Short Communications
Severe Anaplasma phagocytophilum and Babesia divergens concomitant infection in imported captive reindeer (Rangifer tarandus)
Lola Romanos,
Renaud Pierre Maillard*

Mac Vet Rev 2020; 43 (2): i - vii

10.2478/macvetrev-2020-0023

Received: 04 February 2020

Received in revised form: 18 June 2020

Accepted: 14 July 2020

Available Online First: 13 August 2020

Published on: 15 October 2020

Correspondence: Renaud Pierre Maillard, renaud.maillard@envt.fr
PDF

Abstract

Tick-borne diseases are highly prevalent in domestic and wild ruminants and they may be distributed in wide geographical ranges by animal transportation. The aim of the current study was to investigate the presence of European strains of Babesia spp. and/or Anaplasma spp. in oversea imported reindeer specimens. Imported specimens (n=7) were hospitalized with visible tick infestation (Ixodes ricinus) and signs of cachexia, anemia, and hemoglobinuria. Using blood smears, PCR, and BLAST comparisons, it was confirmed that the animals were infected with a French strain of Anaplasma phagocytophilum and Babesia divergens which is considered to be absent in the USA. We conclude that oversea importation of reindeers must be followed with a routine check for geographically-specific strains of pathogens from the place of origin. This monitoring process must be dynamic and according to recent reports of tick-borne pathogens.

Keywords: Anaplasma, Babesia, Ixodes ricinus, reindeer


INTRODUCTION

Babesia divergens, Babesia spp. EU1, Borrelia (B. burgdorferi s.l.), Anaplasma (A. phagocytophilum) and Mycoplasma wenyonii (or other haemoplasma closely related to) are the main pathogens transmitted by Ixodes ricinus to ruminants in Europe (1, 2, 3, 4). These Ixodes-related pathogens are geographically related to forest pastures are therefore most highly present in wild-animal ruminant species (5, 6). Transport of such animals may contribute to wide dispersion of local geographical strains of these pathogens. Babesiosis in reindeer has been related either to Babesia odocoilei (USA) or B. divergens (Europe) (7), or more recently to Babesia spp. EU1 (also known as Babesia venatorum) in the Netherlands (8). Moreover, up to 5 different Babesia species (B. venatorum, B. capreoli, B. capreoli-like, B. odocoilei-like and B. divergens) have been identified in asymptomatic captive reindeer in Germany (9). Infection by Anaplasma spp. is less reported despite the fact that A. phagocytophilum and A. ovis can infect reindeer (10, 11, 12).
We hypothesized that French strains of Babesia spp. or Anaplasma spp. may be diagnosed in imported, tick infested or non-infested reindeer specimens from an oversea geographical origin. Therefore, the aim of the current study was to investigate the correlation between severe clinical manifestation in imported reindeer specimens from the USA infested by Ixodes spp. and concomitant infection with Babesia spp. or Anaplasma spp.

MATERIAL AND METHODS


The study was conveyed on seven adult captive reindeers (Rangifer tarandus tarandus) that were hospitalized with medical history of anorexia, depression, pyrexia, and significant loss of weight. 4 weeks prior the hospitalization. Three were admitted dead, two died within a few hours after admission, and two were successfully treated. The deceased animals were processed on necropsy examination.
Blood smears (Microscope Nikon type 104, 40x) and coprology were performed on the treated animals. Blood cell count, hematocrit and hemoglobin were also performed (Hematology analyser XT 200 iV, Sysmex France, F-95944 Roissy). Multiple PCRs on blood samples was performed for Anaplasma (1, 12, 14, 15) and Babesia (16), followed by sequence analysis and BLAST comparison with sequence databanks.
DNAs were extracted from 200 μl of EDTA blood samples collected from reindeer using a QIAamp DNA Blood Mini Kit (QIAGEN® France-91974 Courtabeuf), eluted with 100 μl of buffer AE and stored at – 20 °C until use. 
For Anaplasma spp., 5 distinct PCR assays were employed through two steps (as described in 12, 14, and 15). First, the DNA extracts were screened with broad-spectrum PCR primers targeting 16S rRNA gene of Anaplasmataceae, and then with primers targeting major surface protein 4 gene: msp4. Secondly, positive samples were subjected to confirmation by 3 amplification assays: PCRs targeting groESL heat shock operon, ankA gene, and citrate synthase gene: gltA. For Babesia a genus-specific PCR based on the amplification of a fragment of an 18S rRNA gene was performed, using BJ1 and BN2 primers (as described in 16).
PCR assays were performed on an Eppendorf® Mastercycler ep-Gradient thermocycler (Eppendorf France-78360 Montesson).
PCR products were analyzed by gel electrophoresis in 2% agarose (SYBR® Safe DNA gel stain, Invitrogen, Carlsbad, USA).
Sequence analysis was performed with NCBI blast tools (see: https://blast.ncbi.nlm.nih.gov/).

