Veterinary Microbiology, 28 ( 1991 ) 313-325 Elsevier Science Publishers B.V., Amsterdam


An Anaplasma centrale D N A probe that differentiates between Anaplasma ovis and Anaplasma marginale D N A Elizabeth S. Visser~, Riccardo E. Ambrosio a and Daniel T. De

Waal b

Molecular Biology Section a and Protozoology Section b, Veterinary Research Institute, Onderstepoort 011 O, South Africa (Accepted 24 January 1991 )

ABSTRACT Visser, E.S., Ambrosio, R.E. and de Waal, D.T., 1991. An Anaplasma centrale DNA probe that differentiates between Anaplasma ovis and Anaplasma marginale DNA. Vet. Microbiol., 28:313-325. An Anaplasma centrale genomic library was constructed in pUC 13. Two clones pAC5 and pAC 137 hybridising to A. centrale and A. marginale DNA were isolated from this library. One of these, pAC5, also hybridised to DNA from A. ovis. The total insert of pAC5 was subcloned into pBR322. This subclone, pAC5-12, could detect 1 ng A. centrale, 0.5 ng A. marginale and 3.9 ng A. ovis DNA. The hybridisation pattern obtained with pAC5-12 on digests ofA. centrale, A. marginale and A. ovis DNA suggests that this probe detects EcoR 1 and Hindl 11-polymorphisms. Probe pAC5-12 could detect A. ovis DNA in 36% of blood samples tested compared to the 33% detectability obtained with microscopy.


Anaplasmosis is a tick-borne disease of cattle, sheep, goats, buffalo and some other wild ruminants and is caused by the rickettsiae Anaplasma marginale, A. centrale and A. ovis (Theiler, 1910; Losos, 1986 ). The disease is characterized by severe anemia associated with intraerythrocytic parasitism. A. marginale is the most pathogenic species and causes losses through mortality, weight loss and decreased milk production..4, centrale causes milder form of bovine anaplasmosis and .4. ovis causes limited disease in intact sheep. Ovine anaplasmosis is characterised by mild fever and variable degrees of anaemia. In splenectomised sheep there is acute clinical infection (Losos, 1986), however, most animals survive without treatment. Neither intact nor splenectomized cattle are susceptible to .4. ovis, but goats often develop a severe clinical response (Splitter et al., 1956; Losos, 1986). A. marginale and .4. ovis have a comparable distribution throughout the tropics and sub-tropics while .4. centrale occurs naturally only in Africa (Losos, 1986 ). 0378-1135/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.



The evaluation of the effectiveness of disease control programs for anaplasmosis requires the ability to monitor tick and host infections. Animals that recover from the acute stage of anaplasmosis may become carriers of the parasite and serve as reservoirs for disease transmission (Zaugg et al., 1986). The detection and quantitation of low numbers of organisms found in infected ticks and in carrier animals has recently been possible using DNA probes. Nucleic acid probes hybridise only to their complementary sequence on the parasite genome and are a specific and sensitive tool for the diagnosis of infectious diseases (Engelberg and Eisenstein, 1984). A DNA probe has been described that can detect A. rnarginale in infected ticks (Goffet al., 1988 ) as well as in carrier cattle (Eriks et al., 1989 ). Recently, an A. marginale DNA probe was described which could detect a parasitaemia of less than 0.01% (Aboytes-Torres and Buening, 1990). A comparison of an A. marginale DNA probe, complement fixation (CF) and indirect immunofluorescence (IIF) tests for the diagnosis of anaplasmosis in carrier cattle showed that the probe and IIF test were more sensitive than the CF test (Goff et al., 1990). We have reported the isolation of DNA probes specific for A. marginale and A. centrale (Visser and Ambrosio, 1987 ). These probes did not hybridise to A. ovis DNA. DNA probes have been used to monitor the course of clinical disease in equine babesiosis (Posnett et al., in press) and a Babesia bigemina probe has been used to identify and isolate infected forms in the tick vector, Boophilus microplus (Hodgson et al., 1989 ). The availability of a probe detectingA, ovis DNA would be of great value in understanding the epidemiology of anaplasmosis, especially when this probe is used together with the A. centrale and A. rnarginale probes. Here we report isolation o f a DNA probe that hybridises to A. centrale, A. marginale and A. ovis DNA. We also show that this probe can differentiate between Anaplasma species and can detect A. ovis DNA in blood from infected sheep. MATERIALS AND METHODS

