Abstract

Eperythrozoon ovis, used thought to be a rickettsia but now identified as a mycoplasma, is an erythrocytic agent that causes hemolytic anemia both in animals and human. To demonstrate the epidemiological status of Eperythrozoon ovis infection in Chinese population, 1458 healthy volunteers, 247 patients with hematologic disorders and 106 susceptible people with direct contact to suspected animals were investigated by classical blood smear examination. The positive samples were identified by a specific PCR assay. The microscopic results showed a lot of small organisms attaching on the surface of erythrocytes (Fig 1). The partial 16s rRNA gene of these organisms was amplified using the conserved primers and confirmed as hemoplasma by sequence alignment analysis. Moreover, complement regulatory protein (CR1, CD35), indicating the function of red cell membrane, was tested by flow cytometry with mouse anti-human CD35 and caprine-anti-mouse IgG-FITC reagents. The expression of CR1 might help to elucidate the mechanism why the Eperythrozoon always lead to anemia and icterus in human being (Fig 2).

The Eperythrozoon infection rate in healthy was 239/1458, 95/247 in hematologic disease patients and 55/106 in susceptible people, respectively in our results. Results of flow cytometry in peripheral blood sample showed the co-relation between the CD35 expression and the eperythrozoon infection status.

The CD35 values increased obviously in the infected cases. In the 4 outbreak cases with FUO (Fever of unknown origin, finally diagnosed as eperythrozoonsis), CD35 reached the peak which might indicate the immuno-defense from the body against the hemotrophic mycoplasma. Gradually, the CD35 value decreased markedly in the long-term infectious cases, which may be explained by anemia and icterus resulting from destroyed membrane of the erythrocyte.

Fig. 1:

Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6).

Fig. 1:

Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6).

Fig. 2:

The expression of CD35 antigen on the surface of RBCs was assessed by flow cytometry. Compared to CD35 expression from the controls, no obvious differences were observed from the healthy people(around 4.69) (Fig 2-1). In the positive samples, the CD35 values increase from the negative 4.69 to 10.61 (Fig 2--2). In the outbreak cases, however, a sharp increase of CD35 antigen expression at the RBC external membrane were observed with the values of 28.76(Fig. 2–3 and 2–4), which were significantly higher than the value of the control samples (p<0.01). The long-term infectious of eperythroon would cause a sharp decrease of the CD35 levels from 28.76 to 2.31 (Fig. 2–5) (p<0.01), which is even much lower than that of the health people (4.61).

Fig. 2:

The expression of CD35 antigen on the surface of RBCs was assessed by flow cytometry. Compared to CD35 expression from the controls, no obvious differences were observed from the healthy people(around 4.69) (Fig 2-1). In the positive samples, the CD35 values increase from the negative 4.69 to 10.61 (Fig 2--2). In the outbreak cases, however, a sharp increase of CD35 antigen expression at the RBC external membrane were observed with the values of 28.76(Fig. 2–3 and 2–4), which were significantly higher than the value of the control samples (p<0.01). The long-term infectious of eperythroon would cause a sharp decrease of the CD35 levels from 28.76 to 2.31 (Fig. 2–5) (p<0.01), which is even much lower than that of the health people (4.61).

Disclosures: No relevant conflicts of interest to declare.

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