Detection and quantitation of Anaplasma marginale in carrier cattle by using a nucleic acid probe

0095-1137/89/020279-06$02.00/0

Copyright C 1989, American Society forMicrobiology

Detection

and

Quantitation of Anaplasma marginale in Carrier

Cattle

by Using

a

Nucleic

Acid

Probe

INGE S. ERIKS,lt* GUY H. PALMER,2t TRAVIS C. McGUIRE,3 DAVID R. ALLRED, AND ANTHONY F. BARBET'

Departments of InfectiousDiseasesland Comparative andExperimental Pathology,2 College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, and Department of Veterinary Microbiology and Pathology, College of

VeterinaryMedicine, Washington State University, Pullman, Washington

99164-70403

Received 20June 1988/Accepted 2 November 1988

Cattle which have recovered from acuteinfection withAnaplasmamarginale, a rickettsialhemoparasite of cattle,frequently remain persistently infected withalow-levelparasitemia andserve asreservoirs fordisease transmission. To fully understand the role of these carriers in disease prevalence and transmission, it is essential that low levels of parasitemiacanbeaccuratelydetectedandquantitated. We have developedanucleic acid probe, derived fromaportion ofa geneencodinga 105,000-molecular-weight surface protein, thatcan detect A. marginale-infected erythrocytes. The probe is specific for A. marginale andcan detect 0.01 ngof genomic DNA and 500 to 1,000 infected erythrocytes in 0.5 ml of blood, which isequivalenttoaparasitemia of0.000025%. This makestheprobeatleast 4,000 timesmoresensitive than lightmicroscopy. Hybridization of theprobe with treated blood from animalsproventobe carriers ofanaplasmosis showed that parasitemia levels were highly variable among carriers, ranging from >0.0025 to <0.000025%. Parasitemia levels of individual animalsondifferentdateswerealso variable.These results implythat,atanygiven time, individuals withinagroupofcattlemaydiffersignificantlyintheir abilities to transmit disease.

Anaplasma marginale is a rickettsial hemoparasite of cattle and other ruminants. The organism istransmitted via biological and mechanical vectors, such as ticks and biting flies, as well as by contaminated fomites, such as needles (23). Anaplasmosis is of economic importance worldwide andrepresentsamajor obstacleto meatand milkproduction in tropical and subtropical regions (24). A conservative estimateof the annual loss due toanaplasmosis in the U.S. aloneamountsto$100 million and includes 50,000to100,000 cattle deaths(18). The acutephase of the disease can cause

severe anemia, abortions, weight loss, and death (1). Ani-mals thatrecover from theacutephase mayremain

persis-tently infected withlow, microscopicallyundetectable levels of theorganism. These animals,knownascarriers, serve as reservoirs fordisease transmission and allow continual

out-breakstooccur (28).

Little information is available about the carrier state of anaplasmosis or the role carrier animals play in disease prevalence and transmission. This lack of knowledgestems

from the inability todetect and quantitate the low levels of parasitemia found in carrier animals. Thecurrentmethodsof detection involving serological tests arelimited in the diag-nosis of carriers by their inability to distinguish between active carriers and animals with antibodies due to prior infections (17). Thetestsalsogive noindication of levels of parasitemia present in carrieranimals. Currently, the most

reliable method of detecting carriers is by subinoculation of infectedblood into splenectomized, susceptible calves (17). However, this procedure is too costly and time-çonsuming forroutineuseandprovides little information concerning the level ofparasitemia.

Todetect andquantitateA.marginale incarriers,anassay

*Correspondingauthor.

tPresent address: Department ofVeterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State Uni-versity, Pullman, WA 99164-7040.

is needed that will accurately detect very low levels of

parasitemia. Nucleic acid probes, which hybridize only to

theircomplementarysequences, canprovideasensitiveand specific tool for diagnosis of infectious diseases (10). In addition, the intensity of the hybridization signals obtained correlates with the number of parasites, thus providing information on the level of parasitemia (5, 6, 13, 27).