Isolation ofAnaplasma DNA A. centrale DNA was isolated from infected bovine blood (28% parasitaemia) as described by Visser and Ambrosio (1987). The initial bodies were lysed with 1% SDS and incubated overnight at 37°C in 10 m M EDTA and Pronase (final concentration 200 pg m l - ~). After phenol-chloroform extractions and ethanol precipitation (Maniatis et al., 1982 ) the DNA was purified further by cesium chloride density gradient centrifugation (Maniatis et al., 1982), dialyzed against TE (10 m M Tris-HC1 pH 7.5, 1 m M EDTA) and ethanol precipitated. The final pellet was resuspended in TE and the concentration determined. A. marginale (Underberg-isolate: Am-U; 40% parasitemia) and A. ovis (32% parasitemia) DNA was isolated from infected blood and A. marginale



(Barkley-West-isolate: Am-BW; Potgieter et al., 1981 ) DNA from frozen initial bodies as described above.

Construction of an A. centrale genomic library in pUC13 A. centrale DNA (3/tg) was digested with EcoR1 (Promega) and ligated into EcoR 1-digested pUC 13 (Vieira and Messing, 1982 ) as described by Maniatis et al. (1982). Competent Escherichia coli JM 105 cells (YanischPerron et al., 1985 ) were transformed and selected on ampicillin plates. DNA from recombinant colonies was extracted using the alkaline lysis protocol described by Birnboim and Doly ( 1979 ). Recombinant DNA was identified by its migration on agarose gels containing pUC 13 as marker. Inserts were recovered from the pUC 13-recombinants by EcoRl-digestion and Southernblotted (Southern, 1975) to nylon membranes (Hybond-N, Amersham). Fourteen clones were selected on the basis of hybridisation intensity to A. centrale and their DNA used as probes. DNA/DNA hybridisation DNA probes were labelled by nick-translation (Rigby et al., 1977 ) or random priming (Feinberg and Vogelstein, 1983) using 32p-dCTP (Amersham). Hybridisation was at 65°C for 16 h in a buffer containing 5 × Denhardt's solution (Maniatis et al., 1982), 6 × SSC ( 1 × SSC: 0.15 M NaC1, 0.015 M Na3citrate) and 0.5% SDS with 1 /tg of each radiolabelled probe (10 v d.p.m./~g-~). Probes were denatured by boiling prior to use. Membranes were washed at room temperature in 2 × S S C ; 0.1% (w/c) SDS followed by two further washes at room temperature and 65 °C in 0.1 × SSC; 0.1% SDS. Autoradiography was overnight at - 70 ° C with intensifying screens (Du Pont Cronex Quanta III ). Subcloning of pAC5 into pBR322 EcoR 1-digested pAC5 was ligated into pBR322 and after transformation of competent E. coli JM 109 cells (Yanisch-Perron et al., 1985 ), recombinant clones were identified by Southern blotting of EcoR 1-digested DNA to nylon membranes and hybridisation to A. centrale genomic DNA. Sensitivity of probe pAC5-12 Dilution series ofA. centrale, A. marginale and A. ovis DNA ranging from 500 ng to 240 pg were prepared in TE. After heat denaturation (Stroop and Schaefer, 1989), the DNA was slot-blotted to a nylon membrane (HybondN +, Amersham ), and hybridised to labelled pAC5-12. Restriction mapping of pA C5-12 After digestion, the insert of probe pAC5-12 was recovered from an agarose gel using NA45 DEAE cellulose (Schleicher & Schuell ) according to the pro-


E.s. v1SSER ET Ak.

tocol described by Dretzen et al. ( 1981 ). (See also Lizardi, P.M. In: Schleicher & Schuell Sequences-Applications Update Nr. 364). 100 ng of the insert was digested with 15 different restriction enzymes and double digests of enzymes that cut the insert were used to construct a restriction map of pACI2-12 (Priefer, 1984).

