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J Clin Microbiol. Apr 2000; 38(4): 1539–1544.
PMCID: PMC86485

Detection of the Agent of Heartwater, Cowdria ruminantium, in Amblyomma Ticks by PCR: Validation and Application of the Assay to Field Ticks

Abstract

We have previously reported that the pCS20 PCR detection assay for Cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector Amblyomma ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of C. ruminantium originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of Ehrlichia chaffeensis, which is closely related to C. ruminantium. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 107 to 104 organisms but dropped to 61 and 28%, respectively, with ticks bearing 103 and 102 organisms. The assay also detected C. ruminantium in collections of Amblyomma hebraeum and Amblyomma variegatum field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for C. ruminantium in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.

Xenodiagnosis has long been the standard technique for the detection in ticks of the tick-borne rickettsia Cowdria ruminantium, the causative agent of heartwater (cowdriosis), an economically important disease of domestic and wild ruminants in Africa and in the Caribbean (11, 12, 13, 44, 45, 50). By inoculating susceptible small ruminants or mice with homogenates of the Amblyomma species tick vectors and monitoring clinical disease or seroconversion, the existence of C. ruminantium infection in the Caribbean has been confirmed (4, 9) and estimates of tick infection prevalence have been obtained (9, 10, 14, 17, 19, 20, 21, 47). However, xenodiagnosis is expensive and cumbersome, particularly in ruminants, and is slow (taking up to 6 weeks to complete). Furthermore, xenodiagnosis in mice has recently been shown to have low sensitivity and is unreliable for the detection of infection (38). More-expedient DNA probe and PCR assays based on the pCS20 and MAP 1 DNA sequences of C. ruminantium have been developed recently and have been used to detect infections in ticks and animals (1, 31, 37, 38, 42, 43, 52, 60, 62). PCR assays, which have greater sensitivity than DNA probe assays (52), may be useful tests for laboratory and field epidemiological investigations which are needed for improved understanding of heartwater epidemiology and the impact of new control measures (49). However, prior to extensive use, PCR assays have to be fully validated and their ability to detect infection in field ticks needs to be assessed. Here we determine the reliability of the pCS20 PCR assay and apply it to the detection of C. ruminantium in Amblyomma hebraeum and Amblyomma variegatum ticks, the major vectors of heartwater, collected from various locations in southern Africa.

MATERIALS AND METHODS

PCR assay.

The PCR assay was performed as previously described (52), with some modifications. Briefly, DNA was extracted from the individual tick tissue samples using the QIA-amp PCR DNA extraction tissue kits (Qiagen, Hilden, Germany). The tissues were digested at 55°C for 16 h, followed by a 1-h incubation at 70°C. DNA was extracted from the digests as recommended except that, for nymphs, the volumes for digestion and extraction were reduced by half, and elution of DNA from the columns (for all ticks) was performed twice with 50 μl of elution buffer at 70°C. The purified DNA eluate was stored at 4°C until analysis.

For PCRs, 5 μl (5%) of the purified DNA of each adult tick and 20 μl (20%) of the DNA from each nymph were used as the template in reactions with the primers AB128 (5′-ACTAGTAGAAATTGCACAATCTAT-3′) and AB129 (5′-TGATAACTTGGTGCGGGAAATCCTT-3′). These primers flank a 279-bp fragment within open reading frame 2 of the 1,306-bp pCS20 sequence of C. ruminantium (43, 60). PCRs were performed for 45 cycles, except that the MgCl2 and primer concentrations were reoptimized for DNA purified from each tick instar by the Qiagen method. For PCRs with adult tick DNA the primer and MgCl2 concentrations were 0.3 μM and 2.0 mM, respectively, and for nymphs the concentrations were 0.5 μM and 2.0 mM, respectively. Additionally, denaturation of sample DNA prior to amplification was achieved by incubation of the reaction mixtures for 1 min at 94°C in the PCR heating block (model 480; Perkin-Elmer Cetus Corp., Norwalk, Conn.) before the first cycle. Each set of PCRs included negative and positive reagent controls (reactions with no DNA and with 0.1 ng of C. ruminantium DNA, Plumtree isolate, respectively) and sample controls containing DNA from laboratory-reared, uninfected male, female, or nymph A. hebraeum or A. variegatum ticks, as appropriate. Amplification of C. ruminantium DNA was detected by dot blotting 40 μl (80%) of NaOH-denatured PCR products onto nylon membranes followed by hybridization with the pCS20 DNA probe, as previously described (42, 52, 60, 62). Hybridization was detected by exposure of the blots to X-ray film (Kodak Biomax) for 1 to 7 days.

