Molecular evidence of Anaplasma bovis in spotted deer (Axis axis): A case report

Introduction

Anaplasmosis is an important tick-borne disease caused by bacteria from the genus Anaplasma, affecting a wide range of domestic and wild animals, and occasionally humans [1] . The disease is caused primarily by species like Anaplasma marginale, Anaplasma phagocytophilum, and Anaplasma bovis. The transmission is majorly through the tick bite and involves Ixodes ticks [2] . Clinical manifestations of anaplasmosis vary depending on the specific Anaplasma species and the host involved. The disease in ruminants is sometimes acute and characterised by severe anaemia, hemoglobinuria, and icterus due to hemolysis [2] . In humans, A. phagocytophilum is reported to cause Human Granulocytic Anaplasmosis (HGA) [3] . Anaplasma bovis has been extensively documented in Asia, the Middle East, and Africa. Japan and South Korea have reported the disease from several species. It has been reported in a variety of domestic animals including dogs [4] , cattle [5] , Horses [6] , Sheep [7] . While in the wild animals, it has been documented in a few species like Apodemus agrarius, Bandicota indica, Crocidura suaveolens, Mus caroli, Mus formosanus, Rattus losea, and Suncus murinus [8] , Cottontail Rabbits (Sylvilagus spp) [9] , Wild Deer (Cervus nippon) [10] , Raccoons (Procyon lotor) [11] , Hokkaido Brown Bears (Ursus arctos yesoensis) [12] , Wildcats (Felis silvestris) [13] . In the free-range wild population, especially cervids, anaplasmosis can compromise the overall fitness of the population by affecting reproductive success, increasing susceptibility to predation and predisposal to opportunistic infection. Both A. phagocytophilum and A. bovis have caused significant deer population declines in endemic areas across the globe [14,15] . India has several deer species that populate the forests of the country. In recent years, studies from India have targeted anaplasmosis in wild ungulates with feeble success [16] . Recently, the north-eastern hill region of the country reported A. marginale in Mithun (Bos frontalis) [17] . Apart from these reports, there are no investigations on anaplasmosis even in the most abundant ungulates of the country, the spotted deers (Axis axis). The infection can impact herd-dwelling species of cervids and can exhibit seasonal surges as the tick population spikes. In the free range, infection is seldom detected and treated as the animals grow weak and are either predated or succumb to the infection. Treatment typically includes antibiotics like tetracyclines to contain the bacteria along with supportive therapy including antistressors, haematinics, liver tonics, minerals and vitamins [2] . Though a few countries like Japan and China have documented the infection extensively, only a few reports of wildlife have emerged from India. Need for continuous monitoring of wildlife population for the disease in many protected areas of the country will help in the generation of the baseline data on the disease. Newer species of the pathogen and host could be identified by intensifying efforts in surveillance. Continued research is essential to develop effective control measures and mitigate the disease's impact on wildlife. The current case report deals with the clinical presentation and diagnosis of anaplasmosis in a wild spotted deer in free range.

Case report

An adult spotted deer (Axis axis), weighing 45 Kg, female was found to be recumbent in the Gorewada Reserve Forest hence a rescue was initiated at the request of the Divisional Manager, Gorewada Project, Nagpur. The animal was tranquilised using Xyalzine-Ketamine anaesthesia due to its timid nature and struggle at the sight of the rescue team. The animal was managed using the Airway-Breathing-Circulation principle [18] . The animal showed signs of respiratory distress and hyperthermia which was managed rationally using injection Analgin® and cold water spray. The animal showed an uneven pattern of respiration and finally collapsed after 45 minutes of darting. Blood and serum were collected during the management of the anaesthesia and peripheral blood smears were made. The Leishman stain was used to stain the slides. The Complete Blood Check revealed a haemoglobin concentration of 3.2 gm/dl, neutrophilia and a fall in Total Erythrocyte Count (TEC). The deer showed pale mucous membranes and severe tick infestation (Table 1) [19,20,21] . To molecularly characterize the infection, DNA was isolated from the blood following the manufacturer's protocol using the QIAamp® Fast DNA Blood and Tissue Kit (Qiagen Inc., MD, USA). Polymerase chain reaction (PCR) for the common prevalent haemoprotozoans was undertaken as in Table 2. An amplification of approximately 680 bp was obtained after agarose gel electrophoresis (Fig 1 .), which was purified using PCR purification Kit (Mfg. Qiagen Inc., MD, USA). The amplicon was sequenced using Genetic Analyser 3500 (Applied Biosystems, CA, USA) using the forward and reverse primers to ensure reliability. The blast analysis revealed 100% similarity with Anaplasma bovis reported from LC066136 (Bangladesh), MH255935 & HQ913644 (China) majorly from goat and cattle. The sequence was submitted to the NCBI GenBank database and was assigned the accession number PQ192496. For phylogenetic analysis, sequences of A. bovis reported from domestic and wild animals in India and other countries were included in the study (Table 3). The analysis was conducted using MEGA XI software, employing the Maximum Likelihood (ML) method with bootstrap values calculated from 1000 replications to ensure the reliability of the phylogenetic tree [22] (Fig. 2). The phylogenetic tree showed three major clades each maintaining intra-clade specificity. The query sequence was found in clade I alongside other sequences of A. bovis from India, China, Zambia and Kyrgyzstan. The deer isolates from Japan (EU697013) were closely identical to the query sequence. The query sequence was placed alongside other sequences of A. bovis from India, indicating the possibility of the common host range.