Hybridisation of pAC5-12 to enzyme digests ofAnaplasma DNA DNA ofA. centrale, Am-U, Am-BW and A. ovis (5/tg for each digestion ) was digested overnight with 12 restriction enzymes. The enzymes were EcoR 1, Xbal, Pstl, SAC1, Kpnl, Bgll 1, BamH1, Hindl 11, Smal, Sall, Sstl 1 and Clal. After gel electrophoresis, the fragments were Southernblotted to nylon membranes and probed with labelled pAC5-12. Hybridisation of pAC5-12 to ovine blood samples Blood samples (in heparin) of 53 randomly selected sheep were obtained from a State Veterinary Laboratory in an A. ovis endemic area. High salt lysares of 100/zl of the red blood cell fraction of the blood samples were prepared according to the method ofZolg et al. ( 1988 ) and stored at 4°C. After deproteinising the samples (Zolg et al., 1988 ), the DNA was applied to nylon membranes (Hybond-N +, Amersham) and probed with the random prime-labelled insert ofpAC5-12. Blood from an uninfected sheep was used as negative control and A. centrale DNA (100 ng) was used as positive control. Blood smears from each of the samples were also examined microscopically. Thin blood smears were prepared from each of the samples on arrival, stained with Giemsa stain and examined microscopically for A. ovis. A smear was considered negative after examination of 100 microscope fields (i.e. 3-5 rain examination ). RESULTS

Hybridisation of pAC5 to Anaplasma DNA A genomic library of A. centrale DNA was constructed in pUC 13. After transformation and plating on LB-plates, recombinants were picked and their DNA extracted using an alkaline lysis protocol. A. centrale DNA hybridised to DNA from 153 of 200 white colonies selected. Of these, 14 clones were investigated further by hybridisation to EcoR ldigested bovine, A. centrale and A. marginale genomic DNA. No hybridisation was obtained to bovine DNA. Two clones, pAC5 and pAC137, hybridised intensely to these digests (not shown). When used as probe on a membrane containing EcoRl-digested A. ovis DNA as well as bovine, A. centrale and A. marginale (Am-U) DNA, probe pAC5 hybridised to a 2.7 kb band on A. centrale and A. marginale DNA and to a 5.8 kb band on A. ovis DNA (Fig. 1, lanes 2,3,4,5). Probe pAC 137 hybridised to A. centrale and A. marginale








1:1 " 0°56-

Fig. 1. Hybridisation of pAC5 to EcoR 1-digested bovine, A. centrale, A. marginale (Am-U) and A. ovis DNA. The membranes were washed in 0.1 × SSC; 0.1% SDS at 65 ° C and exposed for 24 h. DNA sizes are in kilobase pairs. Lane 1: Bovine DNA; Lane 2: Am-U DNA; Lanes 3 and 4: A. centrale DNA; Lane 5: A. ovis DNA.

DNA only (not shown). No hybridisation to bovine DNA was obtained (Fig. 1, lane 1 ). The insert ofpAC5 was approximately the same size as the pUC vector. In order to isolate insert DNA, it was necessary to subclone the insert into a larger vector. EcoRl-digested pAC5 was ligated into pBR322 (Sutcliffe, 1978 ) and recombinants screened by hybridisation to A. centrale DNA. Clone pAC512, containing the correct size insert, as well as pAC5, was hybridised in turn to A. centrale DNA digested with different restriction enzymes. Identical hybridisation patterns were obtained with pAC5 and pAC5-12 (not shown ).

Sensitivity of probe pA C5-12 Slotblots of a dilution series of A. centrale, A. marginale (Am-U) and A. ovis DNA were prepared and probed with labelled pAC5-12. After hybridisation, the membranes were washed at high stringency in 0.1 × SSC; 0.1% SDS at room temperature. Probe pAC5-12 hybridised to 1 ng A. centrale DNA, 0.5 ng A. marginale and 3.9 ngA. ovis DNA (Fig. 2). These membranes were reprobed with DNA extracted from uninfected bovine and sheep blood. A low level of background hybridisation was obtained suggesting that the samples contained host DNA (not shown).

