The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the...

[Full title: The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America]

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INTRODUCTION Heartwater is an acute infectious disease caused by Ehrlichia (Cowdria) ruminantium and is transmitted to domestic and wild ruminants by Amblyomma ticks (Walker & Olwage 1987). In susceptible domestic ruminants, heartwater can cause severe losses through high mortality rates of up to 90 % (Mahan, Smith, Kumbula, Burridge & Barbet 2001). Animals which recover from primary infection become asymptomatic carriers of E. ruminantium and serve as reservoirs of infection for ticks (Andrew 99 Onderstepoort Journal of Veterinary Research, 71:99–105 (2004) The pCS 20 PCR assay for Ehrlichia rruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America S.M. MAHAN 1 *, B.H. SIMBI 1 and M.J. BURRIDGE 2 ABSTRACT MAHAN, S.M., SIMBI, B.H. & BURRIDGE, M.J. 2004. The pCS 20 PCR assay for Ehrlichia rumi-nantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America. Onderstepoort Journal of Veterinary Research , 71:99–105 White-tailed deer are susceptible to heartwater ( Ehrlichia [ Cowdria ] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis , Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehr-lichia ewingii . Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16 S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS 20 PCR assay which has high specificity and sensitivity for the detection of E. rumi-nantium . This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS 20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16 S PCR primers (nested) that amplify a specific 405–412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS 20 PCR assay also does not amplify any DNA products from E. chaf-feensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminan-tium in these deer and other animal hosts Keywords : Ehrlichia (Cowdria) ruminantium , PCR, pCS 20 sequence, white-tailed deer, white-tailed deer ehrlichia * Author to whom correspondence is to be directed Current address: University of Florida Heartwater Control Research Program, Postnet Suite 294, Private Bag X 06, Waterkloof, Pretoria, 0145 South Africa E-mail: sumanmah@mweb.co.za 1 UF/USAID/SADC Heartwater Research Project, Central Veterinary Research and Diagnostic Laboratory, P.O. Box CY 551, Causeway, Harare, Zimbabwe 2 Department of Pathobiology, University of Florida, P.O. Box 110880, Gainesville, Fl. 32610 USA Accepted for publication 23 October 2003—Editor

