Cloned DNA Probes Identify Anaplasma ovis in Goatsand Reveal a High Prevalence of Infection
SANKALE SHOMPOLElt* SURYAKANT D. WAGHELA,l FRED R. RURANGIRWA, 12AND TRAVIS C. McGUIRE2
Immunology Section, Veterinary Research Laboratory Kabete, P.O. Kabete, Kenya,' and Department ofVeterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University,
Pullman, Washington 99164 704O2
Received 22 December 1988/Accepted 28 August 1989
Anaplasma organisms are observed in erythrocytes from goats with anemia and weight loss in Kenya. Threeanaplasmas have been isolated in nature, Anaplasma ovis, Anaplasma marginale, and Anaplasma centrale. Thetwo recognized species, A. ovis and A. marginale, are known to infect goats. Since only A. ovis causes clinicaldisease in goats, the Anaplasma species in goats in Kenya were identified. To detect A. ovis, a 9.6-kilobase-pairsection of genomic DNA was cloned into pBR322 (pAO12A) and was used in conjunction with an A. marginaleDNA probe previously derived from a gene coding for a 105,000-molecular-weight surface protein (Am1O5L)ofA. marginale. In Southern blots, pAO12A DNA hybridized to several at least partially homologous sequences
that were present in A. ovis and A. marginale genomic DNAs. The pAO12A DNA did not hybridize to Babesiabovis genomic or goat leukocyte DNA. The Anaplasma species that infected goats was identified as A. ovis by(i) DNA hybridization with pAO12A, (ià) hybridization of the A. marginale DNA probe to A. centrale and A.marginale genomic DNAs and lack of hybridization to A. ovis genomic DNA from an isolate obtained in Idahoand Anaplasma DNA from infected goats in Kenya, (iii) the intraerythrocytic location ofAnaplasma organismsin infected goat blood, and (iv) the host specificity of the Anaplasma organisms for goats but not for cattle. Also,by using the two Anaplasma DNA probes, the prevalence of A. ovis in goats from seven locations in Kenya was
found to range from 22 to 87%. The pAO12A DNA probe detected a 0.0035% A. ovis parasitemia in infectedblood, an improved sensitivity which is suitable for use in surveillance and epidemiological studies.
Anaplasma species are obligate intraerythrocytic para-sites in the order Rickettsiales which infect domestic andwild ruminants (3, 29). Only two Anaplasma species, Ana-plasma ovis and Anaplasma marginale, are recognized,while a third species, Anaplasma centrale, has been pro-posed (29). Anaplasma organisms are transmitted biologi-cally by ticks and mechanically by fomites and biting insects(10, 15, 26, 30, 33). Anaplasmosis occurs throughout thetropical and subtropical regions of the world, and anaplas-mosis caused by A. marginale is a constraint to improvedcattle production in developing countries (14). The impor-tance of anaplasmosis in small ruminants in Kenya is notknown. However, Anaplasma organisms have been ob-served in goats with anemia and weight loss from Transmara(Rift Valley Province) and other sheep- and goat-rearingregions in Kenya (22). Since in goats, the two recognizedAnaplasma species cause parasitemia (29) and only A. oviscauses clinical disease (13, 18), an effort was made toidentify and determine the prevalence ofAnaplasma speciesin goats in Kenya.
In other parts of the world, A. ovis causes severe diseasein sheep and goats (13, 18, 32). A. ovis infections of goats arecharacterized by an incubation period of 8 to 40 days (13,23). In acute infections, debility, depression, anorexia, fe-ver, dyspnea, and progressive anemia develop and may befollowed by death (13, 18). The severity of the disease varieswith the age, breed, and nutritional status of the infectedgoats. Goats that have recovered from A. ovis disease
* Corresponding author.t Present address: Department of Veterinary Microbiology and
Pathology, College of Veterinary Medicine, Washington State Uni-versity, Pullman, WA 99164-7040.
remain persistently infected and are reservoirs of the organ-isms (13, 32).A. ovis is defined as a distinct species from A. marginale
based on the intraerythrocytic location of inclusion bodiesand host specificity (13, 32). A. ovis inclusion bodies havebeen found to be 60 to 65% marginal and 35 to 40%submarginal or central (13, 23, 32). In comparison, those ofA. marginale have been found to be 90% marginal and 10%submarginal or central (23, 29). A. centrale was first de-scribed by Theiler in 1912 as a variant of A. marginale (29).Since then, it has been used in many parts of the world toimmunize cattle against A. marginale (25). Inoculation ofcattle with A. ovis does not cause clinical disease, but bloodfrom inoculated cattle causes severe anaplasmosis in sheep(17). A. marginale causes a severe disease in cattle (14), butinoculation of goats or sheep causes parasitemia withoutclinical disease (16, 29). A. centrale infects cattle, causing amild or, sometimes, a severe clinical disease (29).
