Nucleic acid probes as a diagnostic method for tickborne hemoparasites of veterinary importance

veterinary parasitology ELSEVIER

Veterinary Parasitology 75 ( 1995 ) 75-92

Nucleic acid probes as a diagnostic method for tickborne hemoparasites of veterinary importance J.V. Figueroa a'*, G.M. Bueningb aCENID-PAVET, INIFAP-SARH, Jiutepec, Morelos 62500, Mexico bDepartment of Veterinary Microbiology, University of Missouri-Columbia, Columbia, MO 65211, USA

Abstract

An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts ofAnaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isotopic detection system, probes have allowed scientists to test for--originally in samples collected from experimentally infected animals and later in samples collected in the field--the presence ofhemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures.

Keywords: Anaplasma spp.; Babesia spp.; Cowdria sp.; Theileria spp.; Diagnosis-Protozoa; Nucleic acid probes

*Corresponding author: Tel. 52 (73) 19-28-60; Fax. 52 (73) 20-55-44. 0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI0304-4017(94)03112-6

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I. Introduction

Anaplasmosis is a biologically and mechanically arthropod-transmitted disease caused by the rickettsia Anaplasma (Ristic, 198 lb). Three species, two infecting cattle (Anaplasma marginale and Anaplasma centrale) and one in sheep and goats (Anaplasma ovis) are well-recognized (Callow, 1984; Shompole et al., 1989). Anaplasma marginale is widely distributed throughout the world. Clinically, acute bovine anaplasmosis is manifested as fever and a progressive anemia (usually without hemoglobinuria) associated with the presence of intraerythrocytic inclusion bodies (Ristic, 1981b; Callow, 1984 ). Anaplasma centrale is found naturally only in Africa, but it has been introduced in Australia, Israel and South American countries for vaccination purposes. Animals infected with Anaplasma centrale rarely develop acute clinical disease and although not completely avirulent, cattle deaths have never been seen (Callow, 1984). Anaplasma ovis can cause severe disease in sheep and goats (Shompole et al., 1989) and it has a comparable distribution to Anaplasma marginale throughout the tropics and subtropics (Visser et al., 1991 ). Babesiosis is a tick-transmitted disease of domestic and wild animals caused by protozoan parasites of the genus Babesia. The disease is usually characterized by hemolytic anemia and the presence of intraerythrocytic piroplasms (Callow, 1984). Although all domestic animals and many wild animals are susceptible to Babesia, only those diseases considered of great economic relevance in veterinary medicine (bovine babesiosis and equine babesiosis) will be reviewed in terms of their diagnosis with nucleic acid probes. Of the Babesia species that infect cattle, Babesia bigemina and Babesia boris are considered as two of the most economically important disease-causing agents of cattle in tropical and subtropical areas of the world (Ristic, 198 la; Callow, 1984). Both Babesia species share the tick Boophilus spp. as the vector for their biological transmission to cattle (Ristic, 1981 a; Callow, 1984 ). Clinical manifestations of an acute presentation of the disease include fever, anorexia, dullness, weakness, ataxia, hemoglobinuria, icterus, anemia, and presence of intraerythrocytic parasites (Callow, 1984). Equine babesiosis caused by Babesia equi and Babesia caballi is endemic in horses throughout the tropics and subtropics and the characteristics of the disease are similar to those of bovine babesiosis and include anemia, fever and death if animals are not treated (Purnell, 1981 ). Heartwater or cowdriosis is an acute rickettsial disease of cattle, sheep, goats and wild ruminants. The disease is caused by Cowdria ruminantium which is transmitted by species of Amblyomma ticks (Uilenberg, 1981 a). The disease occurs in Africa and some Caribbean islands and is clinically manifested by nervous, intestinal and pulmonary disorders. Several species of the protozoan parasite Theileria cause theileriosis in cattle and wild ruminants. Theileriaparva and Theileria annulata are the etiologic agent of two of the most economically important tick-borne diseases of cattle in the tropics: East Coast Fever and tropical theileriosis, respectively (Uilenberg, 1981b;

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Irvin and Cunningham, 1981 ). Theileria parva, transmitted by Rhipicephalus appendiculatus, occurs in east and central Africa and the disease it causes in cattle is characterized by pyrexia, lymph node swelling, anorexia, and death in susceptible animals (Irvin and Cunningham, 1981). Under field conditions, other Theileria species such as Theileria mutans, transmitted by ticks of the genus Amblyomma, and Theileria taurotragi can complicate the epidemiology of East Coast Fever (Irvin and Cunningham, 1981; Allsopp et al., 1991 ). Theileria annulata which is transmitted by Hyalomma ticks occurs in northern Africa, the Middle East, central Asia and southern Europe (Uilenberg, 1981 b; Allsopp et al., 1993 ) and the disease in cattle is characterized by an acute febrile response, anemia,. and high mortality in susceptible cattle. Theileria sergenti and Theileria orientalis occur in eastern Asia and other nonpathogenic theilerias are present in Australia, Asia, northern Africa, Europe and North and South America (Uilenberg, 198 lb). A definite laboratory diagnosis of the hemotropic organisms Anaplasma, Babesia and Theileria in acutely infected animals, is generally based on the microscopic examination of peripheral blood smears for the presence of intraerythrocytic or intralymphocytic bodies which could be differentiated by the morphological and staining properties of the different parasite genera and species involved (Purnell, 1981; Ristic, 1981a,b; Uilenberg, 1981b; Irvin and Cunningham, 1981; Callow, 1984). Cowdria organisms can be demonstrated in acutely infected animals by microscopic examination of brain cortex smears searching for rickettsial colonies in endothelial cells (Uilenberg, 1981 a). A characteristic feature of hemotropic diseases is that animals recovered from a primary acute attack become carders of the respective protozoan and rickettsial organisms (Purnell, 1981; Ristic, 1981a,b; Uilenberg, 1981b; Irvin and Cunningham, 1981; Uilenberg, 1983; Callow, 1984). These carder animals may serve as a reservoir of infection for the tick vectors and cannot be clinically differentiated from uninfected animals, as organisms, when present, are generally in very low numbers in circulating blood and usually cannot be demonstrated by examining blood films or brain smears stained with traditional methods. Despite an existing array of serological assays for each particular hemotropic disease, these immunological assays are indirect methods and do not detect the causal organisms in samples obtained from a suspected infected animal. The consensus of researchers involved with the study of the major tick-borne diseases of domestic animals (properly named 'the big four' by Dr. G. Uilenberg) is that more sensitive and specific diagnostic methods are needed. This review will attempt to highlight the major points of published articles on the development and application of nucleic acid-based probes for the detection ofAnaplasma, Babesia, Cowdria and Theileria organisms in domestic animals. 2. Nucleic acid probes for Anaplasma sp.