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& Norval 1989; Peter, Burridge & Mahan 2002). Several wild game species are affected by E. rumi-nantium . Some wild animal species are refractory to E. ruminantium infection, while some serve as carriers and others, such as white-tailed deer ( Odo-coileus virginianus) , springbok ( Antidorcas marsu-pialis) and kafue lechwe ( Kobus leche kafuensis) suffer high mortality when infected experimentally or naturally (Camus, Barré, Martinez & Uilenberg 1996; Peter et al. 2002). The role played by wild animal species in the epidemiology of heartwater is still not fully understood. Nevertheless, it has been shown that E. ruminantium can be maintained in Amblyomma hebraeum ticks found exclusively in the Kruger National Park in South Africa where there has been no contact between the wild game species and cattle for at least 40 years (Peter, Bryson, Perry, O’Callaghan, Medley, Smith , Mlambo, Horak, Burridge & Mahan 1999), indicating that the ticks maintain their infection by feeding on infected wild animal species found in the Park In the United States of America (USA), the whitetailed deer population plays a key role in the epidemiology of ehrlichial infections which cause serious illnesses in humans (Dawson, Stallknecht, Howerth, Warner, Biggie, Davidson, Lockhart, Nettles, Olson & Childs 1994; Ewing, Dawson, Kocan, Barker, Warner, Panciera, Fox, Kocan & Blouin 1995; Lockhart, Davidson, Stallknecht, Dawson & Howerth 1997). White-tailed deer are reservoirs of Ehrlichia chaffeensis the causative agent of human monocytic ehrlichiosis, Anaplasma (Ehrlichia)phagocytophilum (Dawson, Warner, Baker, Ewing, Stallknecht, Davidson, Kocan, Lockhart & Olson, 1996), the cause of human granulocytic ehrlichiosis (previously known as the HGE agent), Ehrlichiaewingii, one of the causative agents of canine granulocytotropic ehrlichiosis and also causes infections in humans (Yabsley, Varela, Tate, Dugan, Stallknecht, Little & Davidson 2002) and a novel ehrlichial organism, white-tailed deer ehrlichia (WTDE). The WTDE which is distinct from the other ehrlichial agents based on 16 S rRNA sequences (Dawson et al. 1996; Brandsma, Little, Lockhart, Davidson, Stallknecht & Dawson 1999), is closely related to Anaplasma (Ehrlichia) phagocytophila genogroup, which also includes Ehrlichia equi, Ehr-lichia platys and the HGE agent, also based on 16 S analysis (Little, Dawson, Lockhart, Stallknecht, Warner & Davidson 1997). Heartwater, at present, does not occur in the USA but if it did invade the country, white-tailed deer would be a susceptible target population. It is, therefore, important to differentiate infections caused by the various ehrlichial agents. Serological assays available for detection of antibodies to E. ruminantium also detect crossreactions due to other Ehrlichiae hence compromising interpretations of serological results (Mahan, Barbet, Tebele, Nyathi, Wassink, Semu, Peter & Kelly 1993). Consequently, it is critical to have an assay that can be used with confidence for the detection and differentiation of E. ruminantium infection from infections caused by other agents. The pCS 20 PCR assay has been shown to be highly specific and sensitive for detection of E. rumi-nantium DNA in ticks and in clinically ill animals (Mahan, Waghela, McGuire, Rurangirwa, Wassink & Barbet 1992; Mahan et al . 1993; Peter, Deem, Barbet, Norval, Simbi, Kelly & Mahan 1995; Allsopp, Hattingh, Vogel & Allsopp 1999; Peter, Barbet, Alleman, Simbi, Burridge & Mahan 2000). In addition, it has previously been demonstrated that the pCS 20 PCR assay does not amplify any DNA products from E. chaffeensis and Ehrlichia canis DNA (Peter et al . 1995, 2000), the infectious agents most closely related to E. ruminantium by 16 S rDNA analysis and by analysis of genes encoding the outer membrane proteins (Reddy, Sulsona, Barbet, Mahan, Burridge & Alleman 1998; Sulsona, Mahan & Barbet 1999; Yu, McBride, Zhang & Walker 2000; Ohashi, Rikihisa & Unver 2001). The pCS 20 PCR assay was used to differentiate between the WTDE agent and E. ruminantium MATERIALS AND METHODS White-tailed deer ehrlichia and E. rruminantium DNA Blood samples from white-tailed deer infected with the WTDE were collected from deer located in Georgia, Missouri and Florida in USA, and DNA was prepared as described previously (Little et al. 1997). These DNA samples were a kind donation from Dr Susan Little of University of Georgia. Ehr-lichia ruminantium DNA was extracted from bovine endothelial cell cultures infected with the Crystal Springs strain (Byrom, Yunker, Donovan & Smith 1991). Briefly, infected bovine endothelial cell cultures were spun at low speed (400 x g ) to pellet large cellular material; this was followed by highspeed centrifugation (30 000 x g ) of the supernatants to pellet the organisms. The organisms were resuspended in phosphate buffered saline (PBS) and purified through discontinuous Percol gradients ranging from 0–40 % in PBS to separate them from bovine cellular materials at 400 x g . The purified organisms were harvested from the top 100 Erlichial agent in white-tailed deer in USA