Diagnosis of anaplasmosis is based on detection of Ana-plasma inclusion bodies in blood smears, subinoculation ofblood into splenectomized or susceptible animals, and de-tection in serum of antibodies to Anaplasma antigens. De-tection of intraerythrocytic Anaplasma inclusion bodies inblood smears works well when parasitemias exceed 0.1 to0.2%. However, the Anaplasma organisms lack distinguish-ing morphological characteristics, and in low-level para-sitemias they cannot be differentiated from Howell-Jollybodies. Inoculation of blood into splenectomized animals isan effective way to detect Anaplasma organisms, but itcannot be used as a routine procedure because it is expen-sive and time-consuming. Serological diagnosis of anaplas-mosis suffers from the inability to differentiate between A.marginale and A. ovis because of cross-reactivity (32) and
2730
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/j
cm o
n 16
Feb
ruar
y 20
22 b
y 17
7.10
.240
.252
.
DETECTION OF ANAPLASMA OVIS IN GOATS 2731
the inability to differentiate active from persistent infections(11). To overcome these diagnostic problems and to inves-tigate anaplasmosis in goats in Kenya, development of animproved diagnostic method was necessary. Nucleic acidhybridization, a specific and sensitive procedure for theidentification of infectious agents, was used to detect A.marginale, Babesia bovis, and the organisms that causemalaria in blood (5, 7, 21, 24).
In this study a sensitive DNA probe to detect A. ovis ininfected blood was developed. By using differential DNAhybridization with A. ovis and A. marginale DNA probes,the Anaplasma species infecting goats in several locations inKenya was identified as A. ovis. This identification wasfurther confirmed by the intraerythrocytic location and hostspecificity of the Anaplasma isolates. Based on DNA probeanalysis, the prevalence of A. ovis infections ranged from 22to 87% in goats from seven sample locations- in Kenya:
MATERIALS AND METHODS
Isolation of A. ovis organisms. An A. ovis isolate fromIdaho was kindly provided by K. L. Kuttler (HemoparasiticDiseases Research Unit, Agricultural Research Service,U.S. Department of Agriculture, Pullman, Wash.). Thestabilate was inoculated into a splenectomized goat, para-sitemia was monitored by daily examination of bloodsmears, and blood was taken when the parasitemia reached21%. One hundred milliliters of parasitemic blood waswashed three times with phosphate-buffered saline (PBS;0.01 M phosphate, 0.14 M NaCI [pH 7]), and the buffy coatwas removed at each wash. The washed blood was freeze-thawed to lyse the erythrocytes and centrifuged at 19,000 xg for 20 min at 4°C to pellet A. ovis initial bodies anderythrocyte membranes. Hemoglobin was removed bywashing the pellet three times in PBS. A. ovis DNA wasisolated by a previously described procedure (1).
Preparation of recombinant DNA probes. (i) The A. mar-ginale DNA probe was a 2-kilobase-pair portion of a geneencoding a 105,000-molecular-weight surface protein(Am1O5L) of A. marginale (1, 7). (ii) An A. ovis DNA probewas made by cloning genomic DNA fragments and selectinga clone which reacted strongly with an A. ovis genomic DNAprobe, possibly because the genome contained multiplecopies of the cloned gene.A. ovis DNA (1 ixg) was partially digested with Sau3A,
and the fragments were ligated to 200 ng of BamHI-cleavedand dephosphorylated pBR322 by using T4 DNA ligase (19).Escherichia coli HB101 cells were transformed with therecombinant plasmids by a high-efficiency transformationprocedure (9). Colonies resistant to ampicillin were selectedon yeast-tryptone medium (0.8% tryptone, 0.8% yeast ex-tract, 0.5% NaCl, and 1.5% Bacto-Agar [Difco Laboratories,Detroit, Mich.]) containing ampicillin (100 ,ug/ml). The A.ovis genomic library was screened by colony hybridization(8) with A. ovis genomic DNA labeled with biotin-7-dATP bynick translation (28). After hybridization, the nitrocellulosefilters were processed for color development as recom-mended in the kit (BlueGene; Bethesda Research Laborato-ries, Inc., Gaithersburg, Md.), which included incubation ofthe nitrocellulose paper with streptavidin-alkaline phos-phatase conjugate for 10 min, followed by incubation with afreshly prepared dye solution of Nitro Blue Tetrazolium and5-bromo-4-chloro-3-indolylphosphate in a sealed bag. Colordevelopment was allowed to proceed in the dark for 30 minat room temperature. Bacterial colonies with the strongestcolor reaction were selected.