RESULTS


Clinical examinations revealed cachexia, dehydration (estimated >10%), polypnea (>50 movements per minute), tachycardia (>90 beatings per minute), and hemoglobinuria. The color of mucosae was normal to pale, without signs of icterus. Tens to thousands of engorged Ixodes ricinus were found on each animal (from reindeer 172: 34 ticks, to reindeer 282: countless >1000 ticks).
Blood analysis revealed moderate anemia (Table 1). 



Blood smears revealed intra-erythrocytic protozoa morphologically identified as Babesia divergens (Fig. 1).


Figure 1. Multiple protozoa (arrows: pear-shaped intra-erythrocytic parasites) in a blood smear (from reindeer USA Texas 109) identified as Babesia divergens. Microscope Nikon type 104, 40x

Another blood smear a few hours after revealed the presence of intragranulocytic morulae identified as Anaplasma phagocytophilum (Fig. 2).


Figure 2. Anaplasma phagocytophilum morula (arrow: blackberry-shaped intracytoplasmic foreign body) in a neutrophilic granulocyte (reindeer USA Texas 109). Microscope Nikon type 104, 100x

PCR results confirmed the morphological, bacteriological (Table 2) and parasitological findings (data not shown). After sequencing 5 genes of A. phagocytophilum (Genbank accession numbers JX841250 to JX841254, respectively), BLAST analyses of the sequences confirmed that the isolate was 100% identical to a French A. phagocytophilum isolate (strain BOV 10_179 CCXQ01000001 as deposited in the European Nucleotide Archive (see: https://www.ebi.ac.uk/ena/data/view/CCXQ01).



DISCUSSION


We found that all animals suffered from coinfection with the two major pathogens transmitted in Western Europe to ruminants by Ixodes ricinus (17). Poor nutritional condition, parasitism and transport-related stress (such as “shipping fever” documented in cattle) were the factors contributing to the severity of the disease.
Babesia spp. infection in reindeer is not considered to have frequent clinical manifestation. In the USA, B. odocoieli is correlated with а high mortality rate in reindeers (18), but it can affect other wild ruminants as well (elk - Cervus elaphus elaphus, and white-tailed deer - Odocoielus virginianus) (19, 20). A larger Babesia has also been described in one fatal case in a reindeer (20). B. divergens infects ruminants in Europe but has never been reported in the USA. B. divergens-like/MO-1 in the USA is a distinct strain of B. divergens, which has a lower infection rate in cattle, and distinct morphology when grown in vitro (21, 22, 23).
In early reports in Europe, infection of roe deer with Babesia spp. was supposed to result in subclinical to mild symptoms (11, 24). It is possibly due to the lack of exposure to tick bites under natural conditions, as reindeers seem to be extremely sensitive to most tick-borne pathogens (7, 19). In Europe, babesiosis in reindeer is induced by the very common B. divergens (7), but some protozoa close to B. odocoieli have also been detected in European ticks collected on wild cervids (5, 24). A larger Babesia (B. jakimovi) has also been described in naturally infected reindeers in Siberia. The zoonotic large Babesia spp. EU1 has been reported in Ixodes ricinus in a roe deer (3, 25, 26, 27), and in a reindeer (8). B. divergens, Babesia sp. EU1 and B. odocoieli are genetically and/or morphologically similar (3, 26, 27, 28). Many of “Babesia divergens” infections in European cervids may have been also caused by B. capreoli (3, 29). Hence, formal molecular identification of Babesia sp. in wild ruminants is routinely performed (12).
Anaplasma ovis infection may result in a severe disease in reindeer (10). The vectors associated with this bacterium are not Ixodes spp (30). In our case, no specific sequence of the bacterium was found (30).
Disease due to Anaplasma phagocytophilum infection has not been fully described in reindeer, even if the infection seems moderately prevalent (12). The infection in domestic and wild ruminants results in a seldom fatal disease unless complicated by other infections (2, 31). In our clinical observations, we hypothesized that the severe clinical manifestations in reindeer are a result of coinfection with Babesia divergens. Contributing factors were the supposed immune naivety and theshipping stressors.
Anaplasma phagocytophilum and/or Babesia divergens are frequently related to Ixodes ricinus infestations in Europe. The findings for Anaplasma range from 2 to 45% in various European countries (12, 32), for Babesia divergens from 0.9 to 6.7% (33), for both pathogens from to 4 to 8% in Germany (12, 34), and up to 46.7% in Poland (6). These rates are correlated with the tick life-stage and sex, detection method, and geography (country) (4). The tick population in one geographical zone may contain one or more infective agents (6, 17, 35). The transmission cycle of Anaplasma phagocytophilum is not fully elucidated in Europe. If the documented vector is Ixodes ricinus, the reservoir is not precisely known (31, 34, 36, 37).
Tick bites in susceptible or weakened and immunologically compromised animals may therefore result in one or multiple infections. When introducing wild or domestic ruminants in a new pasture, a farm or country, it is important to consider which disease and which parasite they may introduce, but it is also imperative to consider the danger they incur when facing new parasites and new microbial pathogens.