Cloned DNA probes that detect Anaplasma marginale DNA have been reported in the literature (Visser and Ambrosio, 1987; Goff et al., 1988; Aboytes-

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Torres et al., 1989 ). Name, size, analytical sensitivity and specificity data of the

Anaplasma probes are summarized in Table 1. Visser and Ambrosio (1987) constructed an Anaplasma centrale genomic library from which four DNA probes were obtained. AC1 probe was specific for Anaplasma centrale, whereas AC2, Table 1 Nucleic acid probes for Anaplasrna sp. Probe size/label

Analytical sensitivity DNA

Specificity

Ref.

A. centrale

Visser and Ambrosio, 1987, 1989

iEa or PPE b

AC-1 1.6 Kb DNA, RAc

1.25 ng

AC-2 1.0 Kb DNA, RA

0.3 ng 0.6 ng

8.2 X 105 1.6)< 106

A. centrale A. marginale

pAm113 2.0 Kb DNA, RA

0.5 pg

0.001% 2.5)<102

A. marginale

Goffet al., 1988

pGEM-3 2.0 Kb DNA, RA

0.01 ng

500-1000 iE 0.000025%

A. marginale

Eriks et al., 1989

0.0035% 7 × 10* iE

A. ovis A. marginale

Shompole et al., 1989

-

A. marginale

Aboytes-Torres et al., 1989

< 104-106 iE

A. marginale

Kieser et al., 1990

100 pg

< 0.01% 500-1000 iE

A. marginale

Aboytes-Torres and Buening, 1990

pAml 13 2.0 DNA, RA

-

0.0005-0.00005%

A. marginale

Goffet al., 1990

pAC-5 DNA, RA

0.5 ng 1 ng 3.9 ng

A. marginale A. centrale A. ovis

Visser et al., 1991

0.0000001%

A. marginale

Aboytes-Torres, 1992

pAOl 2A 9.6 Kb DNA, NRA a

10 ng 100 ng

pA 6.4 Kb DNA, NRA

1 ng

pGEM-3 2.0 Kb RNA, RA pStCroixA 1 6.4 Kb DNA

pi25 PCR primers 200 bp/151 SSO~, RA

100 ag

-

-

pi25 PCR primers 200 bp/160 bp DNA, NRA

-

0.00009%

A. marginale

Buening et al., 1992

pi25 PCR primers 200 bp/160 bp DNA, NRA

-

0.0001%

A. marginale

Figueroa et al., 1993b

"Infected erythrocytes. bpercent of parasitized erythrocytes. CRadioactive probe. dNonradioactive probe. ~Single stranded oligonucleotide.

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AC3, and AC4 hybridized to both Anaplasma marginale and Anaplasma centrale DNA. AC1 did not hybridize to Anaplasma ovis DNA. In regions or countries where mixed infections (Anaplasma centrale, Anaplasma marginale) occur (whether naturally or by vaccinating cattle with Anaplasma centrale) it is recommended to use the crossreactive AC2-probe to detect Anaplasma infection, whereas the species-specific probe should be used to identify the infecting parasite for epidemiology studies. The AC 1 probe lacks sensitivity (1.25 ng) and would probably not detect carder animals. Nonetheless, the Anaplasma centrale probes AC2 and AC4 hybridized to DNA of Anaplasma marginale from four different USA isolates and one South African isolate, although certain differences were found in the genome of the isolates tested. As serological and microscopic techniques are often not entirely suitable for analysis of infected ticks, Ambrosio et al. ( 1988 ) used the Anaplasma marginale DNA probes to detect the parasite in Dermacentor andersoni ticks. Probes AC2 and AC4 were able to hybridize and thus detect Anaplasma marginale DNA in tick midgut material, but not in salivary glands probably because the probes had relatively low analytical sensitivity. A 2.0 kbp DNA fragment from a cloned Anaplasma marginale gene (Am 105L, Barber et al., 1987) was used by Goff ct al. (1988) as a probe to detect Anaplasma marginale-infected ticks. Dermacentor andersoni, Dermacentor variabilis and Dermacentor occidentalis ticks exposed to Anaplasma marginale-infected cattle were detected positive when their midgut was dissected, DNA extracted and hybridized to the 2.0 kbp probe. As little as 0.4% of the total DNA extracted from midgut tissue provided enough Anaplasma marginale DNA to be detected by the probe. Later, the 2.0 kbp probe was used to identify Anaplasma marginale DNA in salivary glands of Dermacentor spp. male ticks (Kocan et al., 1989), suggesting that the organism multiplies in that tick tissue during tick feeding. Goff et al. (1990) utilized the 2.0 kbp DNA fragment as a radioactive probe to detect 98.5% of apparent Anaplasma marginale carders in a study carried out in an enzootic area of Washington State, USA. They estimated that the cattle tested had parasitemias ranging from 0.0005 to 0.00005%. Eriks et al. (1989) utilized a nucleic acid probe to detect and quantitate Anaplasma marginale in carder cattle to evaluate the role of such cattle in disease prevalence and transmission. By using a radioactive RNA transcript as probe, they were able to show that carder animals had variable parasitemia levels (0.0025-0.000025%) as well as variable individual parasitemia levels when tested on different dates. Such individual variability in parasitemia was later documented in persistently Anaplasma marginale-infected animals (Kieser et al., 1990) and was associated with a rise and decline (cyclic) rickettsemia as quantitated by the RNA probe assay. The rickettsemia levels found in persistently infected cattle fluctuated cyclically approximately every 5 weeks (Eriks et al., 1993). The role of rickettsemia levels of persistently infected cattle on the infection rate of D. andersoni ticks was evaluated by quantitative probe analysis. It was concluded that the higher the parasitemia level in cattle, the higher the tick infection rate.