Selected individual colonies were amplified overnight at37°C in yeast-tryptone medium. Plasmid DNA was extractedby alkaline lysis (4); treated with the restriction endonucle-ase enzymes Aval, BamHI, and ClaI; and electrophoresed ina 0.6% agarose gel to determine the size of the DNA inserts(19). The clone with the largest A. ovis DNA insert wasanalyzed to determine whether multiple copies of the clonedfragment7were present in the A. ovis genome. A. ovis and A.'marginale genomic DNA were digested with SstI and elec-trophoresed on 0.8% agarose. Goat leukocyte DNA wasused as a control. The DNA fragments were transferred tonitrocellulose paper (31) and probed with the biotin-labeledcloned A. ovis DNA probe.Probe specificity and sensitivity. The cloned A. ovis DNA
was labeled with biotin-7-dATP and used in hybridizationreactions with genomic A. ovis, A. marginale, B. bovis, andgoat leukocyte DNAs. Tenfold serial dilutions (100 ng to 1pg) of the various DNA samples were prepared in TE buffer(10 mM Tris hydrochloride, 1 mM EDTA [pH 8]) anddenatured by the addition of 0.3 M NaOH for 5 min at roomtemperature. An equal volume of 1 M ammonium acetatewas added for neutralization. The denatured DNA sampleswere spotted onto a nitrocellulose membrane by using adot-blot manifold (Hybri-Dot Manifold; Bethesda ResearchLaboratories) and baked for 2 h at 80°C in a vacuum oven.Hybridization conditions were those described previously(12). In addition, the specificities of 32P-labeled A. ovis andA. marginale cloned probes were tested against A. centrale,A. ovis, and A. marginale DNAs.To determine the lowest percentage of infected erythro-
cytes that could be detected by hybridization, blood infectedwith an isolate of A. ovis from Idaho was adjusted to apacked cell volume of 42% and a parasitemia level of 3.5%.This adjusted blood was diluted in 10-fold serial dilutionswith normal blood (42% packed cell volume) to obtain aparasitemia range of 3.5 x 100 to 3.5 x 10-6%. Each dilutionwas washed three times by removing the buffy coat eachtime, and the packed cells were frozen. After freezing andthawing three times, 500 ,ul of the packed cells was taken andwashed three times with sterile PBS at 19,000 x g for 20 minat 4°C to remove hemoglobin. DNA was extracted from 250,ul of lysed and packed cells as described previously (2) andevaluated by hybridization.
Identification ofAnaplasma organisms from goats in Kenya.Anaplasma isolates from goats were obtained from Trans-mara, Kiserian, Ngong, and Magadi, all of which are locatedin the Rift Valley Province of Kenya and all have differentecosystems. The Anaplasma parasites were characterizedby their reactivities to DNA probes, intraerythrocytic loca-tions of inclusion bodies, and host specificities.
(i) Reactivities to DNA probes. To characterize the Ana-plasma organisms infecting goats, DNA was extracted fromAnaplasma isolates from goats and A. marginale isolatesfrom cattle. Tenfold serial dilutions of the DNA (100 ng to 1pg) were denatured and spotted onto nitrocellulose paper forhybridization to the cloned A. ovis DNA probe and to an A.marginale DNA probe.
(ii) Intraerythrocytic locations ofAnaplasma parasites. Thinblood smears from four Anaplasma isolates from goats andfour A. marginale isolates from cattle were stained withGiemsa. The location of the intraerythrocytic inclusionbodies for each isolate was determined as marginal, submar-ginal, or central; and the data were analyzed by using theunpaired t test.
(iii) Host specificities. To determine the host specificities ofAnaplasma isolates from goats, 15 goats from Naivasha (Rift
VOL. 27, 1989
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/j
cm o
n 16
Feb
ruar
y 20
22 b
y 17
7.10
.240
.252
.
2732 SHOMPOLE ET AL.
b-4 4 M bq m M m m m igX 0 CI 0 ; Z ZM k Î 0 meiS, \~~~~~~
1 2 3 4 5 6 7 8 9Kilobase pairs
H
1.104c
1
1 2 3 kb
!Is -23.0-9.5-6.5- 4.310 il
FIG. 1. Restriction endonuclease map of the 9.6-kilobase-pair A.ovis DNA cloned into the BamHI site of pBR322 (pAO12A). ThepBR322 ClaI site is 5' and the pBR322 AvaI site is 3' to the BamHIcloning site. The shaded area is PBR322 DNA.
