Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection

Abstract

Background: Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species.

Results: For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level.

Conclusions: These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

Figure 1

Relative expression of immune response genes in naturally A. phagocytophilum -infected and uninfected wild pigs. The expression of selected genes was quantified by real-time RT-PCR in samples of infected (N = 3) and uninfected control pigs (N = 3). Amplification efficiencies were normalized against porcine cyclophlilyn, beta-actin and GAPDH and infected to uninfected average ± S.D. mRNA ratios determined. In all cases, the mean of duplicate values was used and data from infected and uninfected animals were compared using the Student`s t-test (*P < 0.05).

Figure 2

Detection of anti- A. phagocytophilum MSP4 antibodies in experimentally infected and control pigs. Antibody titers were determined by ELISA, expressed as the average ± S.E. OD_450nm (OD_{pig sera} - OD_{PBS control}) and compared between infected and control pigs by ANOVA test (P > 0.05). OD_450nm values for each infected pig are also shown. Arrows show time of pig inoculation with infected and uninfected tick cells.

Figure 3

Expression of immune response genes in experimentally A. phagocytophilum -infected and uninfected domestic pigs. The expression of selected genes was quantified by real-time RT-PCR in samples of infected (N = 3) and uninfected control pigs (N = 3). Amplification efficiencies were normalized against porcine cyclophlilyn, beta-actin and GAPDH and shown in arbitrary units as average ± S.D. mRNA levels. In all cases, the mean of duplicate values was used and data from infected and uninfected animals were compared using the ANOVA t-test (*P < 0.05).

Figure 4

Serum IL-8, IL-1 beta and TNF-alpha levels in experimentally infected pigs. Cytokine levels were determined by ELISA in the sera from infected and uninfected control pigs and infected to uninfected average ± S.D. ratios determined. Results were compared between infected and control pigs by Student’s t-test (*P ≤ 0.05).

Figure 5

Effect of A. phagocytophilum infection on host cells. A. phagocytophilum (Ap) infection causes cytoskeleton rearrangement required for infection, but in pigs it may also promote phagocytosis and autophagy for effective pathogen clearance. Ap delays the apoptotic death of neutrophils to increase infection, but different and complementary mechanisms may operate in human and pig cells. Pathogen infection stimulates innate immune and pro-inflammatory responses in both humans and pigs. IL-8 is likely secreted by infected neutrophils but monocytes, rather than neutrophils, are probably responsible for proinflammatory IL-1 beta and TNF-alpha cytokine production. The expression of genes involved in adaptive immunity was not impaired in pigs. ROS production is inhibited by pathogen infection of human neutrophils but although this mechanism was not found in pigs, upregulation of TGF-beta in infected pigs may inhibits NO production by suppressing STAT1 activation and accelerating iNOS protein degradation. The effect on lipid metabolism required for pathogen infection of human neutrophils was not found in pigs. Data for human neutrophils was obtained from the recent review by Severo et al. [43]