Previously, we developed a DNA probe based upon a portion of the gene encoding a 105,000-molecular-weight surfaceprotein (designated 105L) ofA. marginale (4, 12).It wasdemonstrated thatthis probe could detectA. marginale in infected tick tissue and within erythrocytes (12). Detec-tion of infectederythrocytes indicated thefeasibilityofusing anucleic acidprobetoidentifyA.marginale in carrier cattle. In this report, we describe the development ofan RNA probe basedonthis genefragment and show that the RNA

probe can be used to identify and quantitate the low,

microscopically undetectable levels of parasitemia foundin

cattle proven to be carriers ofanaplasmosis for at least 3

years.

MATERIALS ANDMETHODS

Source of blood samples. Six steers were experimentally infected withA.marginale(Florida isolate)inJuly1984. All

developedanacuteinfectionandrecovered. Parasitescould

not be detected in Wright-Giemsa-stained peripheral blood

smearstakenfrom thesecattle on aweeklybasis for1 year

following recovery. Blood was obtained for hybridization

assaysinMay,July, andNovember 1987. Parasiteswerenot

detectedby microscopic examinationon anyof these dates.

Carrierstatuswas confirmed inNovember 1987by drawing 100ml of blood from each of the sixsteersand subinoculat-ing individually into splenectomized calves. Wright-Giemsa-stained peripheral blood smears from the recipient calves were examined daily until parasitemia was microscopically evident ata levelof1.0%.

Negative-control bloodwasobtained from calvesunder 4 279

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months ofageand demonstrated by splenectomytobefree ofA. marginaleorfromanadultcow proventobenegative foranaplasmosis by subinoculation ofablood sample intoa splenectomized calf.

Positive-control blood with known parasitemia was ob-tained from splenectomized calves that had been experimen-tally infected with isolates ofA. marginale from Florida; Missouri; South Idaho; Texas; Okanogan, Washington (Washington-O); and St. Croix, U.S. Virgin Islands. The origins and characterizations of the Florida, South Idaho, Texas, and Washington-O isolates have been previously described (19). The St. Croix and Missouri isolates were provided by Gerald Buening (Department of Veterinary

Microbiology, University of Missouri-Columbia).

All of the above blood sampleswere washed three times

with phosphate-bufferedsaline (PBS) (0.137 M NaCl, 10 mM Na2HPO4, 3.2 mM KH2PO4) to remove plasma and buffy

coat. They were then either diluted with PBS to provide knownconcentrations of erythrocytesordiluted with sterile

waterandfurther depleted of leukocytes byuseof Whatman CF11 cellulose (Whatman, Inc., Clifton, N.J.) columns (2). All samples were stored at -70°C withoutcryoprotectant.

In vitro cultures of bovine erythrocytes infected with Babesia bovis andBabesia bigeminawereprovided by Terry

McElwain and Steve Hines (Department of Infectious

Dis-eases, University of Florida, Gainesville). Blood from rats

infected with Trypanosoma bruceiwas provided by Sondra Kamper (Department of Infectious Diseases, University of Florida, Gainesville).

Probespecificity. Individual volumes of blood containing5 X 108B. bovis- andB. bigemina-infected erythrocytes and 5

x 108T. brucei organismswereeach addedto 1mlof PBS.

Avolume of blood containing 5 X

108

A. marginale-infected erythrocytes added to 1 ml of PBS was used as a positive control. These samples were treated with sodium dodecyl sulfate and proteinase K by previously described methods (12). The DNAwasdenatured by using NaOH (final

concen-tration, 0.4 N). Tenfold serial dilutions of each samplewere made, and 10 ,I of each dilution, containing from 106to102 T. brucei organisms and from 106 to 102 B. bovis-, B.

bigemina-,orA.marginale-infected erythrocytes,were blot-ted directly onto charged nylon filters (Zetaprobe; Bio-Rad Laboratories, Richmond, Calif.). Filters werebakedfor 2 h

at 80°C ina vacuum oven.

Probereactivity with different A. marginale isolates. PBS-washed blood from calves infected with Florida, Missouri, SouthIdaho, Texas, and Washington-O isolates ofA. mar-ginale was frozen and thawed two times to lysethe

eryth-rocytes.Bloodcontaining 5 x

107

infectederythrocyteswas addedto 1 mlof PBS. Sampleswere treated with SDS and proteinase K by previously described methods (12). A5-,I sample of each dilution, containing from

105

to102 infected erythrocytes, was spotted directly onto nitrocellulose (Schleicher & Schuell, Inc., Keene, N.H.), and the filters were baked for 2 hat80°C in avacuum oven.