Discussion

This is the first record of A. bovis in a spotted deer (Axis axis) from India and with molecular analysis; additional epidemiological data has been generated. Anaplasma bovis is a tick-borne rickettsial pathogen primarily infecting domestic and wild ruminants. The pathogen has been reported in various domestic and wild ruminants, predominantly in tropical and subtropical regions, including India, Bangladesh, China, Japan, and Africa [23] . While A. bovis primarily infects monocytes, leading to anaplasmosis in cattle and small ruminants, its role in wildlife is still under investigation. The detection of A. bovis in a spotted deer suggests potential spillover from domestic livestock to wild ungulates due to shared habitat and vector populations. Studies have indicated that asymptomatic carriers in wildlife may serve as reservoirs for anaplasmosis [24] . In the current case, the deer was found recumbent due to the low haemoglobin concentration and resulting weakness. The complete blood check indicated a low haemoglobin concentration, thrombocytopenia, acute neutrophilia and low Total Erythrocyte Count (TEC). The findings are in complete agreement with the findings of other ruminants like cattle and goats [25] . Xylazine-ketamine anaesthesia has innate complications in cervids, xylazine is a respiratory depressant and can cause hypotension and bradycardia [26] . In animals that have a compromised total erythrocyte count and haemoglobin concentration, the outcomes of the anaesthesia can be unpredictable. During emergency field rescue operations, such complications are encountered and require intense emergency management to save the animal. The clinical presentation characterised by respiratory distress, hyperthermia and eventual mortality highlights the pathogenic potential. Though an isolated case, such infection can compromise overall fitness by inducing severe anaemia and increasing vulnerability to environmental stressors and predation. In endemic areas, domestic animals that graze in the protected areas may serve as a potential reservoir of infection to susceptible wild species. The phylogenetic analysis revealed three major clades each maintaining intra-clade specificity. The sequences in clade I were majorly from cattle reported from India and deer sequences from Japan closely aligned with the query sequence. The phylogenetic analysis indicated the possibility of shared host range and of wild-domestic transmission cycle. Although A. bovis is limited to ruminants and there is no evidence of spillover to humans, the disease can have a great impact on deer populations in the free range. Tickborne pathogens pose a significant threat to wildlife; particularly in protected areas with significant wildlife domestic animal interaction. The spotted deer are an important prey species supporting a population of leopards and tigers in the landscape. The possibility of transmission of A. bovis to large cats is poorly documented, but there is evidence of the infection in cats [27] . The potential consequences for apex predators, particularly those dependent on cervid prey populations, highlight the need for further research into the pathogen's impact across trophic levels. Thus, extensive surveillance of the wild population will provide clarity on whether the infection can impact the deer population and dependent predator species. The current findings highlight the need for continued surveillance and research to elucidate the transmission dynamics, genetic diversity, and ecological impact of A. bovis. Integrating such knowledge into wildlife conservation and management plans is essential to mitigate the effects of such diseases on biodiversity and ecosystem health. Table 1: Haematological parameters of the deer sample conducted on HM5 (Mfg. Abaxis) equipment [28] Sr

Acknowledgement

AcknowledgmentThe authors express their gratitude to the Principal Chief Conservator of Forests (Wildlife) & Chief Wildlife Warden, Maharashtra State, for granting approval to conduct this research. They also acknowledge the support of the Managing Director, Forest Development Corporation of Maharashtra Ltd., Nagpur; the Chief Executive Officer, FGZ Ltd., Nagpur; and the Office of the Divisional Manager, Gorewada Project, Nagpur, for providing the necessary facilities for the study. Special thanks are extended to the Director, Wildlife Research & Training Centre, for facilitating the research work at WRTC, Gorewada, Nagpur.