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Aboytes-Torres et al. (1989) and Aboytes-Torres and Buening (1990) developed a recombinant DNA probe (A1) which had high specificity and sensitivity forAnaplasma marginale DNA. The radioactive probe cross-hybridized to DNA from 12 geographically different Anaplasma marginale isolates from the Caribbean region, Mexico and USA. The probe detected Anaplasma marginale DNA in cattle during the acute phase of disease 2-13 days prior to the detection of marginal bodies by light microscopy. Aboytes-Torres et al. (1989) also utilized the AI probe in a nonradioactive format. The sensitivity, however, was ten-fold lower than that obtained with the radioactive probe. The DNA probe was evaluated on blood samples from experimentally infected cattle 44 days after anaplasmosis treatment with commercial drugs and dexamethasone-induced immunosuppression. Cattle were found positive by DNA probe analysis, indicating that the drug did not completely clear cattle of the infection. DNA probes that identify Anaplasma ovis have also been developed (Shompole et al., 1989). Anaplasma ovis was detected in goats in Kenya, with prevalence rates ranging from 22 to 87% by using a 9.6 kbp nonradioactive DNA probe. Visser et al. ( 1991 ) described an Anaplasma centrale-derivedDNA probe which could differentiate between Anaplasma species and which could be used to detect Anaplasma ovis-infected sheep. The radioactive PAC 5-12 DNA probe detected Anaplasma ovis DNA in 36% of 53 blood samples collected from randomly selected sheep in an endemic area of South Africa. Polymerase chain reaction (PCR) based assays have been developed in an effort to increase the analytical sensitivity, while retaining the high specificity for detection ofAnaplasma marginaleDNA (Stich et al., 1991; Aboytes-Torres, 1992; Buening et al., 1992 ). A PCR assay to detect Anaplasma marginaleDNA in ticks was reported by Stich et al. ( 1991 ). Primers derived from a major surface protein gene (msplB, Barbet and Allred, 1991 ) were utilized to amplify a 406 bp Anaplasma marginale DNA fragment from bisected bodies and salivary glands of male D. andersoniticks. The target fragment was detected in PCR reactions of all infected, bisected ticks and salivary gland preparations that were tested, but not in similar preparations from uninfected ticks. A slightly modified PCR assay, in which a 409 bp target DNA fragment would be amplified, was later adapted for the detection of Anaplasma marginale in the hemolymph of D. andersoni ticks (Stich et al., 1993a). Both female and male ticks exposed as nymphs to Anaplasma marginale-infected donor calves were detected positive to infection when the PCR assay utilized 1/A heat-denatured hemolymph samples. The PCR amplified fragment could also be visualized in reactions carried out on hemolymph of both male and female ticks exposed as adults to an Anaplasma marginale donor. In addition, the 409 bp DNA fragment was determined to be conserved among seven geographical isolates from USA (Stich et al., 1993a). The authors indicated that the hemolymph/PCR test could be very useful for application to field collected ticks, as the assay could detect Anaplasma marginaleorganisms present in very small numbers in the tick vector. Also, since the hemolymph/PCR test does not require the killing of the vector, the ticks could be used in further studies. Stich et al. (1993b) demonstrated that the 409 bp Anaplasma marginaleDNA

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fragment could also be amplified from tick saliva samples. Female and male adult ticks, as well as male ticks exposed as nymphs to Anaplasma marginale-infected donor calves, were stimulated to salivate by dopamine injection. Saliva samples were positive for Anaplasma marginale by PCR assay. This suggested that Anaplasma marginale organisms could be transmitted, via saliva, to cattle by the feeding ticks (Stich et al., 1993b). A set of primers for PCR amplification ofAnaplasma marginale DNA was designed from sequences contained within plasmid A1 (Aboytes-Torres, 1992). Aboytes-Torres (1992) reported high analytical sensitivity and specificity for detection ofAnaplasma marginale DNA by combining the PCR fragment amplification with detection by a radioactive single stranded oligonucleotide (SSO) probe (see Table 1 ). The combined PCR-SSO system has subsequently been used extensively in detecting the carder stage in experimentally or naturally infected Anaplasma marginalecattle. In addition, the PCR-SSO system has been utilized to screen Boophilus microplusticks forAnaplasma marginaleinfection (AboytesTortes, 1992). Buening et al. (1992) modified the PCR-SSO radioactive system to a PCR nonradioactive DNA probe assay to detect Anaplasma marginale carder cattle. Although not as sensitive as the PCR/SSO system, the nonradioactive system identified more carder animals than the commonly used complement fixation test. The advantages of the modified nonradioactive probe assay are that the probe is safe to handle, can be stored for prolonged periods of time (for at least 1 year), can be used repeatedly and no special disposal requirements are needed (Buening et al., 1992). The PCR/nonradioactive assay described by Buening et al. ( 1992 ) was later integrated into a multiplex PCR assay system for the simultaneous detection of Babesia bigemina, Babesia boris and Anaplasma marginale DNA in cattle blood samples (Figueroa et al., 1993b). 3. Nucleic acid probes for Babesia sp.

Nucleic acid probes for Babesia boris detection have been described (MeLaughlin et al., 1986; Holman et al., 1989; Jasmer et al., 1990; Petchpoo et al., 1992) and their sensitivity and specificity are outlined in Table 2. Sequence diversity, as determined by probe analysis of restriction enzyme profiles, was reported to exist between Mexican and Australian isolates of Babesia boris (Jasmer et al., 1990) between Mexican and Thai isolates (Petchpoo et al., 1992), and between Australian geographic isolates (Cowman et al., 1984; Dalrymple et al., 1992). Moreover, DNA probe detection of variable virulence in subpopulations of Babesia boris in isolates has been described (Carson et al., 1990). Hybridization studies using eDNA probes derived from poly(A+ ) RNA obtained from an avirulent strain of Babesia boris revealed polymorphisms both at the RNA level as well as at the DNA level among various Australian isolates of Babesia boris and their derivatives (Cowman et al., 1984). The authors also reported that a differentially expressed gene in avirulent isolates, although not nec-

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Table 2 Nucleic acid probes for Babesia sp. Probesize/label

Analytical sensitivity DNA

Specificity

Ref.

iEa or PPE b

pCB2 DNA RAc

20-100 pg 10 ng

0.01%

B. boris B. bigemina

McLaughlin et al., 1986

Bb-1, Bb-3 DNA, RA

12 pg

1 iE/10 MFd 1 × 103 iE

B. boris

Holman et al., 1989 Wagner et al., 1992

B. boris B. btgemina B. boris

Jasmer et al., 1990

150 iE

B. bigemina

Buening et al., 1990

0.25% 0.5%

B. equi B. equi

Posnett and Ambrosio, 1989

0.0025% 0.0025%

B. equi

Posnett et al., 1991

pBo6 2.75 Kb DNA, RA pBo25 1.2 Kb DNA, RA p16 6.3 Kb DNA, RA pSB20 1.6 Kb DNA, RA pEH21 1.5 Kb DNA, RA