CONCLUSION


This study identified the presence of Babesia divergens absent in the USA and French strain of Anaplasma phagocytophilum in reindeer specimen infested with Ixodes ricinus and imported from the USA. This concludes that tick-borne pathogens can be widely dispersed via vector transmission. The importation and transportation of wild animals is a significant risk factor.
Blood smears and molecular identification methods are effective in detection of tick-borne pathogens in suspect wild ruminants and should be routinely performed and updated.

CONFLICT OF INTEREST


The authors declared that they have no potential conflict of interest with respect to the authorship and/or publication of this article.

ACKNOWLEDGMENTS


The authors cordially thank C. Ferlat, DVM, who referred these cases. This work is part of the research project about haemobacteria in wild and domestic ruminants performed in the Interaction of Host and Pathogen Agents (IHAP) unit. It is also part of LR’s residency program (European College of Bovine Health Management-ECBHM).

AUTHORS’ CONTRIBUTIONS


LR performed the clinical examination, necropsies, blood smears, blood analysis and partially the PCR  analysis. RPM performed the morphological diagnosis from the blood smears and part of PCR and blast analysis. Both authors contributed equally in writing this manuscript.

References

  1. Stoffregen, W.C., Alt, D.P., Olsen, S.C., Waters, S.C., Stasko, J.A. (2006). Identification of a haemoplasma species in anemic reindeer (Rangifer tarandus). J Wildl Dis. 42(2): 249-258. https://doi.org/10.7589/0090-3558-42.2.249 PMid:16870847
  2. Stuen, S. (2007). Anaplasma phagocytophilum - the most widespread tick-borne infection in animals in Europe. Vet Res Commun. 31 (Suppl. 1): 79-84. https://doi.org/10.1007/s11259-007-0071-y PMid:17682851
  3. Bastian, S., Jouglin, M., Brisseau, N., Malandrin, L., Klegou, G., L'Hostis, M., Chauvin, A. (2012). Antibody prevalence and molecular identification of Babesia spp. in roe deer in France. J Wildl Dis. 48(2): 416-424. https://doi.org/10.7589/0090-3558-48.2.416 PMid:22493116   
  4. Becker, C.A., Bouju-Albert, A., Jouglin, M., Chauvin, A., Malandrin, L. (2009). Natural transmission of zoonotic Babesia spp. by Ixodes ricinus ticks. Emerg Infect Dis. 15(2): 320-322. https://doi.org/10.3201/eid1502.081247 PMid:19193284 PMCid:PMC2657642 
  5. Hilpertshauser, H., Deplazes, P., Schnyder, M., Gern, L., Mathis, A. (2006). Babesia spp. identified by PCR in ticks collected from domestic and wild ruminants in southern Switzerland. Appl Environ Microbiol. 72(10): 6503-6507. https://doi.org/10.1128/AEM.00823-06 PMid:17021198 PMCid:PMC1610307             
  6. Asman, M., Nowak, M., Cuber, P., Strzelczyk, J., Szilman, E., Szilman, P., Trapp, G., Siuda, K., Solarz, K., Wiczkowski, A. (2013). The risk of exposure to Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Babesia sp. and co-infections in Ixodes ricinus ticks on the territory of Niepołomice forest (southern Poland). Ann Parasitol. 59(1): 13-19.     
