Molecular Detection of Anaplasma phagocytophilum in Small Mammals and Infesting Ticks in Laikipia County, Kenya

Abstract

Anaplasmosis is a set of disease conditions of various mammals caused by bacteria species of the genus Anaplasma. These are sub-microscopic, Gram-negative, obligate intracellular pathogens that infect both vertebrate and invertebrate hosts. Significant species that infect domestic and wildlife animals include Anaplasma marginale, Anaplasma ovis, Anaplasma mesaeterum, Anaplasma platys, and Anaplasma phagocytophilum. Although A. phagocytophilum has a widespread distribution, there are only a few epidemiological reports from sub-Saharan Africa. This study focused on molecular detection and characterization of A. phagocytophilum in small mammals and their infesting ticks in Laikipia County, Kenya. A total of 385 blood and 84 tick archival samples from small mammals (155 females and 230 males) were analyzed. The blood samples were subjected to a nested PCR-HRM melt analysis using species-specific primers to amplify the 16S ribosomal RNA genes. The ticks were also subjected to nested PCR-HRM involving 16S rRNA gene primers. Anaplasma phagocytophilum DNA was detected in 19 out of 385 samples using species-specific 16S rRNA gene primers giving a prevalence of 4.9% for A. phagocytophilum. Analysis of the tick's samples using 16S rRNA gene species-specific primers also detected A. phagocytophilum in 3 samples from Haemaphysalis leachi ticks (3/84) equivalent to prevalence of 3.6%. Sequencing of 16S rRNA PCR products confirmed A. phagocytophilum in small mammals and ticks' samples. Phylogenetic analysis of the haplotype from this study demonstrated a close ancestral link with strains from Canis lupus familiaris, Alces alces, Apodemus agrarius, and ticks (Haemaphysalis longicornis) reported in Europe, China, and Africa. Comparison was also made with a known pathogenic A. phagocytophilum variant HA and a nonpathogenic variant 1 that were clustered into a distinctive clade different form haplotypes detected in this study. All the haplotype sequences for A. phagocytophilum from this study were submitted and registered in GenBank under the accession numbers OQ308965-OQ308976. Our study shows that small mammals and their associated ticks harbor A. phagocytophilum. The vector competence for H. leachi in A. phagocytophilum transmission should further be investigated.

Figure 1

Map of Laikipia County with the areas selected for sample collection marked by stars and diamonds.

Figure 2

Typical representation of HRM profile plot of Anaplasma phagocytophilum from small mammals in Laikipia County performed in a Rotor-gene Q thermocycler using 16S rRNA gene primers and analyzed by Rotor gene software. Sterile water was used as a negative test control.

Figure 3

Typical representation of HRM profile plot of Anaplasma phagocytophilum amplified from ticks associated with small mammals in Laikipia County performed in a Rotor-gene Q thermocycler using 16S rRNA gene primers and analyzed by Rotor gene software. T3, T9, and T23 denoted different Anaplasma phagocytophilum haplotypes.

Figure 4

Phylogenetic relationship of Anaplasma phagocytophilum haplotypes in blood and tick samples from small mammals in Laikipia County, Kenya, with representatives from other regions. Arrow shows haplotypes from Kenya reported in this study.