Validation of PCR assay (i) Detection of diverse C. ruminantium isolates.

To determine the ability of the PCR assay to detect DNA from geographically diverse C. ruminantium isolates, DNA prepared from cultured C. ruminantium isolated from ticks collected at 13 study sites in southern Africa was tested in the PCR assay (Tables (Tables11 and and2).2). The test was also performed on DNA from 24 other cultured C. ruminantium isolates made previously from Amblyomma species ticks or blood collected in heartwater-endemic regions of 10 countries in sub-Saharan Africa and two islands in the Caribbean (Table (Table2).2). Supernatant (0.5 ml) from terminal bovine endothelial cell cultures of each isolate was centrifuged at 20,000 × g for 20 min, and DNA was extracted from the pellets using QIA-amp kits and tested by PCR, as described above.

TABLE 1
Sites for collection of Amblyomma ticks in southern Africa
TABLE 2
Origin of C. ruminantium isolates tested by PCR

(ii) Specificity.

The specificity of the PCR assay was determined by testing 100 laboratory-reared, uninfected adult A. hebraeum ticks (50 males and 50 females) and 100 uninfected A. hebraeum nymphs. Tissue samples from 10 extra uninfected adult ticks (5 male and 5 female), each spiked with 106 cell culture-derived C. ruminantium organisms (Plumtree isolate) which were purified on Percoll gradients and enumerated by staining with acridine orange (33), were processed concurrently with the adult tick samples to simulate the processing of real samples and determine the potential for cross-contamination under routine conditions. Similarly, 10 extra nymph samples spiked with 106 C. ruminantium organisms were processed together with the uninfected nymphs. The 95% confidence intervals of the specificity estimates for adults and nymphs were calculated by the Bonferroni method.

(iii) Specificity against Ehrlichia chaffeensis.

A specificity test against E. chaffeensis, which is phylogenetically and antigenically closely related to C. ruminantium, was performed (27, 28, 34, 58). PCRs were performed, as described above, with C. ruminantium AB128 and AB129 primers on 1 and 10 ng of E. chaffeensis DNA purified from organisms cultured in vitro in canine DH82 cells and on 1 and 10 ng of C. ruminantium DNA (Zimbabwean Crystal Springs isolate). PCRs were also performed on 1 and 10 ng of E. chaffeensis DNA and on C. ruminantium DNA with the primers 5′-GAGTACGGATCCGCAATTTTTCTAGGATATTCC-3′ and 5′-ATCAGACTGCGGCCGCATGTAATTAGCGATAGAAACACCA-3′, which amplify a 727-bp fragment from E. chaffeensis containing a gene homologous to the C. ruminantium MAP 2 gene (5, 35). Reactions with E. chaffeensis primers were performed in 100 μl consisting of 1× buffer (20 mM Tris-HCL [pH 8.8], 10 mM KCL, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 100-μg/ml bovine serum albumin), 1.25 U of pfu DNA polymerase (Stratagene, La Jolla, Calif.), a 200 μM concentration of each deoxynucleoside triphosphate, and a 1.0 μM concentration of each primer. The reaction mixtures were overlayed with mineral oil and incubated at 94°C for 3 min before undergoing 10 cycles each of 15 s at 94°C, 1 min at 43°C, and 1 min at 72°C followed by 25 cycles each of 15 s at 94°C, 1 min at 49°C, and 1 min at 72°C, and finally 7 min at 72°C. As a further positive control, PCRs were also performed, under conditions described above for C. ruminantium primers, on 1 and 10 ng of E. chaffeensis DNA and C. ruminantium DNA with general Ehrlichia primers E2 (5′-GTGGCAGACGGGTGAGTAATGC-3′) and E3 (5′-GGTAACGTCAATATCTTCCC-3′), which amplify a 350-bp fragment from the conserved region of the 16S rRNA gene of members of the tribe Ehrlichia (L. A. Matthewman, N. Lally, K. Sumption, P. J. Kelly, and D. Raoult, unpublished data; 54). Twenty microliters of the products of the above PCRs were electrophoresed through agarose gels, Southern blotted, and hybridized with the pCS20 DNA probe.