Probe sensitivity. To determine the lowest percentage of infectederythrocytes that could be detected, serial dilutions weremade of known numbers oferythrocytes infectedwith the Florida isolate ofA. marginale. The infected

erythro-cytes were added to 0.5-ml samples of uninfected control blood that had been buffy coatdepleted and restored to a final concentration of 8 x 109 erythrocytes per ml. This

resulted in levels of parasitemia ranging from 0.0025 to

0.00000125%. DNAwasextractedasfollows.Sampleswere washed three times in PBS by centrifugation (30,000X g)to

removehemoglobin and suspended in 500RIof Tris-sodium

chloride buffer

containing

1% SDS and 0.5 mg oflysozyme

per ml.

Samples

wereincubatedat

37°C

for 1 h. Proteinase K

was added to a concentration of 200

,ug/ml,

and

samples

were incubated

overnight

at

37°C.

To ensure

complete

digestion

of

proteins

inthis

larger

volumeof

blood,

protein-aseKwas

again

addedtomakeafinalconcentration of 400

,ug/ml, andsamples were furtherincubatedat

60°C

for 1 h.

Samples

were transferred to siliconized

Microfuge

tubes

(Beckman Instruments,

Inc.,

Fullerton, Calif.)

and

depro-teinized

by using phenol

and

phenol-chloroform.

NaOHwas

addedto afinalconcentrationof 0.4

N,

and the

samples

were

incubated at

37°C

for 1 h to denature the DNA. The denatured

samples

were

applied

to a

charged nylon

mem-brane

(Zetaprobe) by using

a dot blot manifold

(Hybri-Dot

Manifold;

Bethesda Research

Laboratories, Gaithersburg,

Md.).

To determine the lowest amount of

purified genomic

A.

marginale

DNA that could be

detected,

DNAwas isolated

by previously

described methods

(4)

from blood infected

with A.

marginale (Florida isolate)

at

approximately

50%

parasitemia.

Purified DNA was denatured

by using

0.1

volume of 1 N

NaOH,

and 10-fold serial dilutions were

made.A

10-1tl

sample

of each

dilution,

containing

from 102

to10-6ngof

DNA,

was

spotted

directly

onto

nitrocellulose,

and thefilterswerebakedfor2hat

80°C

ina vacuum oven.

Detectionof

ascending parasitemia.

The

probe

was

exam-ined for its

ability

todetect

ascending

levelsof

parasitemia

following

initial

challenge.

A

susceptible,

splenectomized

calfwasinoculated

intramuscularly

withavolume ofblood

containing 1010

A.

marginale-infected erythrocytes

(Wash-ington-O isolate).

Samples

of bloodweretaken from thiscalf

before

infection,

atthetime of

inoculation,

and

daily

there-after until

parasitemia

was

microscopically

detectable at a

level of

0.1%

in

Wright-Giemsa-stained peripheral blood

smears.Blood

samples

were

buffy

coat

depleted,

suspended

inPBSto known

erythrocyte

concentrations,

and frozen at

-20°C.

Avolumeof blood

containing

4 x

109

erythrocytes

wastakenfromeach

sample,

and theDNAwasextracted

by

themethodsdescribed above.DNAwasdenatured

by

using

NaOH

(final concentration,

0.4

N),

and the samples were

applied

to

charged nylon

membranes

(Zetaprobe) by

using

a

dotblot manifold

(Hybri-Dot).

Detection of carrier

cattle.

Blood

samples

were taken on

threedifferentdatesfrom eachofthesix carrier

animals

and

uninfected controls. Bloodwas washed in

PBS,

suspended

to afinal concentration of

approximately

8 x 109

erythro-cytes per ml, and frozen and thawed two times to

lyse

erythrocytes.

Samples (0.5 ml) of

washedbloodwereadded

to

PBS,

and DNAwasextracted and

applied

to

nylon

filters

asdescribed above.

RNAprobe

preparation

and

hybridization.

