Borrelia miyamotoi sensu lato in Père David Deer and Haemaphysalis longicornis Ticks

By sequence analysis of 16S rRNA, flaB, p66, and glpQ, we identified Borrelia miyamotoi in 1 of 4 Père David deer (n = 43) seropositive for Borrelia spp. and 1.2% (3/244) of Haemaphysalis longicornis ticks from Dafeng Elk National Natural Reserve, China. Future studies should assess Borrelia pathogenesis in deer.

By sequence analysis of 16S rRNA, flaB, p66, and glpQ, we identified Borrelia miyamotoi in 1 of 4 Père David deer (n = 43) seropositive for Borrelia spp. and 1.2% (3/244) of Haemaphysalis longicornis ticks from Dafeng Elk National Natural Reserve, China. Future studies should assess Borrelia pathogenesis in deer. P ère David deer (Elaphurus davidianus) are extinct in the wild and found only in captivity, principally in China, England, and the United States. Just 5,000 animals remain, with 40% located in Dafeng Elk National Natural Reserve in China, which attracts >1 million tourists annually. Ticks are common in the Dafeng Elk National Natural Reserve (1), so we investigated the tickborne bacterial pathogens in Père David deer at this reserve.
The institutional animal care and use committee of Yangzhou University College of Veterinary Medicine (Yangzhou, China) (YZU-CVM#2015-076) approved this study. We took whole blood samples from 43 apparently healthy Père David deer (20 males, 23 females), separated out the plasma (1,800 × g for 10 min), and used the plasma to detect antibodies against bacterial pathogens with the SNAP 4Dx kit (IDEXX, Westbrook, ME, USA) (2) according to the manufacturer's instructions. Further, ELISAs and Western blots using Borrelia miyamotoi GlpQ recombinant protein (RayBiotech, Norcross, GA, USA) and peroxidaselabeled rabbit anti-deer IgG (SeraCare, Milford, MA, USA) were performed as described previously (3) to detect GlpQ antibodies specific to B. miyamotoi.

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We collected a convenience sample of Haemaphysalis longicornis ticks (n = 244) from elk in the Dafeng Elk National Natural Reserve during the summer of 2016 and stored the collection at -80°C. We used the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturers instructions to extract DNA from Père David deer whole blood samples and entire H. longicornis ticks. We used published PCR protocols targeting the 16S rRNA (4), flaB (5), and glpQ (6) genes and an in-house p66 PCR (forward primer 5¢-CGATTTTTCTATATTTGGACACAT-3¢, reverse primer 5¢-GATATAGATTCTACAGGTATTG-CATAATC-3¢) to screen blood samples and ticks for B. miyamotoi. We sequenced both strands of PCR products using BGI's (Shanghai, China) services and aligned them using ClustalW in MEGA 7 (http://www.megasoftware. net/) with the nucleotide sequences of 11 relapsing fever group borreliae and 7 Lyme disease group borreliae found in GenBank.
One of the seropositive female deer (2.3% of overall deer population) and 3 (1.2%) of the 244 ticks were positive for the 4 Borrelia genes tested (16S rRNA, flaB, glpQ, p66) by PCR. The sequences obtained from the PCR products showed the 4 animals had identical sets of Borrelia genes. The 16S rRNA, flaB, and p66 sequences were more similar to those of the relapsing fever group borreliae ( (Figure).
B. miyamotoi is a member of the relapsing fever group first isolated in Japan and subsequently found in North America, Europe, and Russia (8). B. miyamotoi has not been reported in deer but can be pathogenic in humans, usually resulting in an acute febrile influenza-like illness but occasionally causing severe disease, including meningoencephalitis (9). Further studies are needed to determine the effects of B. miyamotoi infections in deer, especially because studies on Ixodes scapularis ticks in the United States have indicated that deer might be a sylvatic reservoir (10).
I. persulcatus and I. pavlovskyi ticks are known to be infected with B. miyamotoi in Asia, whereas other Ixodes spp. ticks are vectors in the United States and Europe (9). Tick control in semi-free-ranging animals is challenging; the Père David deer we studied are commonly infested with ticks. The only tick species identified on Père David deer in Dafeng Elk National Natural Reserve was H. longicornis (1), which can reach high densities in the environment (summer 89.5 ± 17.1 ticks/10 m 2 , winter 1.47 ± 0.35 ticks/10 m 2 ) and cause anemia and even death in heavily infested animals. Our finding of B. miyamotoi in H. longicornis ticks adds to the list of organisms reported in this tick, primarily B. burgdorferi sensu lato and unclassified Borrelia spp.
In summary, we have shown that B. miyamotoi sensu lato occurs in Père David deer and H. longicornis ticks in Dafeng Elk National Natural Reserve. Further studies are needed on the pathogenicity of the organism in deer and the role of H. longicornis ticks in the epidemiology of infections in deer and humans.