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layer (0 %) and washed twice with PBS by centrifugation at 30 000 x g and thereafter resuspended in fresh PBS. DNA was extracted from the organisms by the Qiagen technique as recommended by the manufacturers (Qiagen Genomic DNA Extraction kit, Qiagen Corp, Valencia. CA) PCR assay to detect white-tailed deer ehrlichia DNA A diagnostic nested PCR was performed using an established protocol (Little et al. 1997). The PCR primers were kindly provided by Dr Susan Little. A primary reaction using external general Ehrlichia 16 S primers ECB and ECC on white-tailed deer or E. ruminantium DNA was conducted as previously described (Little et al. 1997). A secondary reaction on the primary PCR product was carried out using internal primers DGA and GA 1 UR specific to the WTDE (Little et al . 1997). For the primary amplification, a 50 µl reaction mixture containing 5 µ l of DNA, 10 mM Tris Cl (pH 8.3), 50 mM KCl, 0.001 % gelatine, 2.5 mM MgCl 2 , 200 µm each of deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, 1.25 U Taq DNA polymerase (Perkin Elmer Corporation, Norwalk, Connecticut, USA), and 1 µM each of primers ECB (5’- CGTATTACCGCGGCTGCTGGCA-3’), and ECC (5’-AGAACGAACGCTGGCGGCAAGCC-3’), was overlaid with 50 µ l of mineral oil (Sigma Co., St Louis, Missouri, USA). Amplification was done by denaturation at 94 °C for 1 min, annealing at 45 °C for 2 min and extension at 72°C for 2 min. A total of 40 cycles were performed followed by an automatic extension for 10 min at 72 °C. The secondary nested PCR was performed using 1 µ l of the product from the primary reaction mixed as above but with 5 µm each of the primers DGA 5’- TTATCTCTGTAGCTTGCTACG-3’ and GAIUR 5’- GAGTTTGCCGGGACTTCTTCT-3’. The reaction was amplified for 30 cycles of 1 min denaturation at 94°C, 2 min annealing at 55°C and 2 min extension at 72 °C. The products from the second nested PCR reactions were analyzed by electrophoresis on a 1.5 % agarose gel and viewed with UV light illumination and photography after staining with ethidium bromide. The PCR products were transferred onto a nylon membrane (NEN Life Science Products, Boston, Massachusetts) and hybridized with a 32 Plabelled specific pCS 20 DNA probe or the 16 S WTDE amplified DNA fragment as probe, as described previously (Mahan et al. 1992; Peter etal. 2000). The results of the hybridizations were evaluated by autoradiography. In all the PCR assays, a reagent blank control (no template DNA) and a respective positive control (WTDE or E.ruminantium DNA) reaction were included The E. rruminantium -specific pCS 20 PCR assay A nested PCR, specific for E. ruminantium , was also performed to amplify DNA from E. ruminan-tium and WTDE-infected deer DNA samples. In a primary reaction, primers U 24 (5’- TTTCCCTATGATACAGAAGGTAAC-3’) and L 24 (5’-AAAGCAAGGATTGTGATCTGGACC-3’) were used to amplify from E. ruminantium DNA (350 bp DNA fragment) and from WTDE DNA. In the second or nested PCR assay with primers AB 128 (5’- ACTAGTAGAAATTGCACAATCTAT-3’) and AB 129 (5’-TGATAACTTGGTGCGGGAAATCCTT-3’), a 279 bp E. ruminantium -specific DNA fragment was amplified as described previously (Mahan et al. 1992; Peter et al. 2000). Each PCR reaction contained a volume of 50 µ l containing the following components: 5 µ l of template DNA, 10 mM Tris pH 8.3, 50 mM KCl, 0.001 % gelatine, 3.0 mM MgCl 2 , 0.5 µm each of the primers L 24 and U 24 (Genosys Biotechnologies, Inc. The Woodlands, Texas, USA), 200 µm of deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, and 1.25 U Taq polymerase. Denaturation of DNA was done at 94 °C for 1 min, annealing of primers was at 55 °C for 1 min and extension at 72°C for 2 min. After 45 cycles, an automatic 10 min extension at 72 °C was followed by a soak at 4°C. In the second or nested reaction, 1 µ l of the primary product was used in a 50 µ l reaction mix with the same reaction and amplification conditions as the primary reaction but with the primers: AB 128 and AB 129. In all the PCR reactions, a reagent blank control (no template DNA) and a respective positive control was included. The products from the second nested PCR reactions were analyzed by electrophoresis on a 1.5 % agarose gel and viewed with UV light illumination and photography after staining with ethidium bromide. The PCR products were transferred onto nylon membrane (NEN Life Science Products, Boston. MA) and hybridized with a 32 P-labelled specific pCS 20 DNA probe or the 16 S WTDE-amplified DNA fragment as probe, as described above. The results of the hybridizations were evaluated by autoradiography RESULTS The nested WTDE 16 S specific primers DGA and GAIUR amplified a DNA product of 405–412 bp 101 S.M. MAHAN, B.H. SIMBI & M.J. BURRIDGE