Plasmid DNA

containing

a

previously

describedA.

marginale

gene insert

(pAM97) (4)

was

digested

with

SstI,

anda

2.0-kilobase

(kb)

fragment

was isolated on a

low-melting-point

agarose

gel

(SeaPlaque Agarose;

FMC

Bioproducts,

Rockland,

Me.).

The

fragment

was

ligated

in agarose into the SacI site of

dephosphorylated

plasmid pGEM-3 (Promega Biotec,

Mad-ison,

Wis.). By using

this construct, Escherichia coliHB101

cellsweretransformedto

ampicillin

resistance

(14).

Purified

pGEM-3

DNA

containing

the2.0-kbSstI

fragment

insertof

105LA.

marginale

DNAwaslinearized

by

using

the

restric-tion enzyme

EcoRV,

which cuts close to one end of the insert DNA. The SP6 promoter was used to generate an

[ot-32P]CTP

(DuPont-NEN

Research

Products, Boston,

Mass.)-labeled

RNA

transcript

witha

specific

activity

of1 X

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108 to 2 x 108 dpm/,ug of DNA(Riboprobe Gemini System II; Promega Biotec).

Nitrocellulose filterswereusedforinitialexperiments. We later substituted nylon because of its ease of handling,

potentially increased DNA-binding capacity, and simpler

hybridization and washing conditions.

Nitrocellulose filterswere hybridized and washed as pre-viously described (12). Nylon filters were prehybridized for at least 6 h at42°C inamixture containing50% formamide, 7% SDS, 1 mM EDTA, 0.25 M NaCl, 0.25 M sodium

phosphate (pH 7.2), and 100 ,ug of sheared, denatured

herring sperm DNA per ml. Hybridization was carried out

overnightat42°C. Filterswerewashed at roomtemperature for15min each in 2x SSC (lx SSCis0.15 MNaClplus 15 mM sodium citrate, pH

7.0)-0.1%

SDS, 0.5x SSC-0.1% SDS,and 0. lx SSC-0.1%SDS. Ahigh-stringencywash was

carriedoutfor30minat60°C byusing0.1x SSC-1% SDS.

Afterbeing washed, the filters were rinsed briefly in 0.1x

SSCatroomtemperature,andautoradiographywascarried

outwithCronexLightning-Plusintensifyingscreens(E.I. du Pont de Nemours& Co., Inc., Wilmington, Del.).

RESULTS

Proof of carrier states. Six steers were proven to be

carriers ofA. marginale 3 years after initial infection and recovery by subinoculation of their bloodindividually into

six splenectomized, susceptible calves. A. marginale-in-fected erythrocytes were microscopically detectable at a

1.0%level from 15 to 32daysfollowing subinoculation into

these susceptible calves.

Specificityof the RNA probe. TheprobewasspecificforA.

marginale, as evidenced by lack ofhybridization to blood

infected withB.bovis,B. bigemina, andT. brucei,aswellas to bovine leukocyte DNA from uninfected control blood (datanot shown). Prior studies, usinga DNA probe made

fromthe sameclonedgenefragment, demonstratedalack of hybridization to Anaplasma ovis and B. bovis (12). The

reactivity ofthe probe with differentA. marginale isolates was tested. The probe reacted with approximately equal

intensity to all five U.S. isolates (Florida, Missouri, South

Idaho, Texas, and Washington-O) (Fig. 1). In addition, it wascapable of detecting an isolate fromSt. Croix (data not

shown). Thespecificity ofthe RNAprobe was identicalto thatobtained fromthe DNAprobe.

Sensitivity of the RNAprobe. The sensitivity oftheprobe

was initially determined using 10-fold serial dilutions of purified A. marginale DNA. The probe was capable of detecting0.01ngofgenomicDNA(Fig. 2).Thesensitivity of

the RNAprobewas similar to thatofthe DNA probemade

fromthe sameclonegenefragment. Whenserialdilutions of A. marginale-infected

erythrocytes

were made, the RNA

probewasable todetect 500 to 1,000infectederythrocytesin 0.5 mlofuninfected blood (Fig. 3). This is

equivalent

to a

parasitemialevelofapproximately0.000025%.If the

approx-imate genome size ofasingle rickettsial organismis assumed to be 1,500 kb (15), 0.01 ng is the equivalent of

approxi-mately 6,000

organisms.