Restriction mapping of pAC5-12 The restriction map of pAC5-12 was constructed with the following enzymes: BamH1, Hindl 11, Pstl, Sacl and Sall (Fig. 3). No restriction sites were present for Bgll 1, Clal, Kpnl, Smal, Sstl 1 and Xbal.





Fig. 2. Hybridisation o f probe pAC5-12 to serial dilutions of A. centrale, Am-U and A. ovis DNA. The m e m b r a n e s were washed in 0.1 × SSC containing 0.1% SDS at room t e m p e r a t u r e and exposed tbr 24 h. Lane 1: A. centrale DNA; Lane 2: A m - U DNA; Lane 3: A. ovis DNA. a-j: Twofold dilution series of the D N A ranging from 250 ng to 0.5 rig. a: 250 ng; b: 125 ng; c: 62.5 ng; d: 31.3 ng; e: 15.6 ng; f: 7.8 ng; g: 3.9 ng; h: 1.95 ng; i: 0.98 ng;j: 0.49 ng.

















Fig. 3. Restriction m a p of pAC5-12. Ba: B a m H 1; E: EcoR1; Hd: Hind l 1 1; P: Pst 1; Sa: Sac 1; SI: Sall.

Hybridisation patterns o f pA C5-12 on digested Anaplasma DNA Restriction enzyme digests (using the enzymes described in Materials and Methods) ofA. centrale, Am-U, Am-BW and A. ovis DNA were blotted to nylon and hybridised to nick-translated pAC5-12. Except for the DNA digested with Hindl 11, identical hybridisation patterns were obtained on the digests ofA. centrale and Am-U DNA (Hybridisation to A. centrale DNA not shown; only hybridisation to Am-U DNA is shown in Fig. 4A). On A. ovis DNA (Fig. 4B ) the hybridisation pattern obtained showed both differences and similarities with that ofpAC5-12 on Am-U, Am-BW and A. centrale DNA.







3 4 5 6



10 11 12







e Q em













10 11


23.1 9.4 6.6 4.4 2.0

---- ~ i





Fig. 4. Hybridisation of probe pAC5-12 to enzyme digests of Am-U and A. ovis DNA. The membranes were washed in 0.1 × SSC containing 0.1% SDS at room temperature and exposed for 24 h. The DNA sizes are in kilobase pairs. Panel A: Am-U DNA. Panel B: A. ovis DNA. Lane 1: EcoR 1; Lane 2: Xba 1; Lane 3: Pst l; Lane 4: Sac l; Lane 5: Kpn 1; Lane 6: Clal; Lane 7: BamHl; Lane 8: Bgll; Lane 9: Hindl 11; Lane 10: Sail; Lane 11: Sinai; Lane 12: Sstl. O n H i n d l 11-digested A. ovis D N A , p A C 5 - 1 2 h y b r i d i s e d to the s a m e p a t t e r n as on A m - U D N A (Fig. 4A,B, lane 9) b u t with different intensities. A u n i q u e b a n d p a t t e r n was o b t a i n e d o n the H i n d 111-digest o f A m - B W D N A p r o b e d with p A C 5 - 1 2 ( s h o w n in Fig. 5), as well as a similar p a t t e r n to A. centrale a n d A m - U D N A o n the o t h e r digests ( n o t s h o w n ) . A 17 kb b a n d on the C l a l - d i g e s t , 11 kb o n the S m a l - d i g e s t a n d a 8.1 kb b a n d o n the S s t l 1-






23.1 9.4 6.6 4.4 2.3 2.0




Fig. 5. Hybridisation patterns o f probe pAC5-12 on Hindl 1 l-digested Anaplasma DNA. The m e m b r a n e was washed in 0.1 × SSC containing 0.1% SDS at room temperature and exposed for 48 h. The DNA sizes are in kilobase pairs. An arrow shows the position o f the 5.7 kb band. Lane 1: A. ovis DNA: Lane 2: A. centrale DNA; Lane 3: A m - U DNA; Lane 4: Am-BW. TABLEI Results of hybridisation of probe pAC5-12 and microscopic examination of 53 ovine blood samples No. of samples