(iv) Sensitivity.

To determine the sensitivity of the PCR assay under routine sample-handling conditions, dissected tissues from laboratory-reared, uninfected adult male and female A. hebraeum ticks were individually spiked with 10-fold serial dilutions of cell culture-derived, Percoll-purified C. ruminantium (Plumtree isolate) containing 102 to 107 organisms to simulate these levels of infection. In total, 100 ticks (50 male and 50 female) were spiked at each dilution. These were divided into batches of 8 to 15, which were tested separately. Confidence intervals (CI; 95%) for the sensitivity of the PCR assay at each infection level were calculated by the Bonferroni method, and negative and positive predictive values were determined for each level of tick infection.

Detection of C. ruminantium in field ticks.

Collections of A. hebraeum or A. variegatum adult male and flat adult female ticks were made from cattle, goats, or vegetation at 17 locations. These locations included large-scale commercial livestock ranches, livestock quarantine stations, and traditional farming (communal) areas in heartwater-endemic regions of Botswana, South Africa, Swaziland, Zambia, and Zimbabwe (Table (Table1).1). The ticks were tested for C. ruminantium infection by PCR as described above. To confirm C. ruminantium infection in each collection, attempts were made to isolate C. ruminantium by transmission to small ruminants. Sixty to 120 ticks from each site were fed on C. ruminantium-naive sheep or goats, which were then monitored daily for clinical signs of heartwater. Confirmation of infection was obtained by examination of brain smears prepared from biopsies performed during the febrile reaction or postmortem (53, 56). During the febrile stage of infection, plasma was collected from some of the animals and inoculated into bovine endothelial cell cultures to attempt isolation of C. ruminantium (8). Infection of the cultures was confirmed by examination of cell smears prepared after the development of cytopathic effects in the cell monolayers. Thereafter, the isolates were grown in continuous culture, as previously described (7).

RESULTS

Validation of the PCR assay. (i) Detection of diverse C. ruminantium strains.

The PCR assay amplified a 279-bp fragment from the DNA of each of the 37 isolates of C. ruminantium tested (Table (Table2).2). In each case, the amplified fragment hybridized with the pCS20 DNA probe (data not shown), confirming the conservation of the PCR assay primer target sites and the ability of the assay to detect C. ruminantium from throughout the distribution of heartwater.

(ii) Specificity.

Two of 100 uninfected adult ticks and 2 of 100 uninfected nymph ticks tested positive by PCR after DNA extraction and PCR testing together with tick samples spiked with culture-derived C. ruminantium organisms. The specificity of the PCR assay for both adult and nymph ticks was therefore 98%.

(iii) Specificity against E. chaffeensis.

No amplification was detected from 1 and 10 ng of E. chaffeensis DNA with C. ruminantium primers after agarose gel electrophoresis of the products and hybridization with the pCS20 DNA probe (Fig. (Fig.11 and and2).2). Amplification of a 727-bp product with primers for the MAP 2 gene homologue of E. chaffeensis confirmed the presence of E. chaffeensis DNA in the specificity test. These primers did not produce a product from C. ruminantium DNA that was detectable by gel electrophoresis, though the general Ehrlichia primers amplified a 350-bp product from both C. ruminantium and E. chaffeensis (Fig. (Fig.1).1).

FIG. 1
Specificity of the C. ruminantium pCS20 PCR assay against E. chaffeensis. Agarose gel electrophoresis of products from PCRs with C. ruminantium primers and C. ruminantium DNA (lanes 2 and 3) or E. chaffeensis DNA (lanes 4 and 5), with E. chaffeensis primers ...
FIG. 2
Specificity of the C. ruminantium pCS20 PCR assay against E. chaffeensis. Lanes 1 to 5 are lanes 1 to 5 of the gel in Fig. Fig.1,1, respectively, after Southern blotting and hybridization with the pCS20 DNA probe.

(iv) Sensitivity.