100 pg 1 ng 100 pg 10 pg 0.49 ng 0.97 ng

pSB20 1.6 Kb DNA, RA pSE2 0.95 Kb DNA, RA pBC11 2.65 Kb DNA, RA pBCI91 3.4 Kb DNA, RA

0.25 ng 0.125 ng

0.12% 0.06%

B. caballi B. caballi

Posnett and Ambrosio, 1991

p16 6.0 Kb DNA, NRA c

1 ng

0.001%

B. bigemina

Figueroa et al., 1992a

p16 4.8 Kb DNA, RA

10pg

3× 105iF f

B. bigemina

Hodgson et al., 1992

pMUBI 6.0 Kb DNA, RA

25 pg

0.00025%

B. bovis

Petchpoo et al., 1992

Synthetic oligosto ssrRNA B,RA

20 pg RNA

0.00002%

B. btgemina

Reddy and Dame, 1992

1-10 iE

B. boris

Fahrimal et al., 1992

100

0.000001% 3 iE PCR h

B. bigemina

Figueroa et al., 1992b

< 1 ng

< 460 iE

B. boris

Wagner et al., 1992

0.00001% 30 iE MPCR i

B. bigemina

Figueroa et al., 1993b

0.000001% 3 iE PCR 30 iE MPCR

B. bovis

Figueroa et al,, 1993a,b

PCR/DNA probe, RA 711 bp/642 bp PCR/DNA probe, NRA 278 bp/170bp PCR 600 bp PCR/DNA probe, NRA 278 bp/170bp PCR/DNA probe, NRA 356 bp/291 bp

10 pg

"Infected erythrocytes. bpercent of parasitized erythrocytes. eRadioactive probe, aMicroscope fields magnification × 1000. eNonradioactive probe. qnfective form. SSmall subunit ribosomal RNA. hPolymerase chain reaction. ~Multiplex polymerase chain reaction.

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essarily involved in the attenuation phenomenon, could be utilized as a marker of avirulence. Genomic DNA was collected from a Babesia boris isolate in the progression from a virulent to an avirulent condition (attenuation) during serial passaging through splenectomized cattle (Carson et al., 1990). Southern blot analysis of genomic Babesia boris DNA using a marker of attenuation (eDNA probe) revealed that the signal of hybridization increased in intensity as the isolate was serially passaged in cattle in the progression from virulent parasites to an avirulent population (Carson et al., 1990). Dalrymple et al. (1992) described a eDNA probe, BovA 1 (600 bp) which would discriminate among strains in mixed populations of Babesia boris. They reported that a mixture of genetically distinct subpopulations of parasites are present commonly in Babesia boris infections of cattle, and such subpopulations can be distinguished with the DNA probe by restriction enzyme digestion analysis ofgenomic DNA (Dalrymple et al., 1992). To increase the sensitivity of DNA probes for detection of carrier cattle infected with Babesia boris, researchers have utilized the PCR assay to amplify minute amounts of Babesia boris target DNA (Fahrimal et al., 1992; Wagner et al., 1992; Figueroa et al., 1993a). By PCR, a 711 bp fragment was amplified from Babesia boris DNA samples of isolates from Texas, Mexico and Australia (Fahrimal et al., 1992). In addition, the 711 bp fragment was detected with a radioactive DNA probe which recognized sequences within the 711 bp amplified fragment. When blood samples of proven Babesia boris carders were analyzed by the PCR assay, overall positive PCR detection rates for carder cattle were 85% and over 95% when samples were analyzed once or twice (samples taken 1-2 weeks apart), respectively. Preliminary data on the use of PCR to amplify a 600 bp fragment of Babesia boris DNA has been reported (Wagner et al., 1992). The amplified product was species-specific and it could be visualized when as few as 460 infected erythrocytes were used as starting material for the PCR assay. Similarly, Figueroa et al. (1993a) described a PCR/nonradioactive DNA probe assay to specifically amplify and detect Babesia boris DNA sequences that are present within the gene that codes for the rhoptry protein p60 (Suarez et al., 1991 ). The assay was highly sensitive and utilized in the detection of experimentally infected Babesia boris carder cattle (Figueroa et al., 1993a). This PCR/nonradioactive DNA probe assay has subsequently been incorporated into a multiplex PCR system (Figueroa et al., 1993b) which was recently applied on bovine blood samples collected within a bovine babesiosis endemic zone (Figueroa et al., 1993c) for the simultaneous detection of Babesia bigemina, Babesia boris and Anaplasma marginale DNA. The sensitivity and specificity of nucleic acid probes for Babesia bigemina detection is shown in Table 2. Buening et al. (1990) reported the characterization of a repetitive DNA probe (pl 6) for detection of Babesia bigemina in infected erythrocytes. The radioactive 6.3 kbp insert DNA probe was shown to be speciesspecific and hybridized to Babesia bigemina isolates from Mexico, Texas, Puerto Rico, St. Croix, Costa Rica and Kenya. Hodgson et al. (1992) tested a subfrag-

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ment of the Babesia bigemina cloned DNA (Buening et al., 1990) in order to detect and quantitate Babesia bigemina infective forms in the salivary glands of the tick vector Boophilus microplus. The results indicated that the radioactive probe could detect the DNA of at least 10 3 Babesia bigemina infective forms extracted from the salivary glands of both the nymph and adult stages of infected Boophilus microplus ticks. Although not as sensitive as the radioactive probe, a derivative of p 16 has been evaluated as a nonradioactive DNA probe to monitor Babesia bigemina infection in experimentally inoculated cattle (Figueroa et al., 1992a). It was determined that Babesia bigemina infection in these cattle was more often detected positive by nucleic acid hybridization with the nonradioactive DNA probe than by light microscopy examination of peripheral blood smears. The nonradioactive DNA probe was then used in an epidemiological survey to determine the Babesia bigemina prevalence in indigenous cattle from a babesiosis endemic area (Ramos et al., 1992). Although able to detect infection in very young animals (less than 3 months old), it was found that the analytical sensitivity of the Babesia bigemina probe was too low for its use in large-scale epidemiological studies of the disease. These results indicated that in order to detect Babesia bigemina carrier animals a more sensitive assay would have to be utilized. Figueroa et al. (1992b) developed a PCR/nonradioactive DNA probe assay in which primers derived from a sequence contained in p 16 insert (Buening et al., 1990) were used to amplify a 278 bp fragment of the Babesia bigemina genomic target DNA. With the increased analytical sensitivity reported for the PCR/DNA probe assay (Table 2 ), six experimentally infected cattle were detected as carriers of Babesia bigemina parasites 11 months after infection. As mentioned above, the Babesia bigemina PCR detection system was subsequently incorporated into a multiplex PCR system for the simultaneous detection ofBabesia bigemina, Babesia boris andAnaplasma marginale in experimentally infected cattle (Figueroa et al., 1993b). A field study was conducted by Figueroa et al. (1993c) to evaluate the feasibility of the multiplex PCR system in detecting cattle infected with any of the three microorganisms in a hemotropic diseases endemic area in southern Mexico. The multiplex PCR assay system detected cattle carrying single, double or triple infections with an overall prevalence rate of 66.7%, 60.1% and 59% for Babesia bigemina, Babesia boris and Anaplasma marginaleinfection, respectively. A method based on the detection of small subunit ribosomal RNA of Babesia bigernina was developed by Reddy and Dame (1992), and its sensitivity and specificity is listed in Table 2. A mixture of three synthetic, radioactive oligonucleotides was used to hybridize to total RNA purified from blood samples of three Babesia bigemina experimentally infected calves. The pooled oligonucleotides detected parasite RNA in blood samples as early as 2 days after inoculation, and thereafter until days 14-16 postinoculation. Interestingly, the probes detected infection in one of the calves in the absence of clinical symptoms and in the absence of infected red cells in microscopic examination of thick smears (Reddy and Dame, 1992). This type of result had also been observed in experimentally infected steers in which a PCR-amplified product was detected in several animals