  7. Langton, C., Gray, J.S., Waters, P.F., Holman, P.J. (2003). Naturally acquired babesiosis in a reindeer (Rangifer tarandus tarandus) herd in Great Britain. Parasitol Res. 89(3): 194-198. https://doi.org/10.1007/s00436-002-0737-x PMid:12541061     
  8. Kik, M., Nijhof, A.M., Balk, J.A., Jongejan, F. (2011). Babesia sp. EU1 infection in a forest reindeer, The Netherlands. Emerg Infect Dis. 17(5): 936-938. https://doi.org/10.3201/eid1705.101834 PMid:21529420 PMCid:PMC3321791    
  9. Wiegmann, L., Silaghi, C., Obiegala, A., Karnath, C., Langer, S., Ternes, K., Kämmerling, J., Osmann, C., Pfeffer, M. (2015). Occurrence of Babesia species in captive reindeer (Rangifer tarandus) in Germany. Vet Parasitol. 211(1-2): 16-22. https://doi.org/10.1016/j.vetpar.2015.04.026 PMid:25986326     
  10. Haigh, J.C., Gerwing, V., Erdenebaatar, J., Hill, J.E. (2008). A novel clinical syndrome and detection of Anaplasma ovis in Mongolian reindeer (Rangifer tarandus). J Wildl Dis. 44(3): 569-577. https://doi.org/10.7589/0090-3558-44.3.569 PMid:18689641   
  11. Stuen, S. (1996). Experimental tick-borne fever infection in reindeer (Rangifer tarandus tarandus). Vet Rec. 138(24): 595-596. https://doi.org/10.1136/vr.138.24.595 PMid:8799988    
  12. Sánchez Romano, J., Grund, L., Obiegala, A., Nymo, I.H., Ancin-Murguzur, F.J., Li, H., Król, N., Pfeffer, M., Tryland, M. (2019). A multi-pathogen screening of captive reindeer (Rangifer tarandus) in Germany based on serological and molecular assays. Front Vet Sci. 6, 461. https://doi.org/10.3389/fvets.2019.00461 PMid:31921918 PMCid:PMC6933772
  13. Miller, A.L., Evans, A.L., Os, Ø., Arnemo, J.M. (2013). Biochemical and hematologic reference values for free-ranging, chemically immobilized wild norwegian reindeer (Rangifer tarandus tarandus) during early winter. J Wildl Dis. 49(2): 221-228. https://doi.org/10.7589/2012-04-115 PMid:23568897           
  14. Laloy, E., Petit, E., Boulouis, H.J., Gandoin, C., Bouillin, C., Gounot, G., Bonnet, S., Maillard, R. (2009). Dynamics of natural infection by Anaplasma phagocytophilum in a dairy cattle herd in Brittany, France. Clin Microbiol Infect. 15 (Suppl 2): 24-25. https://doi.org/10.1111/j.1469-0691.2008.02142.x PMid:19298405
  15. Laloy, E., Petit, E., Boulouis, H.J., Lacroux, C., Corbiere, F., Schelcher, F., Bonnet, S., Maillard, R. (2009). First detection of Anaplasma phagocytophilum-like DNA in the French lizard Rupricapra pyrenaica. Clin Microbiol Infect. 15 (Suppl 2): 26-27. https://doi.org/10.1111/j.1469-0691.2008.02143.x PMid:19298404
  16. Lempereur, L., De Cat, A., Caron, Y., Madder, M., Claerebout, E., Saegerman, C., Losson, B. (2011). First molecular evidence of potentially zoonotic Babesia microti and Babesia sp. EU1 in Ixodes ricinus ticks in Belgium. Vector Borne Zoonotic Dis. 11(2): 125-130. https://doi.org/10.1089/vbz.2009.0189 PMid:20575647