The sensitivity of the PCR assay varied with the level of tick infection and was high, ranging from 97 to 88%, with samples containing 107 to 104 organisms (Table (Table3).3). However, the sensitivity dropped to 61% and then to 28% with samples containing 103 and 102 C. ruminantium organisms, respectively. The positive predictive value of the PCR assay was high, greater than 93%, at all levels of infection (Table (Table3).3). The negative predictive value, however, dropped from 93% at the 105 level to 58% at the 102 level.

TABLE 3
Sensitivity and predictive values of the PCR assay for C. ruminantium at different levels of tick infection

Detection of C. ruminantium in field ticks.

The PCR assay detected C. ruminantium DNA in A. hebraeum ticks collected at 13 sites in four southern African countries, Botswana, South Africa, Swaziland, and Zimbabwe (Table (Table4).4). Transmission of heartwater was attempted with ticks and was successful from all but two of these sites (Chinamora and Mhondoro Communal Lands in Zimbabwe), confirming the presence of C. ruminantium infection in these tick batches. The prevalence of infection in the A. hebraeum ticks from different sites ranged from 1.6 to 83.3% in males and from 2.1 to 57% in females. The PCR assay also detected C. ruminantium in A. variegatum ticks collected at two sites in Zambia (Gamela and Lutale) and at two sites in Zimbabwe (Kana E and Zvimba) (Table (Table4).4). Transmission of C. ruminantium with the ticks from Zambia was successful, while transmission with A. variegatum from the Zimbabwean sites failed due to lack of tick attachment. The prevalence of infection within A. variegatum ticks ranged from 6.25 to 39.3% in males and from 2.1 to 14.3% in females.

TABLE 4
Analysis by PCR of field-collected Amblyomma ticks from southern Africa

DISCUSSION

This study expands on a previous preliminary evaluation of the pCS20 PCR diagnostic assay for C. ruminantium in ticks (52) by providing data on its operational efficiency that permit the interpretation of assay results under routine testing conditions. The use of the test to detect infections in widespread collections of field ticks is also described. Positive features of the PCR assay include its high sensitivity, specificity, predictive values, and isolate cross-reactivity. The ability of the PCR assay to detect DNA of C. ruminantium originating from locales throughout the distribution of heartwater and in field ticks collected from diverse sites in southern Africa demonstrates conservation of the primer sequences and the wide applicability of the assay. The test did not detect DNA of E. chaffeensis, which, serologically and on the basis of 16S rDNA analysis, is closely related to C. ruminantium (27, 28, 58). The absence of cross-reactivity with E. chaffeensis is particularly pertinent to the screening of Amblyomma ticks in the United States, which is under threat of the introduction of heartwater and where E. chaffeensis infection occurs naturally in Amblyomma americanum ticks (2, 6) and in a wild host of Amblyomma ticks, white-tailed deer (Odocoileus virginianus) (32). The PCR assay detected infection in both A. hebraeum and A. variegatum field ticks and has also been used to detect experimental infections in Amblyomma gemma, a significant vector of heartwater in east Africa (unpublished observations), and in Amblyomma maculatum (39), the most important potential vector in the United States. The pCS20 PCR assay has also been shown, in an independent evaluation, to be more sensitive than other C. ruminantium PCR assays based on 16S rDNA and the MAP 1 gene (1). It is thus highly suited to laboratory and field studies on C. ruminantium transmission, to investigations in previously unstudied heartwater regions, and for surveillance programs in areas under risk of the introduction of infection or attempting eradication of infection. For example, the assay would be a valuable tool for defining the distribution of C. ruminantium infection in ticks in the Caribbean region, where eradication of the disease is being considered.

