Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

Abstract

Background: Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.

Methods: Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no M73220 and 16S variant 2 which is identical to GenBank no AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.

Results: Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.

Conclusion: Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.

Figure 1

qPCR amplification of the plasmid dilution series. A plasmid dilution series was produced containing 10¹ to 10⁶ copies of the msp2 (p44) gene of A. phagocytophilum. The figure shows the amplification cycles of the dilution series used as standard curve for quantification of the infection. In addition the figure shows the lack of amplification of the non template controls.

Figure 2

Standard curve of the plasmid dilution series. The standard curve was used for the quantification of A. phagocytophilum organisms in the blood from infected lambs.

Figure 3

Melting point analysis of the amplicons. The figure shows the melting point analyses (Tm) of the plasmid, A. phagocytophilum organisms isolated from blood and non template controls. No primer dimers were formed.

Figure 4

qPCR of infection with 16S rRNA gene variant 1 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophilum infection in five lambs (L1-L5) inoculated with the Norwegian 16S rRNA gene variant 1, monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate C_q readings (X) for each sample, calculated as Log₁₀ (1+X).

Figure 5

qPCR of infection with 16S rRNA gene variant 2 of A. phagocytophilum. Cyclic bacteraemia of persistent A. phagocytophilum infection in five lambs (L6-L10) inoculated with the Norwegian 16S rRNA gene variant (GenBank no) AF336220 monitored by qPCR for six months. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate C_q readings (X) for each sample, calculated as Log₁₀ (1+X).

Figure 6

qPCR of uninfected control animal (L11). The uninfected control animal was monitored by qPCR as described for the infected lambs throughout the experimental period. The horizontal line shows the threshold for bacteraemia and represents the lowest (10 copies) plasmid dilution for the standard curve calibration. The results are presented as logarithm transformed means of triplicate C_q readings (X) for each sample, calculated as Log₁₀ (1+X).

Figure 7

Serology of A. phagocytophilum infection. Mean antibody titre ± SD in lambs inoculated with 16S rRNA gene variant 1 (continuous line) and 16S rRNA gene variant 2 (dotted line) of A. phagocytophilum during a six months infection period. * P < 0.05.