Bacteriome in Ticks Collected from Domestic Livestock in Kenya

Background: Metagenomics approaches are increasingly being utilized as “dipstick” for microbial carriage. In this study, 16S rRNA metagenomics was used to probe for microbial community that resides in the ticks, those they pick from the environment, wildlife and livestock and to identify potential tick borne zoonoses. Methods: Tick DNA from 463 tick pools collected from domestic animals between 2007 and 2008 were amplified with primers that target the 16S rRNA V3-V4 domain and then sequenced on Illumina Miseq platform using 300 cycles version 3 kits. Ticks were pooled according to species and animal from which they were collected. A non-target control was used to track laboratory contaminants. Sequence data were analyzed using Mothur v1.3 pipeline and R v3.3.1 software and taxonomy determined using SILVA rRNA database. Shannon diversity index was used to compute bacterial diversity in each tick species before computing the means. Results: A total of 645 bacteria genera grouped into 27 phyla were identified. Four phyla contributed 97.4% of the 36,973,934 total sequences. Proteobacteria contributed 61.2% of these sequences that tarried to 33.8% genera, compared to 15.9% (23.4% genera) for Firmicutes, 15.6% (20% genera) for Actinobacteria and 4.7% (11.6% genera) for Bacteroidetes. The remaining 23 phyla only contributed 2.6% of the sequence reads (11.2% genera). Amongst the 645 genera, three groups were discernible, with the biggest group comprised commensals/symbionts that contributed 93.6% of the genera, but their individual sequence contribution was very low. Group two comprised genera that are known to contain pathogenic species, with Coxiella contributing 15,445,204 (41.8%) sequences, Corynebacterium (13.6%), Acinetobacter (4.3%), Staphylococcus (3.9%), Bacillus (2.7%) and Porphyromonas (1.6%), Ralstonia (1.5%), Streptococcus (1.3%), Moraxella (1.3%), amongst others. Group three comprised genera known to contain tick borne zoonotic pathogens (TBZ): Rickettsiae, Anaplasma, Francisella, Ehrlichia, Bartonella and Borrelia. Individually the TBZ contributed Amblyomma variegatum carried the least diverse bacteria (mean Shannon diversity index of 2.69 ± 0.92) compared to 3.79 ± 1.10 for A. gemma, 3.71 ± 1.32 for A. hebraeum, 4.15 ± 1.08 for other Amblyomma spp, 3.79 ± 1.37 for Hyalomma truncatum, 3.67 ± 1.38 for other Hyalomma spp, 3.86 ± 1.27 for Rhipicephalus annulatus, 3.56 ± 1.21 for Rh. appendiculatus, and 3.65 ± 1.30 for Rh. Pulchellus, but the difference was not significant (p = 0.443). Conclusion: This study illustrates the utility of 16S rRNA metagenomics in revealing the complexity of bacteria communities that reside and/or transit through the tick having been picked from the environment, livestock and/or wild animals, some with potential to cause zoonoses.

A Duo 4-Plex Real Time PCR for Detection of Eight Tick-Borne Zoonoses in Kenya

Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation of multiplex real time polymerase chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was optimized for Rickettsia species (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays can specifically detect all target sequences in the same reaction tube. Assays were assayed eight times to determine assay performance and the limit of detection was determined as the lowest plasmid concentration that was amplified for all the targets. Standard curves of threshold cycle (Ct) versus copy numbers were generated and used to determine linearity and efficiency of the assays. Pairwise comparison of singleplex and multiplex assays was done using Bland-Altman plots. Prevalence was calculated as overall percentage of positive patients to each TBZ tested Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay 2 was less sensitive and on average had a limit of detection of 18 gene copies. In replicate tests, both assays had intra-assay variation of less than two cycles. Multiplex assays performance was comparable to respective singleplex assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from acute febrile illness patients attending hospitals in different counties in Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C. burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high analytical sensitivity suggests potential for the duo 4-plex qPCR for detection of common TBZ.

Zoonotic Pathogens in Ticks Collected from Livestock in Kenya

Ticks are reservoirs of a variety of pathogens including bacteria, viruses and protozoa. We used PCR to detect pathogens of public health importance in ticks collected from diverse regions of Kenya. 503 tick pools were collected from 982 cattle, 300 sheep and 379 goats that were presented for slaughter at major abattoirs in Nairobi and Mombasa. Tick DNA was screened by qPCR optimized for single-plex detection of Babesia microti, two-plex Coxiella burnetii/Ehrlichia chaffeensis or Bartonella henselae/Borrelia recurrentis or three-plex for the non-human Babesia spp/Anaplasma phagocytophylum/Borrelia burgdorferi. Pathogen prevalence was calculated against tick type and the geographical origin. Computational analysis was performed with Graphpad prism 5. Out of 503 tick analyzed, 21% (106) were positive for at least one pathogen. C. burnetii was the most abundant at 70% (74/106), followed by non-human Babesia at 17% (18/106), 5% (5/106) for B. burgdorferi, 7% (7/106) for other Borrelia species and <1% (1/106) for E. chaffeensis and A. phagocytophylum. B. henselae and the human infective B. microti were not detected. Rh. pulchellus was the most promiscuous one in carrying pathogens: C. burnetti, Babesia, B. burgdorferi, E. chaffeensis and A. phagocytophylum. Non-human infective Babesia were detected in all ticks except Amblyomma. Four counties had 70% of the infected ticks: Marsabit 25% (n = 26/106), Kajiado 17% (18/106), Wajir 16% (17/106) and Narok 11% (12/106). This study identified a number of tick-borne pathogens that cause febrile infections often confused with malaria. Follow-up research will be needed to determine prevalence in humans.