The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes

Abstract

ABSTRACT An emerging infectious disease caused by "Anaplasma capra" was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma. Anaplasma capra-positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL, 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright-Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra. Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra-positive, while leukocyte DNA samples were A. capra-negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra-positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., "Anaplasma capra," in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name "A. capra" as Anaplasma capra sp. nov.

Keywords: Anaplasma capra; Rickettsia; erythrocyte; tick-borne; zoonotic.

Figure 1.

Results of multiplex PCR detection assay for the identification of A. capra sp. nov in whole blood, erythrocyte, and leukocyte DNA samples from the same goat whole blood sample. Lane W: Whole blood DNA sample positive for A. capra sp. nov (874 bp), A. bovis (529 bp), and A. phagocytophilum (172 bp). Lanes E1–E3: Erythrocytes DNA samples positive for A. capra sp. nov (874 bp). Lanes L1–L3: Leukocytes DNA samples positive for A. bovis (529 bp) and A. phagocytophilum (172 bp). Lane P: Positive control of multiplex PCR for detecting A. capra sp. nov, A. bovis, A. ovis (347 bp), and A. phagocytophilum. Lane N: Negative control.

Figure 2.

Identification results of different blood cell DNA samples by PCR based on gltA, 16S rRNA and msp4 genes of A. capra. A-C was the result based on gltA, 16S rRNA and msp4 gene, respectively. Erythrocyte DNA samples were A. capra-positive only based on multiplex loci, while leukocytes DNA samples were A. capra-negative.

Figure 3.

Phylogenetic analysis of A. capra sp. nov identified in this study based on 16S rRNA (1261 bp, A) and gltA (594 bp, B) genes. The trees were constructed using NJ method with MEGA 7.0 software and the numbers on the tree indicate bootstrap values for the branch points. Bootstrap analysis was performed with 1000 replicates. Numbers on the branches indicate percent support for each clade. Red font denotes the sequences obtained in this study. Rickettsia raoultii was used as outgroup. The green background represents intraerythrocytic Anaplasma spp.

Figure 4.

Phylogenetic analysis of A. capra sp. nov identified in this study based on msp4 (656 bp, A) and groEL (874 bp, B) genes. The trees were constructed using NJ method with MEGA 7.0 software and the numbers on the tree indicate bootstrap values for the branch points. Bootstrap analysis was performed with 1000 replicates. Numbers on the branches indicate percent support for each clade. Red font denotes the sequences obtained in this study. Rickettsia raoultii was used as outgroup. The green background represents intraerythrocytic Anaplasma spp.

Figure 5.

Photomicrographs of A. capra sp. nov and infected goat erythrocytes. A1–A4, SEM photomicrographs of erythrocytes and A. capra sp. nov. Normoerythrocytes (A1) and infected erythrocytes. Arrows indicate A. capra sp. nov (A2–A4). A2 shows invading pathogen (red arrowhead). B1–B4 and C1–C4, TEM photomicrographs of erythrocytes and A. capra sp. nov. Uranyl acetate and Reynold's lead citrate stain. Morulae are observed beside and within multiple erythrocytes (arrows). B2–B4 and C2–C4 show higher magnification of the morulae. Electron-dense particles (Lysosomes, arrows) are observed in less-dense areas (C2–C4, arrows). D1–D4, TEM photomicrographs of the separated A. capra sp. nov morulae. D1 (arrows) shows lower magnification of the morulae and D2–D4 present higher magnification of the morulae. No Lysosomes are observed in the morulae.

Figure 6.

Wright–Giemsa, CISH, immunocytochemistry, and IFA analyses of uninfected and infected goat erythrocytes. A1–A2, Wright–Giemsa-stained erythrocytes. B1–B2, CISH assay of the erythrocytes smear. A1–D1 are negative controls. The probe was labeled with DIG. C1–C2 and D1–D2, immunocytochemistry and IFA of erythrocytes incubated with positive goat serum. Black and white arrows denote A. capra sp. nov-positive erythrocytes. Red arrows show A. phagocytophilum-positive neutrophilic granulocytes.