  17. Andersson, M.O., Víchová, B., Tolf, C., Krzyzanowska, S., Waldenström, J., Karlsson, M.E. (2017). Co-infection with Babesia divergens and Anaplasma phagocytophilum in cattle (Bos taurus), Sweden. Ticks Tick Borne Dis. 8(6): 933-935. https://doi.org/10.1016/j.ttbdis.2017.08.005 PMid:28869191       
  18. Mathieu, A., Pastor, A.R., Berkvens, C.N., Gara-Boivin, C., Hébert, M., Léveillé, A.N., Barta, J.R., Smith, D.A. (2018). Babesia odocoilei as a cause of mortality in captive cervids in Canada. Can Vet J. 59(1): 52-58.       
  19. Bartlett, S.L., Abou-Madi, N., Messick, J.B., Birkenheuer, A. Kollias, G.V. (2009). Diagnosis and treatment of Babesia odocoilei in captive reindeer (Rangifer tarandus tarandus) and recognition of three novel host species. J Zoo Wildl Med. 40(1): 152-159. https://doi.org/10.1638/2008-0011.1 PMid:19368255
  20. Holman, P.J., Swift, P.K., Frey, R.E., Bennett, J., Cruz, D. Wagner, G.G. (2002). Genotypically unique Babesia spp. isolated from reindeer (Rangifer tarandus tarandus) in the United States. Parasitol Res. 88(5): 405-411. https://doi.org/10.1007/s00436-001-0576-1 PMid:12049456     
  21. Herwaldt, B.L., de Bruyn, G., Pieniazek, N.J., Homer, M., Lofy, K.H., Slemenda, S.B., Fritsche, T.R., Persing, D.H. Limaye, A.P. (2004). Babesia divergens-like infection, Washington State. Emerg Infect Dis. 10(4): 622-629. https://doi.org/10.3201/eid1004.030377 PMid:15200851 PMCid:PMC3323086
  22. Holman, P.J., Spencer, A.M., Telford, S.R 3rd, Goethert, H.K., Allen, A.J., Knowles, D.P., Goff, W.L. (2005). Comparative infectivity of Babesia divergens and a zoonotic Babesia divergens-like parasite in cattle. Am J Trop Med. 73(5): 865-870. https://doi.org/10.4269/ajtmh.2005.73.865 PMid:16282295            
  23. Herc, E., Pritt, B., Huizenga, T., Douce, R., Hysell, M., Newton, D., Sidge, J., Losman, E., Sherbeck, J., Kaul, D.R. (2018). Probable locally acquired Babesia divergens-like infection in Woman, Michigan, USA. Emerg Infect Dis. 24(8): 1558-1560. https://doi.org/10.3201/eid2408.180309 PMid:30016254 PMCid:PMC6056127    
  24. Duh, D., Petrovec, M., Avsic-Zupanc, T. (2001). Diversity of Babesia Infecting European sheep ticks (Ixodes ricinus). J Clin Microbiol. 39(9): 3395-3397. https://doi.org/10.1128/JCM.39.9.3395-3397.2001 PMid:11526189 PMCid:PMC88357        
  25. Bonnet, S., Jouglin, M., L'Hostis, M., Chauvin, A. (2007). Babesia sp. EU1 from roe deer and transmission within Ixodes ricinus. Emerg Infect Dis. 13(8): 1208-1210. https://doi.org/10.3201/eid1308.061560 PMid:17953093 PMCid:PMC2828078    
  26. Duh, D., Petrovec, M., Bidovec, A., Avsic-Zupanc, T. (2005). Cervids as Babesiae hosts, Slovenia. Emerg Infect Dis. 11(7): 1121-1123. https://doi.org/10.3201/eid1107.040724 PMid:16022795 PMCid:PMC3371785    
  27. Duh, D., Petrovec, M., Avsic-Zupanc, T. (2005). Molecular characterization of human pathogen Babesia EU1 in Ixodes ricinus ticks from Slovenia. J Parasitol. 91(2): 463-465. https://doi.org/10.1645/GE-394R PMid:15986627
  28. Zintl, A., Finnerty, E.J., Murphy, T.M., de Waal, T., Gray, J.S. (2011). Babesias of red deer (Cervus elaphus) in Ireland. Vet Res. 42(1): 7. https://doi.org/10.1186/1297-9716-42-7 PMid:21314977 PMCid:PMC3037898    