Thus, eachparasitized

erythrocyte

contained an average of 6 to 12 A. marginale organisms.

Thisis consistent withpreviously publisheddata, wherein 1

to 16

organisms

were detectedper infected

erythrocyte

(12,

25).

Detection ofascending parasitemia. The RNAprobewould beusefulfor clinicalandepidemiologicstudiesif it iscapable

of detecting infections when they are not microscopically

evident. Asplenectomized calfwassubinoculated

intramus-105 5x4 1 104 5x103

F

103 5X102

Id. * *

:...

Mo.

Tx.

i.

l.

_-4

f.

.`_

44

i Wa.

C.

FIG. 1. Reactivity ofthe 2.0-kb probe with five different U.S. isolates ofA. marginale. Erythrocytes infected withFlorida(Fl.), Missouri (Mo.), South Idaho(Id.),Texas(Tx.), andWashington-0 (Wa.) isolates ofA. marginale were treated to lyse initial bodies, serially diluted(105,5x i04, 104, 5 x 103, 103,and5 x 10'infected erythrocytes), andappliedtonitrocellulose.Anegative control (C.), consistingofserially diluted uninfected erythrocytes,wastreatedin the same manner.

cularlywith

1010

A.marginale-infected erythrocytes.Onday

2postinfection,apositive signalwasobtainedwith the RNA

probebyusing0.5-mlblood samples(Fig. 4). Onthe basis of

hybridizationintensity,thisparasitemialevelremainedfairly

constantuntilday9

postinfection,

whenitbegantoincrease. Aparasitemialevelof 0.1%wasdetectedmicroscopicallyon

day14 postinfection.

Detection of carrier infections. Samples (0.5 ml) of blood

were taken from the six carrier animals on three separate

dates, appliedtonylonfilters,andhybridizedwith theprobe. Positivesignalswereobtained for eachanimal on at least one

occasion; animals 2 and 5 were positive on two different

dates, andanimals 1 and 4 were positive on all three dates

(Fig.3).Comparisonwith theinfectederythrocytestandards showed that the number of infected erythrocytes present washighly variable among thecarrieranimals, ranging from

>105

to <500 to 1,000 infected erythrocytes per 0.5 ml of

blood. This correspondsto parasitemia levels ranging from

>0.0025 to <0.000025%. Theparasitemia levelsofan indi-vidual animal on different dates were also highly variable.

Forexample, animal 2 had a parasitemia level of approxi-mately0.00025%in May1987;wasbelow thedetectionlimit

of 0.000025% in July 1987; and in November 1987 had a

parasitemialevel exceeding 0.0025%.

DISCUSSION

Inthisreportwe havedescribed thedevelopmentand use

ofan RNA probe to detect and quantitate the low, micro-scopically undetectable levels of parasitemia found in

chronic carriers of anaplasmosis. This probe is an RNA

transcript made from a 2.0-kb SstI fragment of a gene

encodingaportion ofan A. marginale surface protein.The

sensitivity of the probe compares very well with that of probes developed for detection of otherprocaryotic organ-isms (11, 21, 22, 26). Lightmicroscopy iscapable of

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10ng

10

t

0.1

0.01

t

FIG. 2. Sensitivity of the 2.0-kb probe with A. marginale genomic DNA. DNAwas isolated fromA. marginale-infected erythrocytes (Florida isolate), and 10-fold serial dilutionswere madeandappliedtonitrocellulose. Atright isahybridizationwith0.01 ngofDNA.

ingA. marginale parasitemii

atleast4,000timesless sens

probewasshown todetect F

priortomicroscopic detectic postinoculation requiredtoc varywith theinitial dose and

early detection of infections(

inherdoutbreaks of anaplas

The carrier state of anap

understood phenomenon. Ui

5-87

1

2

7-87 i

1 2

1 2

11-87

o5 1

Standards 4

FIG. 3. Detection of carrier carriers andnegative controls (C extractedfrom 0.5-mlsampleso

filters byusing adot-blot appa

extractedfrom knownnumbers beenserially diluted(105,5x 10 addedto0.5 mlofuninfected, ^

alevelsof0.1to0.2%, whichis to determine the levels of

parasitemia

present in carrier sitivethan the RNAprobe. The animals and whether these levels remain stableor riseand