References

1. Stuen S, Granquist EG, Silaghi C. Anaplasma phagocytophilum-a widespread multi-host pathogen with highly adaptive strategies. Frontiers in Cellular and Infection Microbiology. 2013 Jul 22;3:31.
2. Kocan KM, de la Fuente J, Guglielmone AA, Meléndez RD. Antigens and alternatives for control of Anaplasma marginale infection in cattle. Clinical Microbiology Reviews. 2003 Oct;16(4):698-712.
3. Bakken JS, Dumler JS. Human granulocytic anaplasmosis. Infectious Disease Clinics of North America. 2015 Jun;29(2):341-55.
4. Sakamoto L, Ichikawa Y, Sakata Y, Matsumoto K, Inokuma H. Detection of Anaplasma bovis DNA in the peripheral blood of domestic dogs in Japan. Japanese Journal of Infectious Diseases. 2010 Sep 30;63(5):349352.
5. Ooshiro M, Zakimi S, Matsukawa Y, Katagiri Y, Inokuma H. Detection of Anaplasma bovis and Anaplasma phagocytophilum from cattle on Yonaguni Island, Okinawa, Japan. Veterinary Parasitology. 2008 Jul 4;154(3-4):360-364.
6. Seo MG, Kwon OD, Kwak D. Anaplasma bovis infection in a horse: First clinical report and molecular analysis. Veterinary Microbiology. 2019 Jun 1;233:4751.
7. Yang J, Han R, Niu Q, Liu Z, Guan G, Liu G, Luo J, Yin H. Occurrence of four Anaplasma species with veterinary and public health significance in sheep, northwestern China. Ticks and Tick-borne Diseases. 2018 Jan 1;9(1):82-85.
8. Masuzawa T, Uchishima Y, Fukui T, Okamoto Y, Pan MJ, Kadosaka T, Takada N. Detection of Anaplasma phagocytophilum and Anaplasma bovis in small wild mammals from Taichung and Kinmen Island, Taiwan. Japanese Journal of Infectious Diseases. 2014;67(2):111-114.
9. Goethert HK, Telford III SR. Enzootic transmission of Anaplasma bovis in Nantucket cottontail rabbits. Journal of Clinical Microbiology. 2003 Aug;41(8):3744-3747.
10. Wu D, Yoshikawa Y, Kawamori F, Ikegaya A, Ohtake M, Ohashi M, Shimada M, Takada A, Iwai K, Ohashi N. A molecular and serological survey of Rickettsiales bacteria in wild Sika deer (Cervus nippon nippon) in Shizuoka Prefecture, Japan: high prevalence of Anaplasma species. Japanese Journal of Infectious Diseases. 2015;68(5):434-437.
11. Myśliwy I, Perec-Matysiak A, Hildebrand J. Invasive raccoon (Procyon lotor) and raccoon dog (Nyctereutes procyonoides) as potential reservoirs of tick-borne pathogens: data review from native and introduced areas. Parasites & Vectors. 2022 Apr 11;15(1):126.
12. Moustafa MA, Lee K, Taylor K, Nakao R, Sashika M, Shimozuru M, Tsubota T. Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan. Infection, Genetics and Evolution. 2015 Dec 1;36:268-274.
13. Hornok S, Boldogh SA, Takács N, Sándor AD, TuskaSzalay B. Zoonotic ecotype-I of Anaplasma phagocytophilum in sympatric wildcat, pine marten and red squirrel-Short communication. Acta Veterinaria Hungarica. 2022 Aug 26;70(3):363-367.
14. Barlough JE, Madigan JE, DeRock E, Bigornia L. Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). Veterinary Parasitology. 1996 Jun 1;63(3-4):319-329.
15. Egenvall A, Bonnett BN, Gunnarsson A, Hedhammar Å, Shoukri M, Bornstein S, Artursson K. Seroprevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991-94.
16. Scandinavian Journal of Infectious Diseases. 2000 Jan 1;32(1):19-25.
17. Nimisha M, Devassy JK, Pradeep RK, Pakideery V, Sruthi MK, Pious A, Kurbet PS, Amrutha BM, Chandrasekhar L, Deepa CK, Ajithkumar KG. Ticks and accompanying pathogens of domestic and wild animals of Kerala, South India. Experimental and Applied Acarology. 2019 Sep;79(1):137-155.
18. Chamuah JK, Jacob SS, Sumi AA, Awomi LT, Ezung L, Singh M, Patil SG. Report of Anaplasma marginale in Mithun (Bos frontalis) from North Eastern Hilly Region of India. Acta Parasitologica. 2025 Feb;70(1):61-66.
19. Smith T, Pinnock C, Lin T, editors. Fundamentals of anaesthesia. Cambridge: Cambridge University Press; 2009 Feb 10. p. 1-726.
20. Criado-Fornelio A, Martinez-Marcos A, Buling-Saraña A, Barba-Carretero JC. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe: part I. Epizootiological aspects. Veterinary Parasitology. 2003 May 1;113(3-4):189-201.