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from the deer samples containing WTDE DNA from Florida, Georgia and Missouri, but not from E. rumi-nantium DNA (Fig. 1). The general Ehrlichia primers ECB and ECC amplified a product from the E. ruminantium DNA which was of higher molecular weight than the product from the nested PCR on WTDE DNA, and appeared as a diffuse band when visualized by agar gel electrophoresis following the nested PCR (Fig. 1, lane 8). In comparison, the (nested) primers AB 128 and AB 129 specific for the pCS 20 sequence of E. ruminantium , amplified the 279 bp fragment from E. ruminantium DNA but not from the WTDE-infected deer DNA samples originating from Florida, Georgia and Missouri (Fig. 1). Further confirmation of the specificity of the two PCR reactions was done by southern hybridization. The labelled pCS 20 probe of E. ruminantium hybridized only to the products from the pCS 20 PCR assays on E. ruminantium DNA and not to any of the samples which were amplified from WTDE DNA. The 405–412 bp probe of WTDE hybridized only to the products from the nested PCR done with the WTDE primers on WTDE DNA and not to products from E. ruminantium DNA (Fig. 2 A and B) DISCUSSION White-tailed deer play an important role in the epidemiology of infections which affect humans, as they are reservoirs of E. chaffeensis, E. ewingii and Anaplasma (Ehrlichia) phagocytophilum (the HGE agent) (Little, Stallknecht, Lockhart, Dawson & Davidson 1998). The role, if any, of the recently identified WTDE in the pathogenesis of disease remains to be determined because, it has not yet been isolated and characterized in-vitro . However, based on the 16 S sequences, the WTDE has been shown to be unique and different from other ehrlichial agents so far characterized, but it is closely allied to E. phagocytophila genogroup , which includes E. platys, E. equi and Anaplasma (Ehrlichia)phagocytophilum (the HGE agent) (Dawson et al 1996) The phylogenetic classification of the Ehrlichia and Rickettsia spp. has been recently revised based on sequence similarities of the 16 S gene, the citrate synthase gene, the GroEL sequences and genes encoding structural outer membrane proteins of these organisms that were previously classified 102 Erlichial agent in white-tailed deer in USA 405–412 bp ª FIG. 1 A pCS 20 PCR assay for E. ruminantium does not detect DNA from white tailed deer Ehrlichia . Agarose gel electrophoresis of PCR products with primers specific for WTDE show amplification of a DNA product of 405–412 bp from WTDE DNA (lanes 1, 3, 4, 5 and 6) but not from E. ruminantium Crystal Springs strain DNA (lane 8) Lanes 11 and 18 represent the results of the E. ruminantium -specific PCR assay No amplification was detected when the pCS 20 PCR was done on WTDE DNA (lanes 13–16) but amplification of a 279 bp fragment was seen with E. ruminantium DNA (lanes 11 and 18). Lane M is the 123 bp DNA markers. WTDE DNA samples are from white tailed deer from the following locations: lanes 1 and 6 are OS 82 (Ossabaw Island Georgia), lane 3 is MO (Missouri), lane 4 is Florida 2 and lane 5 is Florida 1. Lanes 2 and 17 are reagent blanks and 7, 9, 10, 12 and 17 are empty M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M 279 bp ª

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under different Families or Tribes (Dumler , Barbet, Bekker, Dasch, Jongejan, Palmer, Ray, Rikihisa & Rurangirwa 2001). Due to its genetic similarity to E.chaffeensis and E. canis , Cowdria ruminantium has been placed in the genus Ehrlichia and renamed Ehrlichia ruminantium . This genus now includes E.ruminantium , E. chaffeensis, E. canis, E. ewingii, and Ehrlichia muris . Because of the close genetic and structural relationship of these Ehrlichia spp., and their related epidemiology, it is important to differentiate them from each other using reliable detection assays. Serological assays are unable to differentiate definitively between E. ruminantium and several of the related ehrlichial agents because of marked cross-reactions between immunogenic proteins of these organisms. The PCR assay has proved to be more specific and sensitive in differentiating these related organisms. The pCS 20 PCR assay, which is specific for E. ruminantium , is the method of choice for its detection and differentiation from the other ehrlichial agents (Peter et al. 1995, 2000; Allsopp et al. 1999). It was also able to differentiate between the WTDE organism and E.ruminantium in this study. This was shown by both agarose gel electrophoresis and Southern hybridizations. The nested PCR assay for the WTDE was also found to be specific. However, the fragment that was amplified after the primary PCR using ECB and ECC primers on E. ruminantium might be confused as being a true amplification, and hence Southern hybridization with the 405–412 bp fragment of WTDE DNA as probe would assist in determining whether the agent being detected is E. rumi-nantium or the WTDE agent. The pCS 20 PCR assay would be the method of choice for the detection of E. ruminantium infections in the white tailed deer, if heartwater did spread to the USA. This assay will also not detect infections caused by other related ehrlichial agents and, hence, can be applied reliably for surveillance of heartwater in animals and vectors. The risks of the introduction of heartwater onto the American mainland associated with animal movements are increasing (Burridge, Simmons, Peter & 103 S.M. MAHAN, B.H. SIMBI & M.J. BURRIDGE 405–412 bp ª FIG. 2 A Southern Hybridization of pCS 20 32 P-labelled probe with the PCR products as in Fig. 1, to show positive hybridization of the pCS 20 E. ruminantium probe to the 279 bp products from E. ruminantium DNA. No hybridization occurred with the WTDE DNA samples FIG. 2 B Southern Hybridization of 405–412 bp WTDE PCR product 32 P-labelled as probe with the PCR products as in Fig. 1 and show positive hybridization of the probe to the WTDE DNA samples but not to the samples from E. ruminantium DNA 1 2 3 5 4 6 7 8 9 10 11 12 13 14 15 16 17 18 279 bp ª A B