Valley Province, Kenya) and 6 calves from Kabete (CentralProvince, Kenya) were selected and screened for Ana-plasma parasites by using cloned A. ovis and A. marginaleDNA probes. Thereafter, the goats were treated with long-acting oxytetracycline (Terramycin/LA.; Pfizer LaboratoriesLtd., Inc., New York, N.Y.), at 30 mg/kg of body weight, toeliminate Anaplasma infections (27); an anthelminthic (AI-bendazole [10%; wt/vol]; Smith Kline Animal Health Ltd.,London, United Kingdom), to reduce worm burden; andamprolium (Amprolium [20%; wt/vol]; Merck Sharp &Dohme, Rahway, N.J.), to control coccidia infestations. Ofthe 15 goats, 12 were selected for inoculation with Ana-plasma isolates from goats. Each of three Anaplasma iso-lates from goats were inoculated into four goats and twocalves 23 days after the last treatment with oxytetracycline.Parasitemia was monitored by using the cloned A. ovis DNAprobe and by examination of Giemsa-stained blood smears.
Prevalence of Anaplasma in goats. The prevalence of Ana-plasma organisms infecting goats was determined at sevenmajor goat-rearing locations in Kenya. Blood samples wereobtained from 98 goats from Maseno (Western Province), 16from Ngong, 15 from Naivasha, 111 from Subukia, 139 fromRumuruti, and 38 from Magadi, all of which are in the RiftValley Province, Kenya; 22 blood samples were obtainedfrom goats in Ukunda (Coast Province, Kenya). For evalu-ation, 10 ml of blood was washed three times with sterilePBS, and the buffy coat was removed with each wash. Onemilliliter of packed erythrocytes was frozen, and after it wasfreeze-thawed three times, it was washed three times withsterile PBS at 10,000 x g for 30 min at 4°C to removehemoglobin. Parasite DNA was extracted as described pre-viously (2) and suspended in 20 ,ul of TE buffer. Tenmicroliters of each DNA sample was spotted onto nitrocel-lulose paper for hybridization with the cloned A. ovis and A.marginale DNA probes. Blood samples from goats fromSubukia were not tested with the A. marginale DNA probe.
RESULTS
Evaluation of cloned A. ovis DNA. Ninety-four transformedE. coli colonies hybridized with various intensities to abiotin-labeled A. ovis genomic DNA probe. The bacterialcolony with the strongest reaction was isolated and ampli-fied, and the plasmid (pAO12A) DNA was purified. Analysisof pAO12A DNA with restriction endonucleases revealedthat the A. ovis DNA insert was 9.6 kilobase pairs. Arestriction endonuclease map is shown in Fig. 1. To deter-mine whether multiple copies of cloned A. ovis DNA frag-ment were present in the Anaplasma genome, biotin-labeledpAO12A DNA was hybridized to A. ovis and A. marginalegenomic DNAs digested with SstI, which did not have
FIG. 2. Southern blot of Anaplasma genomic DNAs hybridizedwith biotin-labeled pAO12A DNA. Lanes 1 and 2, SstI-restricted A.marginale and A. ovis genomic DNAs, respectively; lane 3, SstI-restricted goat leukocyte DNA. Positions of HindIII-digestedlambda DNA standards are indicated on the right, in kilobase pairs.
restriction sites in the cloned A. ovis DNA. The pAO12ADNA probe hybridized at different intensities to at least 11bands in the A. ovis genomic DNA and to only 5 bands in theA. marginale genomic DNA (Fig. 2).
Specificity of the DNA probe. The pAO12A DNA probehybridized to DNA from Anaplasma isolates from goats inKenya, to DNA from an isolate ofA. ovis from Idaho, and toDNA from an isolate of A. marginale from Kenya (Fig. 3).Also, the pAO12A DNA probe hybridized to A. centralegenomic DNA but did not hybridize to B. bovis genomicDNA (Fig. 3). A pBR322 DNA probe did not hybridize toeither A. ovis or A. marginale DNA (data not shown). TheA. marginale DNA probe (1, 7) hybridized to the DNA ofA.marginale obtained from cattle in Kenya and to the DNA ofA. centrale obtained from an experimentally infected calf inIsrael, but failed to hybridize to A. ovis DNA from an isolate
1 2 3 4 5 6
a ****
b * fs
c
d
e
g
FIG. 3. Hybridization of the 32P-labeled pAO12A DNA probe to
Anaplasma genomic DNAs. Column 1, A. avis Idaho isolate;
column 2, A. avis Kenya isolate; column 3, A. marginale; column 4,
A. centrale; column 5, B. bovis DNA; column 6, pAO12A DNA
control. DNA amounts on each row (a to g.) were 100, 10, 1, 0.1,
0.01, 0.001, and 0.0001 ng, respectively.