The PCR assay demonstrated high sensitivity (88 to 97%) and negative predictive values (89 to 97%) at tick infection levels between 104 and 107 organisms. This reliability was, however, not observed at lower infection levels and dropped substantially to a 28% sensitivity and 57% negative predictive value in ticks carrying 100 organisms. This was not unexpected as the sample aliquot used for PCR from such ticks contained, on average, five organisms (1/20 of the total). The success of the PCR assay with this level of template frequency is likely to be significantly influenced by inefficiencies in DNA extraction (loss or damage of DNA) or by PCR failure resulting from mispriming or the presence of PCR inhibitors. Amblyomma tick tissue has been shown previously to contain PCR-inhibitory elements which are not always removed during DNA purification, which may partially account for the occurrence of PCR-negative results on DNA probe-positive samples (52). The reduced sensitivity of the PCR assay at low infection levels is likely to affect estimates of field tick infection prevalence, though the full implications are difficult to assess. The pCS20 DNA probe has a detection limit of approximately 70,000 organisms (52) and provides semiquantitative information on the intensity of infection above this level. The distribution of infection intensities below this level is presently not known; therefore it is not possible to determine the proportion of infections that are missed by the PCR assay. Further studies, potentially with quantitative PCR techniques, may help elucidate the nature of low-level infections.

Under sample handling conditions that mimicked the processing of real infected samples, the specificity of the PCR assay was 98%, suggesting that minor sample-to-sample contamination occurred. In situations where no infected samples are present, the specificity appears to be closer to 100% (38), thus improving the positive predictive value when uninfected tick populations are screened for the introduction of infection.

The PCR assay was more effective than tick transmission trials for the detection of C. ruminantium in field-collected ticks. Four of the 17 tick batches failed to feed or failed to transmit infection. These batches were analyzed by PCR and found to be infected. PCR can also be applied to dried and fixed ticks (52), obviating the need to transfer ticks to recipient animals quickly after collection. However, in situations where proof of infection requires direct demonstration of C. ruminantium infection or isolation of the agent, xenodiagnosis should be used. This should preferably be done in small ruminants instead of mice due to easier ante- and postmortem confirmation of infection and the availability of simple techniques for isolation of the organism in cell culture from ruminant plasma (8). In addition, certain C. ruminantium isolates appear to be noninfective for mice (18), though other conclusive experiments on this aspect are required.

The PCR analysis of field ticks provided a wide range of infection rate estimates for both male and female ticks. In most cases, the ticks were collected from livestock. These infection rates, therefore, cannot be considered representative of host-seeking tick populations due to the potential for feeding ticks to acquire infection intrastadially (30). This effect is particularly important for male ticks, which can feed for prolonged periods (weeks to months) (26) and do not lose infection during feeding. The flat female ticks examined in this study may, however, be more representative due to their short attachment period prior to the start of engorgement (1 to 2 days) and thus limited exposure to infection. The infection prevalence of the unfed A. hebraeum ticks collected from vegetation ranged from 2.8 to 9.8%, providing better estimates of the magnitude of the vector infection reservoir, though the sample sizes were small.

The pCS20 PCR assay is presently the most reliable and best-characterized test for C. ruminantium infection in ticks, exceeding previous assays in reliability and speed (38). The lack of cross-reaction with closely related organisms such as E. chaffeensis demonstrated here and with Ehrlichia canis (52) increases the value of this test, particularly as C. ruminantium serologic assays developed to date are limited by either poor specificity or low sensitivity (5, 16, 24, 27, 28, 40, 42, 59). Application of the PCR assay in other heartwater-endemic regions of Africa and the Caribbean will provide valuable and accurate information on infection rates of the Amblyomma tick vectors. Adaptation of the assay to detect C. ruminantium in carrier animals will further facilitate the understanding of an area of epidemiology of heartwater which is currently not completely understood, and this would significantly improve surveillance and control for heartwater.

ACKNOWLEDGMENTS

This study was supported by U.S. Agency for International Development grant LAG-1328-G-00-3030-00 awarded to the University of Florida.

We thank the following for assistance in tick collections: Ian and Margueritte Swannack (Finale Farm, Zimbabwe), Boetie and Hendrik O'Neill (Vlakfontein Estates, Zimbabwe), L. Modisa of the Botswana Veterinary Services, G. Axsel and P. van der Reit of the South African Department of Veterinary Services, N. Bryson of the Medical University of South Africa, L. van der Merwe of Warmbaths Farm RSA, P. Dlamini of the Swaziland Department of Veterinary Services, and L. Makala and G. Munyama of the Zambian Department of Veterinary Services. We are grateful for the technical assistance provided by Gillian Smith, Godfree Mlambo, Fidelis Mugova, and Lovemore Kativu.

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