Proportion of samples (%)

3 13 31 6

+ + -

+ +

5.7 24.5 58.5 11.3

digest were obtained on Am-BW D N A while the band sizes obtained on A. centrale and A m - U D N A for these digests were 15.1, 19.3 and 9.4 kb respectively. A. centrale, A. ovis, A m - U and Am-BW D N A was again digested with Hind 111, blotted and probed with pAC5-12. The identical hybridisation pattern described above was obtained (Fig. 5). With A. ovis and A m - U DNA, two identical bands of 1.78 and 0.9 kb hybridized (Fig. 5, lanes 1 and 3), whereas a 2.6 kb band forA. centrale and 1.18, 0.9 and 0.65 kb bands for AmBW were obtained (Fig. 5, lanes 2 and 4). Two bands of 5.7 and 24 kb hybridised on A. centrale, A m - U and Am-BW, but very faintly on A. ovis DNA. The 3.7 kb band on A m - U D N A resulted from cross-hybridisation between the vector and bovine D N A present in the A m - U D N A preparation (unpublished results).



Hybridisation of pAC5-12 to ovine blood samples The parasitemia of the 53 ovine blood samples was determined by microscopic examination of Giemsa stained thin blood smears. In the sample with the highest parasitemia, one to three parasites were detected in approximately 1 × 104 red blood cells. In seven samples, less than one parasite in approximately 1 × 104 red blood cells was detected while in eight samples, only one to three parasites were observed during a microscopic examination lasting 3 to 5 min. DNA from high salt lysates of these blood samples, and one uninfected control, was extracted and deproteinized before application to a nylon membrane. Probe pAC5-12 was labelled and hybridised to the membrane as described in Materials and Methods. These results together with the results obtained by microscopy are summarized in Table 1. Thirteen samples were positive and 31 samples negative with both microscopy and hybridisation, resulting in a correlation of 83%. Taking microscopy as standard, 5.7% false-negative and 11.3% false-positive samples were recorded. DISCUSSION

In this report we describe the isolation and use of a DNA probe to detect A. ovis DNA. Diagnosis and identification of Anaplasma infection has been limited to microscopy and serological approaches. Diagnostic serology suffers from lack of specificity, and cross-reactivity between A. marginale, A. centrale andA. ovis has been reported (Splitter et al., 1956; Kuttler, 1967). Probes specific for A. marginale (Visser and Ambrosio, 1987; Goff et al., 1988; Aboytes-Torres and Buening, 1990) and A. centrale (Visser and Ambrosio, 1987) have been isolated which range in sensitivity from 127 ng for A. centrale and 8 ng to 0.01 ng for A. marginale (Visser and Ambrosio, 1987; Goffet al., 1988 ). An A. marginale probe has also been used to follow experimentally induced parasitemias in cattle (Eriks et al., 1989 ) and in a comparison to existing serological tests in the detection of carrier cattle (Goff et al., 1990). Recently, Shompole et al. (1989) described a probe that could detect a 0.0035% A. ovis parasitemia in blood from infected goats. Not much information is available about the epidemiology of A. ovis and of the role of carrier animals in spreading the disease. Such studies are complicated by the low parasitemias found in carriers which makes their identification difficult. We have shown that the A. ovis DNA probe can detect low levels of parasites in infected blood from field samples and could be applied to detect carriers. Of the blood samples tested, 5.7% were false negative and 11.3% false positive, using microscopy as standard. The false positives could have resulted from non-specific binding to other blood components, although no hybridi-