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Mahan 2002). One measure to reduce these risks is the use of improved tests, such as the PCR assay, for the screening of animals to prevent the entry of carriers of E. ruminantium . For these tests to be used reliably, it is important that they do not produce cross-reactions with native ehrlichial parasites. This report demonstrates that the pCS 20 PCR assay for E. ruminantium does not cross-react with the novel WTDE organism, making it a valuable tool in monitoring and prevention of introduction of heartwater into the USA and elsewhere ACKNOWLEDGEMENTS This study was funded by the United States Agency for International Development grant number LAG- 1328-G-00-3030-00 to the University of Florida. We thank Dr Susan Little for the donation of the samples infected with the WTDE and the PCR primers specific for WTDE REFERENCES ALLSOPP, M.T.E.P., HATTINGH, C.M., VOGEL, S.W. & ALLSOPP, B.A. 1999. 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Development and evaluation of a polymerase chain reaction assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe. Journal ofClinical Microbiology, 33:166–172 PETER, T. F., BRYSON, N.R., PERRY, B.D., O’CALLAGHAN, C.J., MEDLEY, G.F., SMITH , G.E., MLAMBO, G , HORAK, I.G., BURRIDGE, M.J. & MAHAN, S.M. 1999. Cowdria rumi-nantium infection in ticks in the Kruger National Park. TheVeterinary Record , 145:304–307 PETER, T.F., BARBET, A.F., ALLEMAN, A.R., SIMBI, B.H., BURRIDGE, M.J. & MAHAN, S.M. 2000. Detection of the agent of heartwater, Cowdria ruminantium, in Amblyomma ticks by PCR: validation and application of the assay to field ticks. Journal of Clinical Microbiology , 38:1539–1544 104 Erlichial agent in white-tailed deer in USA

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PETER, T.F., BURRIDGE, M.J. & MAHAN, S.M. 2002 Ehrlichia ( Cowdria ) ruminantium infection (heartwater) in wild animals. Trends in Parasitology 18:214–218 REDDY, G.R., SULSONA, C.R., BARBET, A.F., MAHAN, S.M., BURRIDGE, M.J. & ALLEMAN, A.R. 1998. Molecular characterization of a 28 kDa surface antigen gene family of the tribe Ehrlichiae. Biochemical and Biophysical ResearchCommunications , 247:636–643 SULSONA, C.R., MAHAN, S.M. & BARBET, A.F. 1999. The map 1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes Biochemical and Biophysical Research Communications, 257:300–305 WALKER, J.B. & O LWAGE, A. 1987. The tick vectors of Cow-dria ruminantium (Ixodoidea, Ixodidae, genus Amblyomma ) and their distribution. Onderstepoort Journal of VeterinaryResearch , 54:353–379 YABSLEY, M.J., VARELA, A.S., TATE, C.M., DUGAN, V.G., STALLKNECHT, D.E., LITTLE, S.E. & DAVIDSON, W.R 2002. Ehrlichia ewingii infection in white-tailed deer ( Odo-coileus virginanus) . Emerging Infectious Diseases . 8:668– 671 YU, X., MCBRIDE, J.W., ZHANG, X. & WALKER, D.H. 2000 Characterization of the complete transcriptionally active Ehr-lichia chaffeensis 28 kDA outer membrane protein multigene family. Gene, 248:59–68 105 S.M. MAHAN, B.H. SIMBI & M.J. BURRIDGE

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