Molecular detection of Anaplasma phagocytophilum DNA in Olive Baboons and Vervet monkeys in Kenya

8 Background 9 Nonhuman primates (NHPs) occupy an important place in zoonotic spill-overs, serving as either 10 reservoirs or amplifiers of multiple neglected tropical diseases including tick-borne infections. 11 Anaplasmosis is caused by obligate intracellular bacteria of the family Anaplasmatacae. They are 12 transmitted by Ixodid tick species and have a wide host range including wild animals, domestic 13 animals and humans. The aim of this study was to establish the presence of Anaplasma 14 phagocytophilum in Olive baboons and Vervet monkeys in Laikipia County, Kenya.


Background
There has been a rise in the frequency of emerging infectious diseases (EIDs), among which zoonotic tick-borne infections especially rickettsial diseases such as anaplasmosis, are implicated [1].What most of the recent pandemics have proven is that emerging infectious diseases are mainly of animal origin, particularly wildlife [2].The complex interaction among wildlife, livestock and human populations is a factor that contributes to their emergence [3].
Among wildlife species, non-human primates are often proprietors to different microbial agents some which have zoonotic potential.Primates are closely related to humans phylogenetically and ecologically [4] and they can indirectly transmit infectious agents to humans through intermediate hosts, arthropod vectors or directly through bush meat consumption and accidental bites [5].
Certain factors such as forested tropical regions experiencing land-use changes and encroachment as well as those with a high wildlife biodiversity facilitate the spread of these diseases to livestock and man [6].Others include adoption of new technology in farms, destruction of habitats, climate change, travel and encroachment into new habitats [2].
This study focused on Anaplasma phagocytophium, a pathogenic bacterium of zoonotic potential often spread among wildlife then to livestock and man [7].Anaplasma hemoparasites belong to the family of Anaplasmataceae, order of Rickettsiales, class Alphaproteobacteria and genus Anaplasma [8].However, reclassification of Anaplasma happened recently resulting in A. phagocytophilum being included in the genus whose members were only pathogens with host specificity to ruminants such as A. marginale.This followed advancement in genetic analyses of 16S rRNA genes, groESL and surface protein genes.There is a considerable strain variation with A. phagocytophilum such that there appears to be existence of serological cross-reactivity [9], a minor degree of variation in the nucleotide sequences of the 16S rRNA, groESL, gltA, ank, and msp2 genes [10] and a difference in the host infectivity [11].
This bacterium has a wide host range including domestic animals, wildlife and man [12].In man the disease is known as human granulocytic anaplasmosis (HGA) [13].The disease often presents with influenza-like symptoms in animal and human hosts which include fever, anorexia, diarrhea, leukopenia and thrombocytopenia [7].Ixodid ticks are important in their maintenance as vectors [14].The emergence of Anaplasmataceae as human pathogens has gained the attention of scientific community.A recent surveys have shown human infection with anaplasmosis including in Venezuela [15] and one in Morocco where dog owners were infected with A. phagocytophilum [16].
Recent reports on animal anaplasmosis are available from France, Massachusetts, Brazil, Zambia Ethiopia and Kenya in both domestic and wild animals [17][18][19][20][21][22].While Anaplasma in NHPs has been reported in some countries, its importance in NHPs in Kenya is not yet known which has therefore led us to investigate the occurrence of this bacteria in NHPs in Laikipia, Kenya.
The investigation focused on blood DNA presence of A. phagocytophilum in Olive baboons (Papio anubis) and Vervet monkeys (Chlorocebus pygerythrus) in Laikipia County, Kenya.Laikipia is part of Kenya's rangelands mainly inhabited by trans-human pastoralists.It also has a large wildlife population including primate species.These animals are found close to human settlements.
The objectives of this present study were (i) to assess the prevalence of A. phagocytophilum in Olive baboons and Vervet monkeys and (ii) to establish whether the bacteria is genetically diverse.