sation was obtained to negative controls. The false positives could also have been due to a parasitemia too low to visualize. There was an 83% correlation between the samples found positive and negative with both microscopy and hybridisation. More samples will have to be evaluated to establish the accuracy and sensitivity of detection ofA. ovis DNA by probe pAC5-12. We have not been able to remove all host DNA from our Anaplasma DNA preparations. This was confirmed by the hybridisation obtained with bovine and ovine DNA on control blots (not shown). The sensitivity of probe pAC512 that we reported here was calculated from hybridisation to slot blots containing dilutions prepared from this DNA and is therefore approximate. Nevertheless, this probe is still sensitive enough to detect low parasitaemias. Probes that hybridise to all Anaplasma species and provide a specific pattern for each isolate would be most suitable for diagnosis and epidemiology. Probe pAC5-12 was isolated from an A. centrale genomic library. The similar band patterns obtained on digests ofA. centrale, Am-U and Am-BW DNA, except on the Hindl 1 l-digests, suggests that the pAC5-12 sequence is conserved between these species. The sizes of the 1.18, 0.9 and 0.65 kb Hind 11 ldigested Am-BW bands and the 1.78 and 0.9 kb Hind 111-digested Am-U and A. ovis bands each total approximately the size of the most intense band on Hindl 11-digested A. centrale DNA (2.6 kb). On the EcoRl-digests, pAC512 hybridised to a band of 5.8 kb on A. ovis DNA and to 2.7 kb bands on A. centrale, Am-U and Am-BW DNA. The probe therefore detects Hindl 11 and EcoR 1-polymorphisms and could be a useful tool in epidemiological studies. The variation in intensity of hybridisation of pAC5-12 between Am-U and A. ovis (Fig. 4 ) can be attributed to a lower A. ovis DNA concentration in the blot as well as a lower sensitivity of this probe for A. ovis DNA (See Fig. 2, lane 3 ). DNA probes have been used to differentiate between species and isolates. The Anaplasma-specific probe AC2 (Visser and Ambrosio, 1987 ) hybridised to different bands on A. marginale isolates from the USA (Ambrosio et al., 1988). A cloned DNA probe was used to differentiate between the human and flying squirrel isolates of Rickettsia prowazekii (Regnery et al., 1986). These authors also used DNA restriction endonuclease analysis to differentiate between strains of R. prowazekii. The same technique was used for differentiation between isolates of other bacteria, including Coxiella (O'Rourke et al., 1985), Chlarnydia (Campbell et al., 1987), Escherichia (Marshall et al., 1985 ) and Mycobacteriurn (Collins and DeLisle, 1986). The restriction endonuclease cleavage patterns of five A. marginale isolates from the USA were studied by Krueger and Buening (1988) and significant differences between the isolates were found. Phenotypic differences between Anaplasma-species have been reported. These include variation in virulence, position in the red blood cells and some serological diversity (Losos, 1986). Of the two South African A. marginale-isolates, Am-U has a particularly high



virulence (F.T. Potgieter, personal communication, 1989). Such changes would be reflected in the genetic material of the different species and isolates. Restriction endonuclease analysis as well as hybridisation of a probe such as pAC5-12 to restriction digests for detection on polymorphisms, could be useful to differentiate between Am-U, Am-BW and isolates ofA. centrale and A. ovis present in southern Africa as well as to identify new isolates. With a D N A probe detecting polymorphisms on restriction analysis patterns, fewer bands than the cleavage band patterns on agarose gels would be produced, simplifying differentiation between isolates. The development ofA. centrale and A. marginale in tick vectors as well as their role in the epidemiology of bovine anaplasmosis has been well documented (Potgieter, 1979; Kocan et al., 1980a, b; Potgieter and van Rensburg, 1987). However, little is known about the vector(s) and their role in the transmission ofA. ovis. Since this parasite has the same geographical distribution as A. marginale, it is possible that the same vectors may be involved. D N A probes have been used to detect parasite D N A in vectors (Goff et al., 1988; Hodgson et al., 1989 ). The probe reported here could be a useful tool in such a study. ACKNOWLEDGEMENTS

The authors thank E. Kirkbride for technical assistance.

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An Anaplasma centrale DNA probe that differentiates between Anaplasma ovis and Anaplasma marginale DNA.

An Anaplasma centrale genomic library was constructed in pUC13. Two clones pAC5 and pAC137 hybridising to A. centrale and A. marginale DNA were isolat...
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