Nm j bd
*g
93m zc
o
-2.3-2.0
J. CLIN. MICROBIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/j
cm o
n 16
Feb
ruar
y 20
22 b
y 17
7.10
.240
.252
.
DETECTION OF ANAPLASMA OVIS IN GOATS 2733
1 2 3 4 5 6
a .
b
C
d
e
f
g
FIG. 4. Hybridization reactions of a 32P-labeled A. marginaleDNA probe with Anaplasma genomic DNAs. Column 1, A. ovisisolate from Idaho; column 2, A. ovis isolate from Kenya; column 3,A. marginale; column 4, A. centrale; column 5, B. bovis DNA;column 6, pAO12A DNA control. DNA amounts in each row (a tog) were 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 ng, respectively.
from Idaho (7) or to DNA extracted from Anaplasma iso-lates from infected goats in Kenya (Fig. 4).
Sensitivity of the DNA probe. On dot blots, the biotin-labeled pAO12A DNA probe hybridized to a minimum of 1ng of the 9.6-kilobase-pair A. ovis DNA insert frompAO12A, 10 ng of A. ovis genomic DNA, and 100 ng -of A.marginale genomic DNA (Fig. 5). When serial dilutions ofA.ovis-infected erythrocytes were made, the pAO12A DNAprobe was able to detect a parasitemia level of 0.0035%,which was equivalent to approximately 7 x 104 infectederythrocytes in 250 ,ul of blood (Fig. 5, column 7). This levelof sensitivity is higher than the 0.1 to 0.2% parasitemia levelthat is easily detectible by examination of blood smears.
Identification ofAnaplasma organisms from goats in Kenya.The Anaplasma organisms that infected goats were identi-fied as A. ovis by three characteristics.
1 2 3 4 5 6 7
a 0
b *
c ..
d
.
TABLE 1. Intraerythrocytic location of Anaplasma organismsisolated from goats and A. marginale isolates from cattle
% (mean + SD) organisms from:Anaplasma source
Marginal Submarginal Central
Goats in Kenya 73 3b 21 +2b 7+ lbA. marginale from 83 5 14 3 3 2
cattlea A total of four isolates were examined from each source.b Significantly different (P < 0.025) from A. marginale.
(i) Reactivities to DNA probes. The pAO12A DNA probehybridized to DNA from all Anaplasma isolates from goats,while the A. marginale DNA probe hybridized to DNAextracted from A. marginale and A. centrale isolates fromcattle but did not hybridize to DNA extracted from Ana-plasma isolates from infected goats (Fig. 4).
(ii) Intraerythrocytic locations of Anaplasma parasites. Theinclusion bodies of four Anaplasma isolates from goats andfour A. marginale isolates from cattle were classified intothree intraerythrocytic locations, and the mean values of thenumber of isolates from each location for each species werecompared. Significant differences (P < 0.025) were observedfor ail three intraerythrocytic locations between the Ana-plasma organisms from goats and the A. marginale isolatesfrom cattle (Table 1).
(iii) Host specificities. The pAO12A DNA probe detectedAnaplasma DNA in 13 of 15 goats tested before either drugtreatment or inoculation with the Anaplasma isolates (Fig. 6,rows 1 and 2). However, after treatment with oxytetracy-cline, all the goats were negative for Anaplasma DNA and 12were selected for inoculation with Anaplasma isolates fromgoats (Fig. 6, rows 3 and 4). At 24 days postinoculation, thepAO12A DNA probe detected Anaplasma DNA in 9 of 12goats that were positive for Anaplasma species, while only 4of 12 goats were positive for Anaplasma organisms on bloodsmear examination (Fig. 6, row 5). At the end of theexperimental period, the number of goats remaining wasnine; seven were positive for Anaplasma organisms by bloodsmear examination, while all nine were positive for Ana-plasma DNA when the DNA probe detection method wasused. The maximum levels of parasitemia observed by bloodsmear examination ranged from 1 to 3% (Table 2).
.a b c d e f 9 h i J k 1
1- *o ID O a * O O * a
2- ei
e 3-
4-
5-- * * O *
9
FIG. 5. Reaction of a biotin-labeled pAO12A DNA probe withAnaplasma genomic DNAs. Column 1, pBR322 DNA; column 2,goat leukocyte DNA; column 3, B. bovis DNA; column 4, pAO12ADNA; column 5, A. marginale DNA; column 6, A. ovis DNA;column 7, A. ovis-infected blood diluted with whole blood resultingin a parasitemia range of 3.5 x 10° to 3.5 x 10-r%. DNA amountsin each row (a to g) were 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 ng,respectively.
