent is often no #onger detecta(#e in chronic infections0 additiona#

sero#ogic tests are necessary in cases of suspicion$ A#ternative#y0 direct testing for the agent in

the (one marro!0 #ymph node aspirate0 or synovia is advisa(#e$


9/12

A com(ination of PC) and sero#ogic detection methods0 together !ith hemato#ogic and

chemica# #a(oratory tests0 is the safest !ay to ma8e a diagnosis$

In case of tic8 infestation and arthritic or neuro#ogic symptoms0 anap#asmosis shou#d (e

considered as a diagnostic differentia# in our #atitudes$

$c&noledgments

7e than8 Mrs$ A$ >ahmann=M##er0 Mrs$ $ arte#t and Dr$ )ainer Oehme

from the andesgesundheitsamt in tuttgart for performing the DNA se6uencing$ 7e a#so

than8 Dr$ F$ )ohner &C>=Niederg#att' and Dr$ P$ Enge#hardt &Tier8#ini8 >ofheim0 D=>ofheim am

Taunus' for critica# reading of the manuscript$ 7e than8 MegaCor for financia# support$ Part of

the materia# for this study !as financed (y pri"e money from the Dr$=Ernst=:orschner=

GedSchtnispreis -++. of the Ar(eits8reises fr veterinSrmedi"inische Infe8tionsdiagnosti8

&AHID' der DHG &German Heterinary :oundation'$

REFERENCES

$


10/12

,$ Dongus >0 ah#er M0 Gothe )$ Die unde"ec8e0 'i%ice%'alu!

!a,uieu! &I9odidae'0 in Deutsch#and% eine epidemio#ogische tudie und

unde in Deutsch#and% ur Epidemio#ogie0

Diagnose0 Therapie und Prophy#a9e$ Tierärztl Prax *11,; -/ &F'% 41/?3+*$

*-$ Gothe )$ um Hor8ommen von 'i%ice%'alu! !a,uieu! &atrei##e *,+/' in

Deutsch#and$ Tro%ee" Para!itol *1/,; *1 &4'% 4+.?4+5$

*4$ Groves MG0 Dennis G0 Amy9 >0 >u9so## D$ Transmission of *'rlic'ia cai! to dogs (y

tic8s &'i%ice%'alu! !a,uieu!'$ + 4 et e! *15.; 4/ &5'% 145?13+$

*3$ >arte#t F0 Oehme )0 :ran8 >0 ass#er D0 Fimmig P$ Pathogens and

sym(ionts in tic8s% preva#ence of +a%la!a %'a,oc$to%'ilu &*'rlic'ia !%$'0 ol#ac'ia

!%$0 ic$ Mo#ecu#ar evidence of

coinfection of tic8s !ith orrelia #ur,"oreri sensu #ato and the human granu#ocytic ehr#ichiosis

agent in !it"er#and$ 4 li icro#iol *111; 45 &*+'% 441+?441*$

*1$ e!is GE0 Vr$0 )istic M0 mith )D0 inco#n T0 tephenson E>$ The (ro!n dog

tic8'i%ice%'alu! !a,uieu! and the dog as e9perimenta# hosts of *'rlic'ia cai!$ + 4 et

e!*155; 4, &*-'% *1.4?*1..$


11/12

-+$ ie(isch G0 Thiet 70 ie(isch A$ Die canine mono"ytSre &CME' und die canine

granu#o"ytSre Ehr#ichiose &CGE'0 "!ei durch ec8en (ertragene Infe8tions8ran8heiten (ei

>unden in Deutsch#and$ Pra$ Er#ichiose in der ch!ei" ?

$ Granu#ocytic ehr#ichiosis

in t!o dogs in !it"er#and$ 4 li icro#iol *115; 4. &1'% -4+5?-4+1$

-1$ Puster#a N0 >uder Vuder V


12/12

4-$ Puster#a N0 Puster#a Vegarty

diagnostic and clinical aspects of canine anaplasmosis and ehrlichiosis october 2007

Documents