  29. Malandrin, L., Jouglin, M., Sun, Y., Brisseau, N., Chauvin, A. (2010). Redescription of Babesia capreoli (Enigk and Friedhoff, 1962) from roe deer (Capreolus capreolus): isolation, cultivation, host specificity, molecular characterisation and differentiation from Babesia divergens. Int J Parasitol. 40(3): 277-284. https://doi.org/10.1016/j.ijpara.2009.08.008 PMid:19733572   
  30. Cabezas-Cruz, A., Gallois, M., Fontugne, M., Allain, E., Denoual, M., Moutailler, S., Devillers, E., Zientara, S., Memmi, M., Chauvin, A., Agoulon, A., Vayssier-Taussat, M., Chartier, C. (2019). Epidemiology and genetic diversity of Anaplasma ovis in goats in Corsica, France. Parasit Vectors 12(1): 3. https://doi.org/10.1186/s13071-018-3269-7 PMid:30606253 PMCid:PMC6318933         
  31. Woldehiwet, Z. (2010). The natural history of Anaplasma phagocytophilum. Vet Parasitol. 167(2-4): 108-122. https://doi.org/10.1016/j.vetpar.2009.09.013 PMid:19811878       
  32. Blanco, J.R., Oteo, J.A. (2002). Human granulocytic ehrlichiosis in Europe. Clin Microbiol Infect. 8(12): 763-772. https://doi.org/10.1046/j.1469-0691.2002.00557.x PMid:12519349    
  33. Oines, O., Radzijevskaja, J., Paulauskas, A., Rosef, O. (2012). Prevalence and diversity of Babesia spp. in questing Ixodes ricinus ticks from Norway. Parasit Vectors. 5, 156. https://doi.org/10.1186/1756-3305-5-156 PMid:22862883 PMCid:PMC3439691       
  34. Silaghi, C., Woll, D., Hamel, D., Pfister, K., Mahling, M., Pfeffer, M. (2012). Babesia spp. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the parasitized rodents - Analyzing the host-pathogen-vector interface in a metropolitan area. Parasit Vectors. 5, 191. https://doi.org/10.1186/1756-3305-5-191 PMid:22950642 PMCid:PMC3480827
  35. Lempereur, L., Lebrun, M., Cuvelier, P., Sépult, G., Caron, Y., Saegerman, C., Shiels, B., Losson, B. (2012). Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium. Parasitol Res. 110(4): 1525-1530. https://doi.org/10.1007/s00436-011-2657-0 PMid:21947341         
  36. Overzier, E., Pfister, K., Herb, I., Mahling, M., Böck, G. Jr, Silaghi, C. (2013). Detection of tick-borne pathogens in roe deer (Capreolus capreolus), in questing ticks (Ixodes ricinus), and in ticks infesting roe deer in southern Germany. Ticks Tick Borne Dis. 4(4): 320-328. https://doi.org/10.1016/j.ttbdis.2013.01.004 PMid:23571115   
  37. Lagrée, A.C., Rouxel, C., Kevin, M., Dugat, T., Girault, G., Durand, B., Pfeffer, M., Silaghi, C., Nieder, M., Boulouis, H.J., Haddad, N. (2018). Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle. Parasit Vectors. 11, 163. https://doi.org/10.1186/s13071-018-2661-7 PMid:29523202 PMCid:PMC5845262


Copyright

© 2020 Romanos L. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

The authors have declared that no competing interests exist.

Citation Information

Macedonian Veterinary Review. Volume 43, Issue 2, Pages i-vii, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2020-0023, 2020