* *e .FIG. 6. Detection of A. ovis DNA in blood samples by using a
biotin-labeled pAO12A DNA probe. Processed blood samples fromgoats were spotted onto nitrocellulose. Rows 1 (columns a to 1) and2 (columns a to c), blood samples that were obtained before the 15goats were treated with oxytetracycline (blood samples in la and 2cwere negative for Anaplasma DNA); rows 3 (columns a to 1) and 4(columns a to b), blood samples that were obtained after 14 goatswere treated with oxytetracycline; row 5 (columns a to 1), bloodsamples that were obtained 24 days after 12 goats were inoculatedwith A. ovis isolates.
VOL. 27, 1989
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/j
cm o
n 16
Feb
ruar
y 20
22 b
y 17
7.10
.240
.252
.
2734 SHOMPOLE ET AL.
TABLE 2. Infectivity of Anaplasma isolates for calves and goats
No. infected/no. Days to 1% % MaximumAnaplasma inoculated parasitemia parasitemia
Calves Goats (range) (mean ± SD)
Kiserian 0/2 2/4 10, 19- 2, 2aNgong 0/2 4/4 19-25 3 + 2Transmara 0/2 3/4 26-34 1 + 0.5a Since only two goats were infected with the Kiserian isolate, both values
are given.
Blood samples from all six calves inoculated with the threeAnaplasma isolates from goats were screened for Ana-plasma organisms during the experimental period of 50 days.Anaplasma organisms were not detected by the pAO12ADNA probe, the A. marginale DNA probe, or examinationof blood smears.
Prevalence of Anaplasma species in goats. The pAO12ADNA probe, the A. marginale DNA probe, and blood smearexamination were used to determine the prevalence ofAnaplasma organisms in major sheep- and goat-rearinglocations in Kenya. A total of 439 goat blood samples fromseven locations were tested for Anaplasma infections. ThepAO12A DNA probe detected A. ovis DNA at parasitemialevels below the 0.1 to 0.2% detected by examination ofGiemsa-stained blood smears. The prevalence ranged from22 to 87% in the samples from the goats from the sevenlocations surveyed (Fig. 7). In one of the sample locations(Ngong), the pAO12A DNA probe detected A. ovis in 10goat kids (age, 2 to 4 weeks).
DISCUSSION
A DNA probe was developed and evaluated for use indetecting A. ovis in goats. The detection method with theDNA probe, a 9.6-kilobase-pair insert of A. ovis genomicDNA in pBR322 (pAO12A), was 30 to 60 times more
100
90
0
0.
cc
0.
50
70
MONA Probe
od Smw Exriénation
60
50
40
30
20
10 ~~~~~ ,e
Location of Goats SanpledFIG. 7. Prevalence of A. ovis in goats from seven locations in
Kenya determined by hybridization of blood with the biotin-labeledpAO12A DNA probe and the A. marginale DNA probe and byexamination of Giemsa-stained blood smears of the organisms.
sensitive than the light microscopic method for detection ofA. ovis parasitemias. The biotin-labeled pAO12A DNAprobe detected 10 ng of purified A. ovis genomic DNA andA. ovis parasitemia levels of 0.0035%, which was equivalentto approximately 7 x 104 infected erythrocytes in 250 ,ul ofblood. The sensitivity of the biotin-labeled DNA probe wasless than that obtained by radioactive-labeled DNA probesdeveloped to detect other hemoparasites (7, 21, 24). How-ever, biotinylated DNA probes circumvent the safety andshelf-life problems associated with the use of radioactiveprobes.