Study area, sample population and sample size
This study was part of the USAID Predict II project whose aim was to collect targeted information to support the interventions to mitigate spread of zoonotic viruses with pandemic potential.The focus was on highest risk locations and interfaces, where animals and people share changing landscapes.The study area, Laikipia County (Figure 1) located in the Rift Valley of Kenya with co-ordinates of 005'N 36040'E, was picked as one of the locations.There is a diverse range of wildlife in the area, including NHPs with 8 species of them found in the area.

DNA extraction
Whole blood samples stored in TRIzol reagent at -80 o c were retrieved and allowed to thaw at room temperature.Extraction of genomic DNA from each of the whole blood sample was done using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, California, USA) following manufacturer's instructions.

Nested PCR
First set of primers (p3709-p4257) were used in a nested PCR run for amplifying the p44 gene of Anaplasma species in Olive baboons and Vervet monkeys (Table 1).Using the second set of primers (p3761-p4183), 1ul of the product from the first amplification was used in a 25ul reaction mixture.Reactions were performed in a final volume containing 18.25ul double distilled RNase/DNase free water, 2.5ul PCR buffer, 0.75ul 1.5Mm Mgcl2, 0.5ul of 10um DNTPs, 0.5ul of 10um forward primer, 0.5ul of 10um reverse primer, 0.1ul of 2.5U Taq DNA polymerase and 1.0ul of the DNA template.Thermal cycling profiles were as previously described [23].

Conventional PCR
The primer set EHR16SD/R was used in a simple PCR to amplify 345bp fragment of Anaplasma 16S rRNA gene on the same samples with thermo-cycling profile as previously described [24].PCR products were electrophoresed on 1% agarose gel to check the size of amplified fragments by comparison with a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega, Madison, USA).

DNA sequencing and data analysis
Ten selected positive Anaplasma spp.PCR products obtained with primers EHR16SD/R were purified with Thermo Scientific GeneJET PCR Purification Kit#K0701, #K0702 Protocol according to the manufacturer's instructions.Purified DNA fragments were sequenced using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, USA), using the same forward and reverse primers (Table 1) of each PCR assay.Sequence assembly for forward and reverse primers was done using DNA Sequence Assembler v4 (2013), Heracle BioSoft [25].The sequences were matched to those deposited in the GenBank database using BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Multiple alignment of the sequences was done using BioEdit Sequence Alignment Editor (Hall, T.A. 1999).Construction of phylogenetic tree was done using Muscle 3.8 using the neighbor-joining method and visualization of the trees with FigTree v1.4.4 [26].
All methods were carried out in accordance with relevant guidelines and regulations.

Molecular survey of Anaplasma species
A total of 164 blood samples 146 from Olive Baboons and 18, Vervet monkeys where screened for A. phagocytophilum through nested PCR employing the two sets of primers for p44 genes (Table 1).However, all these samples tested negative.A re-run and troubleshooting was done to confirm the results still turned out negative.With the same samples, a different assay and conventional PCR using primers targeting 16S rRNA gene (Table 1), it amplified a specific band of approximately 345bp.The overall prevalence for anaplasmosis was 18.3% (30/164) as estimated by EHR16SD/R PCR (Table 1) with 17.8% and 22.2% in Olive baboons and Vervet monkeys respectively.

Molecular characterization of Anaplasma phagocytophilum. 16S rRNA genotypes
Nine of the 10 PCR products were successfully sequenced on both DNA strands and generated nucleotide sequences with primers EHR16SD/R targeting 345bp of the 16S rRNA gene of Anaplasma spp.Multiple alignment of Anaplasma nucleotide sequences of 4 A. phagocytophilum isolates revealed that all the sequences were conserved except one 15A which was the most divergent in comparison to the rest with a lot of accumulated mutations and numerous insertions.
They all shared 99% to 100% nucleotide similarity except 15A at 81% (Table 2).2).Phylogenetic analysis revealed that the isolates from Japan, South Korea, France, China, South Africa and Denmark belonged to clade I but have recent common ancestor with the Kenyan isolates clustered into one clade II except isolate 15A which appeared separately and represented an outgroup (Figure 3).