)arasitizederythrocytes 12days fall over time. The variation seen in hybridization intensity Dn. Clearly,thenumber ofdays impliesthat, at any given time, individualswithin a group of detectaninfected animal would cattle may differ significantly in their abilities to transmit

routeofinoculation. However, disease. These results have important epidemiologic

impli-couldreducelossessignificantly cations. It is possible that below a certain level of para-mosis. sitemiaindividual cattle are no longer capable of transmitting

lasmosis is presently a poorly anaplasmosis via biological or mechanical vectors. Both ntil now, it hasbeenimpossible externaland internal factors may be capable of altering the levelofparasitemiainanindividual. For

example,

immuno-suppression of infected cattle (8), environmental or nutri-tionalstress(7),andconcurrentinfection with other diseases

3

4

5

6

C can

provoke

arecrudescence of clinical

anaplasmosis.

These factors may contribute to the fluctuations in

parasitemia

à ,

levels observed in the six carriers.

A key epidemiologic question is the importance and

prevalenceof carriers inenzooticallystableregions.Usinga

3 4 6 C test thatwilldistinguish infected animals from those thatare

immune will allow us to better define the

relationship

be-..

_tween herd

immunity

and disease transmission. This

infor-mation may allow forthe design ofa model to

predict

the

impact of control measures, including vaccination, on

en-3 4 5 6 C zooticstability (3).

To fully evaluate the role carriers play in maintaining

enzootic stability, it is necessary that the probe be able to

detect allcarrier animalsregardlessof

parasitemia

level. We

103

2 have not yetobtained this

degree

of

sensitivity.

To further

104

103

1

0

increase the

sensitivity

of thisassay,several alternativesare

available. A more

highly

reiterated sequence could be

sought,suchasthose usedtodetect Plasmodium

falciparum

(20). However, restrictionanalysisofA. marginalegenomic

cattle.Blood was

obtained

fromsix DNA has failed to reveal highly reiterated sequences

(un-')on three different dates. DNA was

fwashed blood andappliedtonylon

published

data). Another possibility is the development of a ratus. Standards consisted of DNA probe to detect rRNA (9). However, ribosomal genes are of infectederythrocytes,which had oftenhighlyconserved among similarorganisms,and ensur-4,104, 5 x103, 103, and 5 x102) and ingadequate specificitymaybe difficult. Arecentinnovation washed blood. is a polymerase chain reaction which uses athermostable

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À

DAY -1

DAY

9

DAY

14

FIG. 4. Detection of ascending parasitemia with the 2.0-kb

probe.Blood wastakendailyfromasplenectomized calfbefore(day

-1) and after subinoculation with A. marginale (Washington-O

isolate). DNA wasextracted from 0.5-mlportionsof these samples

andappliedtonylonfiltersby usingadotblotapparatus. Samples

are arranged chronologically from left to right. On day 14,

para-sitemiawas observed microscopicallyat a 0.1%level.

DNApolymeraseto specifically

amplify

thetargetsequence

of a probe. This technique, which can amplify a target sequenceup to200,000-fold, has

recently

beenmodifiedfor usein Iysedcells, withoutarequirementforisolation of the

DNA (16). By using this technique to amplify the target

sequence of the RNAprobe, we could

substantially

reduce

thetime necessaryforsample preparation while gainingthe

potential to detect even the lowest levels of parasitemia

found incarriercattle.

ACKNOWLEDGMENTS

WethankTeresa G. Harkins,SondraKamper,and Ann

Schnei-derforexcellent technicalassistance.

This work receivedsupportfromU.S. DepartmentofAgriculture grants 87-CRSR-2-3106, 85-CRCR-1-1908, 85-CRSR-2-2619,and

86-CRCR-1-2247; United States-Israel Binational Agricultural

Re-search and DevelopmentFundgrant US-846-87;andU.S. Agency

for International Development grantDAN-4178-A-00-7056-0.

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