Selection of the DNA probe was based on a stronghybridization signal obtained during colony hybridization.This suggested that multiple copies of the cloned DNAfragment may occur in the A. ovis genome, as has beendescribed for other parasites (21, 24). This suggestion wasstrengthened when the cloned A. ovis DNA fragment hybrid-ized with various intensities to 11 bands in the A. ovisgenomic DNA. Hybridization of the cloned A. ovis DNA toA. marginale and A. centrale genomic DNAs demonstratedat least partially homologous sequences in these organisms.The Anaplasma species from goats in Kenya was identi-
fied as A. ovis by using the pAO12A DNA probe in conjunc-tion with the 2-kilobase-pair A. marginale DNA probe. Theidentification was based on the hybridization of the A.marginale DNA probe to A. centrale and A. marginalegenomic DNAs and its failure to hybridize to AnaplasmaDNAs from infected goats. Also, differences in the in-traerythrocytic locations and host specificities between A.ovis and A. marginale observed here are consistent withresults of previous studies, in which these latter criteria wereused to differentiate the two species (13, 23, 32). In additionto these criteria, monoclonal antibodies to A. marginalewhich fail to react with A. ovis may also be useful fordifferentiation (20).The prevalence of A. ovis infections in goats in Kenya
ranged from 22 to 87%, as determined with blood samplesfrom goats from seven locations. This high prevalence wasattributed to the presence of a variety of possible vectorssuch as ticks and other biting insects. Goat kids (age, 2 to 4weeks) from Ngong infected with A. ovis may have con-tracted infections from ticks during suckling (D. Stiller,unpublished data) and, to a lesser extent, through transpla-cental transmission (6, 34).
In conclusion, the Anaplasma species infecting goats inKenya was identified as A. ovis by hybridization to a clonedA. ovis DNA probe but not to an A. marginale DNA probewhich hybridized to A. marginale and A. centrale genomicDNAs. The identification as A. ovis was further confirmedby the intraerythrocytic location of inclusion bodies and hostspecificity. The pAO12A DNA probe used to detect A. ovisrepresents a major improvement over current diagnosticmethods, as it identifies a 0.0035% level of parasitemia,which is a higher level of sensitivity than the 0.1 to 0.2%obtained by light microscopic examination of blood smears.The two DNA probes revealed a high prevalence of A. ovisinfection in goats from several locations in Kenya. TheseDNA probes can be used extensively in epidemiologicalstudies to determine the incidence and prevalence of A. ovisin various ruminant hosts and biological vectors such asticks, as well as to monitor progress in vaccination programsto control anaplasmosis.
ACKNOWLEDGMENTSThis research was done under grant AID/DAN 1328-G-SS-4093-00
from the U.S. Agency for International Development Title XII
J. CLIN. MICROBIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/j
cm o
n 16
Feb
ruar
y 20
22 b
y 17
7.10
.240
.252
.
DETECTION OF ANAPLASMA OVIS IN GOATS 2735
Small Ruminant Collaborative Research Support Program, in col-laboration with the Ministry of Livestock Development, Govern-ment of Kenya. This paper is published with the permission of theDirector of Veterinary Services of Kenya.We are grateful to Guy H. Palmer for suggestions on the experi-
mental design; Varda Shkap for providing the A. centrale DNA; andJoseph Buyu, Abu Oriko, and Paul Lacy for technical assistance.
LITERATURE CITED1. Barbet, A. F., G. H. Palmer, P. J. Myler, and T. C. McGuire.
1987. Characterization of an immunoprotective protein complexof Anaplasma marginale by cloning and expression of the genecoding for polypeptide Am 105L. Infect. Immun. 55:2428-2435.
2. Barker, R. H., L. Suebsaeng, W. Rooney, G. A. Alecim, H. V.Dourado, and D. F. Wirth. 1986. Specific DNA probe for thediagnosis of Plasmodium falciparum malaria. Science 231:1434-1436.
3. Bevan, L. E. W. 1912. Anaplasmosis of sheep. Vet. J. 68:400-401.
4. Birnboim, H. C. 1981. A rapid alkaline extraction method for theisolation of plasmid DNA. Methods Enzymol. 100:243-255.
5. Eriks, I. S., G. H. Palmer, T. C. McGuire, D. R. Alired, andA. F. Barbet. 1989. Detection and quantitation of Anaplasmamarginale in carrier cattle by using a nucleic acid probe. J. Clin.Microbiol. 27:279-284.
6. Fowler, D., and B. L. Swift. 1975. Abortion in cows inoculatedwith Anaplasma marginale. Theriogenology 4:59-67.
7. Goff, W., A. Barbet, D. Stiller, G. Palmer, D. Knowles, K.Kocan, J. Gorham, and T. McGuire. 1988. Detection of Ana-plasma marginale infected tick vectors by using a cloned DNAprobe. Proc. Natl. Acad. Sçi. USA 85:919-923.
8. Grunstein, M., and D. S. Hogness. 1975. Colony hybridization: amethod for the isolation of cloned DNAs that contain specificgene. Proc. Natl. Acad. Sci. USA 72:3961-3965.
9. Hanahan, D. 1983. Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166:557-580.
10. Howell, D. E., G. W. Stiles, and L. H. Moe. 1941. The hereditarytransmission of anaplasmosis by Dermacentor andersoni Stiles.Am. J. Vet. Res. 19:165-166.