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which had inapparent clinical manifestations and which were serologically and microscopically negative (Figueroa et al., 1992b). Repetitive DNA probes for Babesia equi were constructed by Posnett and Ambrosio (1989 ). Their sensitivity and specificity for Babesia equi is described in Table 2. The application of two repetitive DNA probes which were specific for Babesia equi DNA was later reported by Posnett et al. ( 1991 ). Artificially infected horses were monitored by probing blood samples collected before, during and after the infection with Babesia equi. It was found that the probes could detect low, microscopically undetectable levels of parasites in the infected horse blood. Owing to their superior sensitivity, compared with the microscopic examination of blood smears, the ability of the probes to detect Babesia equi in field infections was tested. The probes detected Babesia equi DNA in blood samples collected from clinically infected horses as well as from carrier animals. Moreover, the Babesia equi DNA probes did not hybridize to blood collected from horses infected with Babesia caballi (Posnett et al., 1991 ).

4. Nucleic acid probes for Cowdria rumlnantium

Conventional diagnostic techniques such as the light microscopy examination of stained blood smears, or stained preparations from the tick vector will not detect C. ruminantium infection. The development of diagnostic DNA probes for Cowdria organisms were first reported by Ambrosio and Wilkins (1989). Recombinants carrying Cowdria DNA sequences were tested for their suitability as specific rickettsial probes. Waghela et al. ( 1991 ) described the construction of recombinant clones containing DNA sequences of C. ruminantiurn (Table 3 ). A specific DNA probe (pCS20) was utilized to detect Cowdria DNA in the midgut ofAmblyomma variegatum adult ticks which had been fed as larvae or nymphs on goats experimentally infected with C. ruminantium. The pCS20 probe was later shown to cross-hybridize to purified DNAs of C. ruminantium from endemic areas of Zimbabwe, South Africa, Nigeria, and the Caribbean island of Guadeloupe (Mahan et al., 1992). In addition, it was shown that C. ruminantium DNA could be detected with the pCS20 probe in preparations made from plasma samples collected from infected sheep. Mahan et al. (1993) have also shown that a 279 bp C. ruminantium DNA fragment can be PCR-amplified from sheep plasma samples by using a specific set of oligonucleotides to prime the reaction. Yunker et al. (1993) showed that the pCS20 DNA probe could also be used to detect C. rurninantium infection in Amblyomma hebraeum tick midgut and tick salivary glands. They suggested that by using a sensitive, efficient and accurate DNA-based method for detection of infection, the estimation of tick infection rates and endemic stability can now be reliably established.

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Table 3 Nucleic acid probes for Cowdria ruminantium

Probe size/label

Sensitivity

Specificity

Ref.

C ruminantium ( + ) TM b

Waghela et al., 1991

C. ruminantium

Mahan et al., 1992

DNA (ng) pCS20 1.3 kb DNA, RA a pCR9 754 bp DNA, RA pCS20 1.3 kb DNA, RA

1

(+)SP c pCS20 1.3 kb DNA, RA

1

C. ruminantium

Yunker et al., 1993

( + ) TM, TSG d PCR e 279 bp, AGE f

C. ruminantium

Mahan et al., 1993

(+)SP

aRadioactive probe. bDNA extracted from tick midgut. CDNA extracted from sheep plasma. dDNA extracted from tick salivary gland. ePolymerase chain reaction. rAgarose gel electrophoresis.

5. Nucleic acid probes for Theileria sp. Conrad et al. ( 1987, 1989) (also see Table 4) have described the use of DNA probes to detect genome diversity in Theileria parva, to distinguish among T. parva stocks derived from infected cattle, and to characterize T. parva parasites derived from infected buffalo. By using DNA probes on restriction enzyme digested DNA from cloned Theileria-infected lymphoblastoid cell lines, they showed there was a marked restriction fragment length polymorphism (RFLP) in antigenically different T. parva subclones which had been derived from the same cloned organisms. RFLPs were also observed in T. parva isolates obtained from infected buffalo when reacted with the probes. This indicated that there was considerable genome diversity in the populations derived from buffalo. It was suggested that the differences could have resulted either from deletions and/or rearrangements in the parasite DNA, by genetic recombination, and by the presence of a mixed parasite population which may have been transmitted from buffalo to cattle. It was concluded that parasites collected from cattle which had been infected with buffalo-derived T. parva lawrenci did not show genotypic characteristics which could be used to distinguish the latter species from cattle-derived T. parva parva and T. parva bovis parasites. The same probes (Conrad et al., 1989) were tested on samples collected from salivary glands of Theileria-infected R. appendiculatus ticks (Chen et al., 1991 ). When evaluated, the probes detected T. parva sporoblast and sporozoite DNA in salivary glands of adult ticks which had been fed as nymphs on calves infected

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Table 4 Nucleic acid probes for Theileria sp. Probe size/label

Sensitivity

Specificity

Ref.

pgTpM-23 2.3 kb DNA, RA" lgTpM-58 5.8 kb DNA, RA

-

T parva genome diversity

Conrad et al., 1987,

1 iAb/Salivary gland

T. parva

Chen et al., 1991

T. sergenti

Hirano et al., 1991

pgTpM-23 2.3 kb DNA, RA lgTpM-58 5.8 kb DNA, RA

- 5 × 104 Sporozoites

pTsl I 2.0 kb DNA, NRA c

2 pg

1989

O.0OO8%a P C R / R A probe 405 bp/20-mer oligo 233 bp/p67 DNA probe

Single parasite

T. parva ( + ) CC ~

Bishop et al., 1992

P C R / N R A probe 875 bp/C-2 eDNA 17mer 1347 oligo RA 18mer 1348 oligo RA 17mer I11264 oligo RA 18mer 1519 oligo RA

0.5 lag 0.00009% d 100-1000 iC e 100-1000 iC f 100-1000 iCf 4000 iC

T. sergenti T. buffeli T. annulata T. parva

Tanaka et al., 1993

T. taurotragi

Theileria sp. (buffalo)

Allsopp et al., Allsopp et al., AUsopp et al., Allsopp et al.,

1993 1993 1993 1993

"Radioactive probe. bInfected acinus. • lonradioactive probe. °Percent parasitemia. •Carder cattle. qnfected cells.