Discussion
An epidemiological surveillance of pathogens in NHPs found at the human-animal interface is important in developing strategies on prevention and control of emerging and re-emerging zoonotic diseases.
Therefore, this study reports the detection of A. phagocytophilum DNA within the studied localities with an overall prevalence of 18.3%.A. phagocytophilum, an obligate intracellular bacterium, is the agent of human granulocytic anaplasmosis, formerly known as human granulocytic ehrlichiosis [13]. A. phagocytophilum has been previously reported to infect a wide range of animal hosts in various parts of the world [17][18][19], in Africa, [20,27] while in Kenya [21,22,28,29].This study adds further evidence on the occurrence of A. phagocytophilum in Olive baboons and Vervet monkeys in Kenya.
In this study, a total of 164 samples were tested and an overall prevalence for anaplasmosis was 18.3% and 17.8% and 22.2% in Olive baboons and Vervet monkeys respectively which was similar to that reported in a survey on cattle in Ethiopia [27].This is however inconsistent with reports by other authors.For example, in Zambia reported a 13% prevalence in baboons and Rhesus macaques [20].In Kenya, a study on Anaplasma hemoparasites in wild animals revealed high seroprevalence [29] while another reported 35.5% sero-prevalence in cattle in Nairobi, Kenya [22].
Studies have shown that A. phagocytophilum can persist for the lifetime of animals due to its effect on the glucose metabolic pathways for maintenance of infection and multiplication [30].This explains the high detection rates of Anaplasma infection in certain animal populations.Different host species, sample sizes, diagnostic technique, study areas demography and endemicity disease status in each study region could also explain it.
In this study, primers targeting p44 and 16S rRNA genes of A. phagocytophilum were identified from previous studies and used for detection of A. phagocytophilum [23,24].However, there was a significant difference in the performance of these two assays.The primers targeting p44 gene did not yield any positive bands on PCR.This is in agreement with several studies in cattle [27], wild birds and bats [31] and in ruminants and ticks [32].This can be explained by the high variability of p44 gene, which includes intra-species variability, with consequent protein polymorphism and the generation of antigenic variations [33,34].The 16S rRNA gene yielded positive bands and therefore reinforced that it is a good marker for detection of Anaplasma as reported by other studies [20,27,35].
Further sequencing analysis revealed that the sequences of 16S rRNA were very conserved not only between African isolates but also between the other isolates of world-wide origin agreeably with previous studies [36].The sequences of the A. phagocytophilum isolates from Kenya were identical to those from Japan, South Korea, France, China, South Africa and Denmark.The species isolates were from human, cattle, ticks, dogs and rodents (Figure 3).This is consistent with a study on baboons in Zambia [20].Multiple alignment of Anaplasma nucleotide sequences of the A.phagocytophilum isolates revealed that all the sequences of samples from baboons were conserved except for 15A.It was the most divergent in comparison to the rest with a lot of accumulated mutations and numerous insertions (Figure 2).
Extensive molecular studies have enabled understanding of the genetic profiles, level of genetic relatedness or variations among isolates across the different geographical regions and animal species.The isolates from Japan, South Korea, France, China, South Africa and Denmark belonged to clade I but have recent common ancestor with the Kenyan isolates clustered into one clade II except isolate 15A which appeared separately and represented a kind of outgroup (Figure 3).
Previous studies have reported genetic relatedness between A. phagocytophilum infecting animals and humans [15,35].Similarly, in this study isolates from Olive baboons appear to be genetically related to those of human (MF582329.1)(Figure 3).This, to the best of our knowledge, this is among the first molecular detection and estimate of the prevalence of Anaplasma phagocytophilum in Kenya's Olive baboons and Vervet monkeys.Nonhuman primates have been shown to host different pathogens, including several Anaplasma species [34,20].We assume that they could serve as a good indicator of bacteria circulation in ecosystem and explain the persistence of anaplasmosis in domestic animals despite consistence control.Phylogenetic tree of the 16S rRNA gene from A.phagocytophilum isolates.

Figure 1 :
Figure 1: Map of the sampling sites.

Figures Figure 1
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Figure 2 Multiple
Figure 2

Table 1 :
Primers used for detection and/or characterization of Anaplasma species in the present study

Table 2 :
BLASTn analysis results using 16S rRNA sequences of isolates from Olive baboons and Vervet monkeys.