Anaplasma phagocytophilum in Marmota himalayana

Abstract

Background: Human granulocytic anaplasmosis is a tick-borne zoonotic disease caused by Anaplasma phagocytophilum. Coinfections with A. phagocytophilum and other tick-borne pathogens are reported frequently, whereas the relationship between A. phagocytophilum and flea-borne Yersnia pestis is rarely concerned.

Results: A. phagocytophilum and Yersnia pestis were discovered within a Marmota himalayana found dead in the environment, as determined by 16S ribosomal rRNA sequencing. Comparative genomic analyses of marmot-derived A. phagocytophilum isolate demonstrated its similarities and a geographic isolation from other global strains. The 16S rRNA gene and GroEL amino acid sequence identity rates between marmot-derived A. phagocytophilum (JAHLEX000000000) and reference strain HZ (CP000235.1) are 99.73% (1490/1494) and 99.82% (549/550), respectively. 16S rRNA and groESL gene screenings show that A. phagocytophilum is widely distributed in marmots; the bacterium was more common in marmots found dead (24.59%, 15/61) than in captured marmots (19.21%, 29/151). We found a higher Y. pestis isolation rate in dead marmots harboring A. phagocytophilum than in those without it (² = 4.047, p < 0.05). Marmot-derived A. phagocytophilum was able to live in L929 cells and BALB/c mice but did not propagate well.

Conclusions: In this study, A. phagocytophilum was identified for the first time in Marmota himalayana, a predominant Yersinia pestis host. Our results provide initial evidence for M. himalayana being a reservoir for A. phagocytophilum; moreover, we found with the presence of A. phagocytophilum, marmots may be more vulnerable to plague. Humans are at risk for co-infection with both pathogens by exposure to such marmots.

Keywords: Anaplasma phagocytophilum; Anaplasmosis; Coinfection; Marmota himalayana; Plague; Yersinia pestis.

Fig. 1

M. himalayana infected with A. phagocytophilum and Y. pestis. A and B marmot A and marmot B found shortly after they had died. C Enlarged pulmonary nodules in marmot A

Fig. 2

Neighbor-joining trees based on 16S rRNA gene nucleotide and GroEL amino acid sequences. A tree based on 16S rRNA gene sequences of Anaplasma and Ehrlichia genera, and on R. rickettsii species. B tree based on GroEL amino acid sequences within the Anaplasma genus

Fig. 3

Polymorphisms of the groEL gene and GroEL protein between marmot-derived A. phagocytophilum. Yellow highlights and red letters: nonsynonymous mutations. * 216 nucleotide mutation confirmed twice by sequences of primary PCR and nested PCR

Fig. 4

A. phagocytophilum propagated in L929 cells (A, C) and control cells (B, D). A, B 40X objective (direct observation). C, D oil immersion objective (Giemsa staining)

Fig. 5

Genome overview and phylogenetic tree of A. phagocytophilum. A Sequence similarity between marmot-derived A. phagocytophilum (in the center) and other compared strains. The legend shows the GC content and GC skew for the marmot-derived isolate, and the sequence identity on a sliding scale. B: Phylogenetic tree of A. phagocytophilum from various countries and sources