with one of six different stocks of T. parva. The degree of correlation between the results obtained by microscopic examination and DNA probe hybridization ranged from 84 to 100%. The two probes had sensitivities of 91.6-94.1% and speeificities of 92.1-97%. In addition, the probes detected one infected acinus in a salivary gland. Restriction fragment length polymorphisms have also been detected among T. parva stocks using different nucleic acid probes (Bishop et al., 1993). Twenty different RFLPs were observed among 18 DNA samples from schizont-infected lymphocytes and six samples from piroplasm DNA when blots containing EcoR1 digested DNA were hybridized with a repetitive DNA probe. Heterogeneity in T. parva stocks was also found when restriction enzyme digests were subjected to Southern blotting and hybridized with a small subunit ribosomal RNA gene and telomedc oligonudeotide probes (Bishop et al., 1993). Eight T. parva stocks could be separated into four groups according to their size polymorphism. The authors suggested that these T. parva DNA probes could be a useful means for determining the genetic similarities of field isolates to reference stocks of T. parva. They also mentioned that monitoring of the genetic composition of T. parva stabilates used as immunizing agents as well as the characterization of parasites isolated from cattle immunized against East Coast Fever, could be accomplished by using the T. parm nucleic acid probes.

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A practical method for carrier state detection of cattle infected with T. parva in Africa has been developed by Bishop et al. ( 1992 ). Amplification of parasite DNA by PCR, using specific oligonucleotide primers for T. parva repetitive DNA sequences or gene specific primers, demonstrated parasites present at low parasitemias in carrier cattle. In addition, Bishop et al. (1992) were able to design a system in which probes specific for a particular T. parva stock, would differentially hybridize to a homologous PCR-amplified DNA fragment. Thus, stock-specific oligonucleotide probes which differed in sequence from other stocks and which more strongly hybridized to DNA of a homologous stock, would be of practical use for detection of, for example, cattle that had been vaccinated with a stabilate of a particular T. parva stock. The authors also showed that the PCR/ specific oligonucleotide probe system could be used on bovine lymph node biopsies taken from schizont-positive T. parva parva-infected animals as well as on dissected R. appendiculatus salivary glands containing T. parva infected acini. More recently, species-specific oligonucleotide probes directed to the small subunit ribosomal RNA (ssrRNA) sequences have been used by Allsopp et al. (1993). With this system, each of six species of Theileria that infect domestic cattle could be distinguished. By using species-specific oligonucleotide probes either to directly detect ssrRNA gene sequences or to detect PCR amplified products from Theileria ssrRNA genes, T. parva, T. annulata, T. mutans, T. taurotragi, Theileria sp. (buffalo) and a Theileria sp. (cattle) could be readily distinguished by nucleic acid hybridization. The authors suggested that these speciesspecific probes could be of great value for detecting the presence of different species in mixed Theileria infections in cattle. It was also reported that buffalo-derived T. parva (previously designated as T. parva parva) could not be differentiated from cattle-derived T. parva (previously T. parva lawrencei) with the use of the T. parva oligonucleotide probe (Allsopp et al., 1993 ). Nucleic acid probes have also been described for detection of Theileria sergenti infection in cattle (Hirano et al., 1991 ). Sensitive probes were selected and evaluated for genome diversity analysis of T. sergenti DNA in Japan (Matsuba et al., 1992). The comparison of patterns of restriction enzyme digested piroplasm DNA obtained when hybridized with the specific probe, revealed RFLPs between the stocks of T. sergenti analyzed, thus identifying DNA diversity among the different parasite stocks collected in geographically different areas, but not between stocks isolated from the same grazing area. Tanaka et al. ( 1993 ) went further and described a nonradioactive PCR/DNA probe method for the detection of T. sergenti infection in cattle. The method was proved to be significantly more sensitive as a diagnostic tool than the microscopic examination of blood smears. Owing to its sensitivity the PCR/DNA probe method was tested on T. sergentiinfected cattle, detecting cattle persistently infected (Tanaka et al., 1993 ). References Aboytes-Torres, R., 1992. Development and practical applications of a PCR-based diagnostic system for the study of chronic bovine anaplasmosis. Ph.D. Dissertation, University of Missouri-Columbia.

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Aboytes-Torres, R. and Buening, G.M., 1990. Development of a recombinant Anaplasma marginale DNA probe. Vet. Microbioi., 24:391-408. Aboytes-Torres, R., Buening, G.M., Alvarez, J.A. and Vega, C.A., 1989. Recombinant DNA probe construction and application in anaplasmosis research. Proc. of the 8th National Veterinary Hemoparasite Disease Conference, St. Louis, MO. Veterinary Hemoparasite Disease Research Workers Organization, St. Louis, MO, 10-12 April 1989, pp. 305-316. Allsopp, B.A., Baylis, H.A., Allsopp, M.T.P.E., Cavalier-Smith, T., Bishop, R.P., Carrington, D.M., Sohanpal, B. and Spooner, P., 1993. Discrimination between six species of Theileria using oligonucleotide probes which detect small subunit ribosomal RNA sequences. Parasitology, 107:157165. Ambrosio, R.E. and Wilkins, S.C., 1989. The isolation of nucleic acid sequences specific for Cowdria ruminantium. Proc. of the 8th National Veterinary Hemoparasite Disease Conference, St Louis, MO, 10-12 April 1989, pp. 335-340. Ambrosio, R.E., Visser, E.S., Koeldaoven, Y. and Kocan, K.M., 1988. Hybridization of DNA probes to A. marginale isolates from different sources and detection in Dermacentor andersoni ticks. Onderstepoort J. Vet. Res., 55: 227-229. Barbet, A.F. and Allred D., 1991. The mspl multigene family ofAnaplasma marginale: nucleotide sequence analysis of an expressed copy. Infect. Immun., 59: 971-976. Barbct, A.F., Palmer, G.H., Myler, P.J. and McGuire, T.C., 1987. Characterization of an immunoprotective protein complex ofAnaplasma marginale by cloning and expression of the gene coding for polypeptide Am 105L. Infect. Immun., 55:2428-2435. Bishop, R., Sohanpal, B., Kariuki, D.P., Young, A.S., Nene, V., Baylis, H., Allsopp, B.A., Spooner, P.R., Dolan, T.T. and Morzaria, S.P., 1992. Detection of a carrier state in Theileriaparva-infected cattle by the polymerase chain reaction. Parasitology, 104:215-232. Bishop, R., Sohanpal, B., AUsopp, B.A., Spooner, P.R., Dolan, T.T. and Morzaria, S.P., 1993. Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies. Parasitology, 107:19-3 I. Buening, G.M., Barbet, A., Myler, P., Mahan, S., Nene, V. and McGuire, T.C., 1990. Characterization of a repetitive DNA probe for Babesia bigemina. Vet. Parasitol., 36:11-20. Buening, G.M., Aboytes-Torres, R., Figueroa, J.V. and Allen, L.W., 1992. A PCR amplification/nonradioactive DNA probe assay to detect Anaplasma marginale carriers. Proc. 96th Annual Meeting of the United States Animal Health Association, Louisville, KY, 31 October-6 November 1992. Carter Printing Company, richmond, VA, pp. 287-294. Callow, L.L., 1984. Protozoan and rickettsial diseases. In: Australian Bureau of Animal Health, Animal Health in Australia, Vol. 5, Australian Government Publishing Service, Canberra, Australia, pp. 121-216. Carson, C.A., Timms, P., Cowman, A.F. and Stewart, N.P., 1990. Babesia bovis: Evidence for selection of subpopulations during attenuation. Exp. Parasitoi., 70:404-410. Chen, P.P., Conrad, P.A., Ole-MoiYoi, O.K., Brown, W.C. and Dolan, T.T., 1991. DNA probes detect Theileria parva in the salivary glands of Rhipicephalus appendiculatus ticks. Parasitol. Res., 77: 590-594. Conrad, P.A., Iams, IC, Brown, W.C., Sohanpal, B. and Ole-MoiYoi, O.K., 1987. DNA probes detect genomic diversity in Theileriaparva stocks. Mol. Biochem. Parasitol., 25:213-226. Conrad, P.A., Ole-MoiYoi, O.K., Baldwin, C.L., Dolan, T.T., O'Callaghan, C.J., Njamunggeh, R.E.J., Grootenhuis, J.G., Stagg, D.A., Leitch, B.L. and Young, A.S., 1989. Characterization of buffaloderived tbeilerial parasites with monoclonal antibodies and DNA probes. Parasitology, 98: 179188. Cowman, A.F., Timms, P. and Kemp, D.J., 1984. DNA polymorphisms and subpopulations in Babesia bovis. Mol. Biochem. Parasitol., 11: 91-103. Dalrymple, B.P., Jorgensen, W.K., de Vos, A.J. and Wright, I.G., 1992. Analysis of the composition of samples of Babesia boris and the influence of different environmental conditions on genetically distinct subpopulations. Int. J. Parasitol., 22:731-737. Eriks, I.S., Palmer, G.H., McGuire, T.C., Allred, D.R. and Barbet, A.F., 1989. Detection and quanti-