11. Kuttler, K. L. 1965. Serological survey of Anaplasmosis inci-dence in East Africa, using the complement fixation test. Bull.Epizoot. Dis. Afr. 13:257-262.
12. Leary, J. J., D. J. Brigati, and D. C. Ward. 1983. Rapid andsensitive colorimetric methods for visualizing biotin labeledDNA probes hybridized to DNA or RNA immobilized onnitrocellulose: bio-blots. Proc. Natl. Acad. Sci. USA 80:4045-4049.
13. Lestoguard, F. 1924. Deuxieme note sur les piroplasmoses dumouton en Algerie. L'Anaplasmose: Anaplasma ovis nov. sp.Bul. Soc. Pathol. Exot. 17:784-787.
14. Losos, G. J. 1986. Anaplasmosis, p. 742-795. In G. J. Losos(ed.), Infectious tropical diseases of domestic animals. ChurchillLivingstone, Inc., New York.
15. Lotze, J. C. 1944. Carrier cattle as a source of infective materialfor horsefly transmission of anaplasmosis. Am. J. Vet. Res.5:164-165.
16. Maas, J., and G. M. Buening. 1981. Characterization of Ana-
plasma marginale infection in splenectomized domestic goats.Am. J. Vet. Res. 42:142-145.
17. Magonigle, R. A., W. P. Eckbald, S. D. Lincoln, and F. W.Frank. 1981. Anaplasma ovis in Idaho sheep. Am. J. Vet. Res.42:199-201.
18. Mallick, K. P., S. K. Dwivedi, and M. N. Malhotra. 1979.Anaplasmosis in goats: report on clinical cases. Indian Vet. J.56:693-694.
19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual, p. 125, 150. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.
20. McGuire, T. C., G. H. Palmer, W. L. Goff, M. I. Johnson, andW. C. Davis. 1984. Common and isolate-restricted antigens ofAnaplasma marginale detected with monoclonal antibodies.Infect. Immun. 45:697-700.
21. McLaughlin, G. L., T. D. Edlind, and G. M. Ihler. 1986.Detection of Babesia bovis using DNA hybridization. J. Proto-zool. 31:125-128.
22. Muenstermann, S., S. P. Shompole, and A. Lohding. 1985.Diseases of sheep in Lolgorien, Narok district, Kenya. Reportto the Department of Veterinary Services. Ministry of Live-stock Development, Nairobi, Kenya.
23. Neitz, W. O. 1939. Ovine anaplasmosis: the transmission ofAnaplasma ovis and Eperythrozoon ovis to the Blesbuck (Da-maliscus albifrons). Onderst. J. Vet. Sci. Anim. Indust. 13:9-16.
24. Pollack, Y., S. Metzger, R. Shemer, D. Landau, D. T. Spira, andJ. Golenser. 1985. Detection ofPlasmodiumfalciparum in bloodusing DNA hybridization. Am. J. Trop. Med. Hyg. 34:663-667.
25. Potgieter, F. T., and L. Van Rensburg. 1987. Tick transmissionof Anaplasma centrale. Onderst. J. Vet. Res. 54:5-7.
26. Rees, C. W. 1930. The experimental transmission of anaplasmo-sis by Rhipicephalus sanguineus. North Am. Vet. 11:17-20.
27. Richey, E. J., W. E. Brock, I. O. Kliewer, and E. W. Jones. 1977.Resistance to anaplasmosis after elimination of latent Ana-plasma marginale infections. Am. J. Vet. Res. 38:169-170.
28. Rigby, P. W., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labelling deoxyribonucleic acids to high specific activity invitro by nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.
29. Ristic, M., and J. P. Kreier. 1984. Family III. Anaplasmata-ceae, p. 719. In R. E. Buchanan and N. E. Gibbons (ed.),Bergey's manual of determinative bacteriology, 8th ed. TheWilliams & Wilkins Co., Baltimore.
30. Sanborn, C. E., G. W. Stiles, and L. H. Moe. 1938. Anaplasmo-sis: transmission by naturally infected Dermacentor andersonimale and female ticks. North Am. Vet. 19:31-33.
31. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.
32. Splitter, E. J., H. D. Anthony, and M. J. Twiehaus. 1956.Anaplasma ovis in the United States: experimental studies withsheep and goats. Am. J. Vet. Res. 17:487-491.
33. Stiles, G. W. 1936. Mechanical transmission of anaplasmosis byunclean instruments. North Am. Vet. 17:39-41.
34. Zaugg, J. L. 1987. Ovine anaplasmosis: in utero transmission asit relates to stage of gestation. Am. J. Vet. Res. 48:100-103.