21. Inokuma H, Okuda M, Ohno K, Shimoda K, Onishi T. Analysis of the 18S rRNA gene sequence of a Hepatozoon detected in two Japanese dogs. Veterinary Parasitology. 2002 Jun 26;106(3):265-271.
22. Motoi Y, Satoh H, Inokuma H, Kiyuuna T, Muramatsu Y, Ueno H, Morita Ç. First detection of Ehrlichia platys in dogs and ticks in Okinawa, Japan. Microbiology and Immunology. 2001 Jan;45(1):89-91.
23. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution. 2021 Jul 1;38(7):3022-3027.
24. Rymaszewska A, Grenda S. Bacteria of the genus Anaplasma-characteristics of Anaplasma and their vectors: a review. Veterinary Medicine. 2008 Nov 1;53(11):573-584.
25. Luo S, Bao F, Wu H, Ma W, Zhu L, Huang X, et al. Global prevalence of Borrelia burgdorferi and Anaplasma phagocytophilum coinfection in Ixodes tick populations: protocol for a systematic review and metaanalysis. BMJ Open. 2024 Jun 1;14(6):e083052.
26. Silaghi C, Nieder M, Sauter-Louis C, KnubbenSchweizer G, Pfister K, Pfeffer M. Epidemiology, genetic variants and clinical course of natural infections with Anaplasma phagocytophilum in a dairy cattle herd. Parasites & Vectors. 2018 Dec;11:614-616.
27. Sontakke SD, Umapathy G, Patil MS, Shivaji S. Tolazoline antagonises ketamine-xylazine anaesthesia in an endangered Black buck (Antilope cervicapra). European Journal of Wildlife Research. 2009 Aug;55:357-361.
28. Oliveira AC, Luz MF, Granada S, Vilhena H, NachumBiala Y, Lopes AP, et al. Molecular detection of Anaplasma bovis, Ehrlichia canis and Hepatozoon felis in cats from Luanda, Angola. Parasites & Vectors. 2018 Dec;11:1-6.
29. Thrall MA, Weiser G, Allison RW, Campbell TW, editors. Veterinary Hematology and Clinical Chemistry. Hoboken (NJ): John Wiley & Sons; 2012. p. 776.
30. Ghosh S, Patra G, Borthakur SK, Behera P, Tolenkhomba TC, Das M, et al. Prevalence of hard tick infestations in cattle of Mizoram, India. Biological Rhythm Research. 2019 Jul 4;50(4):564-574.
31. Jilintai SN, Hayakawa D, Suzuki M, Hata H, Kondo S, Matsumoto K, et al. Molecular survey for Anaplasma bovis and Anaplasma phagocytophilum infection in cattle in a pastureland where sika deer appear in Hokkaido, Japan. Japanese Journal of Infectious Diseases. 2009 Jan 28;62:73-75.
32. Altay K, Erol U, Sahin OF. The first molecular detection of Anaplasma capra in domestic ruminants in the central part of Turkey, with genetic diversity and genotyping of Anaplasma capra. Tropical Animal Health and Production. 2022 Apr;54(2):129-135.
33. Pradeep RK, Nimisha M, Sruthi MK, Vidya P, Amrutha BM, Kurbet PS, et al. Molecular characterization of South Indian field isolates of bovine Babesia spp. and Anaplasma spp. Parasitology Research. 2019 Feb 14;118(2):617-630.
34. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, et al. Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Applied and Environmental Microbiology. 2006 Feb;72(2):11021109.
35. Athira K, Vijayakumar K, Adithya S, Athira KS, Davis KJ, Gleeja VL, et al. Molecular Detection and Phylogenetic Analysis of Babesia vogeli Infections in Dogs of Thrissur, Kerala, India. Journal of Indian Veterinary Association. 2024 Dec 1;22(3):134-138.
36. Kumar S, Sudan V, Shanker D. Molecular characterization and phylogenetic analysis of Anaplasma marginale major surface protein (MSP5) gene. Journal of Veterinary Parasitology. 2020;34(2):89-94.
37. Mwale R, Mulavu M, Khumalo CS, Mukubesa A, Nalubamba K, Mubemba B, et al. Molecular detection and characterization of Anaplasma spp. in cattle and sable antelope from Lusaka and North-Western provinces of Zambia. Veterinary Parasitology: Regional Studies and Reports. 2023 Apr 1;39:100847.
38. Masuzawa T, Uchishima Y, Fukui T, Okamoto Y, Muto M, Koizumi N, et al. Detection of Anaplasma phagocytophilum from wild boars and deer in Japan. Japanese Journal of Infectious Diseases. 2011 Jul 29;64(4):333-336.