90

J. II..Figueroa, G.M. Buening / VeterinaryParasitology 57 (1995) 75-92

tation ofAnaplasma marginale in carrier cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Eriks, I.S., Stiller, D. and Palmer, G.H., 1993. Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission. J. Clin. Microbiol., 31:2091-2096. Fahrimal, Y., Goff, W.L. and Jasmer, D.P., 1992. Detection ofBabesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA. J. Clin. Microbiol., 30:1374-1379. Figueroa, J.V., Chieves, L.P., Byers, P.E., Frerichs, W.H. and Buening, G.M., 1992a. Evaluation of a DNA-based probe for the detection of cattle experimentally-infectedwith Babesia bigemina. Ann. N.Y. Acad. Sci., 653: 131-145. Figueroa, J.V., Chieves, L.P., Johnson, G.S. and Buening, G.M., 1992b. Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification. J. Clin. Microbiol., 30: 25762582. Figueroa, J.V., Chieves, L.P., Johnson, G.S., Goff, W.L. and Buening, G.M., 1993a. Polymerase chain reaction-based diagnostic assay to detect cattle chronically infected with Babesia boris. Rev. Latamer. Microbiol., 36: 47-55. Figueroa, J.V., Chieves, L.P., Johnson, G.S. and Buening, G.M., 1993b. Multiplex polymerase chain reaction based assay for the detection ofBabesia bigemina, Babesia boris and Anaplasma marginale DNA in bovine blood. Vet. Parasitol., 50:69-81. Figueroa, J.V., Alvares, J.A., Ramos, J.A., Vega, C.A. and Buening, G.M., 1993c. Use of a multiplex polymerase chain reaction-based assay to conduct epidemiological studies on bovine hemoparasites in Mexico. Rev. Elev. Med. Vet. Pays Trop., 46( 1-2): 71-75. Goff, W.L., Barbet, A.F., Stiller, D., Palmer, G.H., Knowles, D., Kocan, K.M., Gorham, J.R. and McGuire, T.C., 1988. Detection ofAnaplasma marginale-infected tick vectors by using a cloned DNA probe. Proc. Natl. Acad. Sci. USA, 85:919-923. Goff, W.L., Stiller, D., Roeder, R.A., Johnson, L.W., Falk, D., Gorham, J.R. and McGuire, T.C., 1990. Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle. Vet. Microbiol., 24:381-390. Hirano, A., Kirisawa, R., Matsuba, T., Komatsu, R., Tanaka, M., Takahashi, K., Kawakami, Y. and Onuma, M., 1991. Evaluation of high sensitive DNA probe for the detection of Theileria sergenti infection in cattle. J. Vet. Med. Sci., 53: 933-935. Hodgson, J.L., Stiller, D., Jasmer, D.P., Buening, G.M., Wagner, G.G. and McGuire, T.C., 1992. Babesia bigemina: Quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe. Exp. Parasitol., 74:117-126. Holman, P.J., Tripp, C.A., Rice-Ficht, A.C. and Wagner, G.G., 1989. A specific DNA probe for detection ofBabesia bovis in blood samples. Proc. of the 8th National Veterinary Hemoparasite Disease Conference, St Louis, MO. Veterinary Hemoparasite Disease Research Workers Organization, St. Louis, MO, 10-12 April 1989, 407 pp. Irvin, A.D. and Cunningham, M.P., 1981. East Coast Fever. In: M. Ristic and I. Mclntyre (Editors), Diseases of Cattle in the Tropics. Martinus Nijhoff, Boston, MA, pp. 393-410. Jasmer, D.P., Reduker, D.W., Goff, W.L., Stiller, D. and McGuire, T.C., 1990. DNA probes distinguish geographical isolates and identify a novel molecule of Babesia bovis. J. Parasitol., 76: 834841. Kieser, S.T., Ericks, I.S. and Palmer, G.H., 1990. Cyclic rickettsemia during persistent Anaplasma marginale infection of cattle. Infect. Immun., 58:1117-1119. Kocan, K.M., Stiller, D., Goff, W.L., Edwards, W., Wickwire, K.B., Stich, W., Yellin, T.N., Ewing, S.A., Palmer, G.H., Barron, S.J., Hair, J.A. and McGuire, T.C., 1989. A review of the developmental cycle ofAnaplasma marginale in Dermacentor spp. ticks. Proc. of the 8th National Veterinary Hemoparasite Disease Conference, St Louis, MO, 10-12 April 1989, pp. 129-148. Mahan, S.M., Waghela, S.D., McGuire, T.C., Rurangirwa, F.R., Wassink, L.A. and Barbet, A.F., 1992. A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep. J. Clin. Microbiol., 30:981-986. Mahan, S.M., Tebele, N., Mukwedeya, D., Semu, S., Nyathi, C.B., Wassink, L.A., Kelly, P.J., Peter, T. and Barbet, A.F., 1993. An immunoblotting diagnostic assay for heartwater based on the ira-

J. V. Figueroa, G.M. Buening / Veterinary Parasitology 57 (1995) 75-92

91

munodominant 32-kiiodalton protein of Cowdria ruminantium detects false positives in field sera. J. Clin. Microbiol., 31: 2729-2737. Matsuba, T., Kawakami, Y., lwai, H. and Onuma, M., 1992. Genomic analysis of Theileria sergenti stocks in Japan with DNA probes. Vet. Parasitol., 4 l: 35-43. McLaughlin, G.L., Edlind, T.D. and Ihler, G.M., 1986. Detection ofBabesia bovis using DNA hybridization. J. Protozool., 33: 125-128. Petchpoo, W., Tan-ariya, P., Boonsaeng, V., Brockelman, C.R., Wilairat, P. and Panyim, S., 1992. A specific DNA probe which identifies Babesia boris in whole blood. Vet. Parasitol., 42:189-198. Posnett, E.S. and Ambrosio, R.E., 1989. Repetitive DNA probes for the detection of Babesia equi. Mol. Biochem. Parasitol., 34: 75-78. Posnett, E.S. and Ambrosio, R.E., 1991. DNA probes for the detection of Babesia caballi. Parasitology, 102: 357-365. Posnett, E.S., Fehrsen, J., de Waal, D.T. and Ambrosio, R.E., 1991. Detection of Babesia equi in infected horses and carrier animals using a DNA probe. Vet. Parasitol., 39: 19-32. Purnell, R.E., 1981. Babesiosis in various hosts. In: M. Ristic and J.P. Kreier (Editors), Babesiosis. Academic Press, New York, pp. 25-64. Ramos, J.A., Alvarez, J.A., Figueroa, J.V., Solis, J., Rodriguez, R.I., Hernandez, R., Buening, G.M. and Vega, C.A., 1992. Evaluation of a colorimetric Babesia bigemina-DNA probe within an epidemioiogical survey. Mem. Inst. Oswaido Cruz. 87 (Suppl. III): 213-217. Reddy, G.R. and Dame, J.B., 1992. rRNA-based method for sensitive detection of Babesia bigemina in bovine blood. J. Clin. Microbiol., 30:1811-1814. Ristic, M., 1981 a. Babesiosis. In: M. Ristic and I. McIntyre (Editors), Diseases of Cattle in the Tropics. Martinus Nijhoff, Boston, MA, pp. 443-468. Ristic, M., 1981b. Anaplasmosis. In: M. Ristic and I. McIntyre (Editors), Diseases of Cattle in the Tropics. Martinus Nijhoff, Boston, MA, pp. 327-344. Shompole, S., Waghela, S.D., Rurangirwa, F.R. and McGuire, T.C., 1989. Cloned DNA probes identify Anaplasma ovis in goats and reveal a high prevalence of infection. J. Clin. Microbiol., 27: 2730-2735. Stich, R.W., Bantle, J.A., Kocan, K.M., Eriks, I.S. and Palmer, G.H., 1991. Preliminary development of a polymerase chain reaction assay for Anaplasma marginale in ticks. Biotechnol. Tech., 5: 269274. Stich, R.W., Bantle, J.A., Kocan, K.M. and Fekete, A., 1993a. Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in hemolymph of Dermacentor andersoni (Acari: Ixodidae ) with the polymerase chain reaction. J. Med. Entomol., 30:781-788. Stich, R.W., Sauer, J.R., Bantle, J.A. and Kocan, K.M., 1993b. Detection ofAnaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae ) with the polymerase chain reaction. J. Med. Entomol., 30: 789-794. Suarez, C.E., Palmer, G.H., Jasmer, D.P., Hines, S.A., Perryman, L.E. and McElwain, T.F., 1991. Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes. Mol. Biochem. Parasitol., 46: 45-52. Tanaka, M., Onoe, S., Matsuba, T., Katayama, S., Yamanaka, M., Yonemichi, H., Hiramatsu, K., Baek, B., Sugimoto, C. and Onuma, M., 1993. Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA. J. Clin. Microbiol., 31: 25652569. Uilenberg, G., 1981 a. Heartwater disease. In: M. Ristic and I. McIntyre (Editors), Diseases of Cattle in the Tropics. Martinus Nijhoff, Boston, MA, pp. 345-360. Uilenberg, G., 1981b. Theileria infections other than East Coast Fever. In: M. Ristic and I. McIntyre (Editors), Diseases of Cattle in the Tropics. Martinus Nijhoff, Boston, MA, pp. 411-427. Uilenberg, G., 1983. Heartwater (Cowdria ruminantium infection): Current status. Adv. Vet. Sci. Comp. Med., 27: 427-480. Visser, E.S. and Ambrosio, R.E., 1987. DNA probes for the detection of Anaplasma centrale and Anaplasma marginale. Onderstepoort J. Vet. Res., 54: 623-627. Visser, E.S. and Ambrosio, R.E., 1989. Characterization of DNA probes for Anaplasma centrale and

92

J.V. Figueroa, G.M. Buening / VeterinaryParasitology 5 7 (1995) 75-92

Anaplasma margmale. Proc. of the 8th National Veterinary Hemoparasite Disease Conference, St. Louis, MO, 10-12 April 1989, pp. 323-333. Visser, E.S., Ambrosio, R.E. and De Waal, D.T., 1991. An Anaplasma centrale DNA probe that differentiates between Anaplasma ovis and Anaplasma rnarginale DNA. Vet. Microbiol., 28: 313325. Waghela, S.D., Rurangirwa, F.R., Mahan, S.M., Yunker, C.E., Crawford, T.B., Barber, A.F., Burridge, M.J. and McGuire, T.C., 1991. A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks. J. Clin. Microbiol., 29: 2571-2577. Wagner, G., Cruz, D., Holman, P., Waghela, S., Perrone, J., Shompole, S. and Rurangirwa, F., 1992. Nonimmunologic methods of diagnosis of babesiosis. Mem. Inst. Oswaldo Cruz, 87 (Suppl. III ): 193-199. Yunker, C.E., Mahan, S.M., Waghela, S.D., McGuire, T.C., Rurangirwa, F.R., Barbet, A.F. and Wassink, L.A., 1993. Detection of Cowdria ruminantium by means ofa DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in South Africa. Epidemiol. Infect., 110: 95-104.