Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. .ItrainlCnr fIsc hsooyadEooy( Ecology and Physiology Insect of Centre International 1. : Affiliations Villinger douwe Kenya Chiuya in Tatenda pathogens tick-borne mar- Zoonotic livestock title: KenyaRunning at western virus, in fever slaughterhouses haemorrhagic and Crimean-Congo kets including pathogens, surveillance Tick-borne continuous for calls and diseases zoonotic livestock under-diagnosed measures. at of preventative livestock risk of the from development highlights ticks the humans, in a and in importance virus, rate CCHF veterinary fatality of case and Confirmation high health a Kenya. public western of in pathogens slaughterhouses several and markets of presence the show findings as in such virus through CCHF identified of detected and pools We analysis 96 melting products. high-resolution PCR incorporating of workflows keys. PCR-sequencing molecular morphological established using using identified presence and pathogen Kenya livestock were western from species of carried collected felis tick counties therefore were Ctenocephalides sampled three We ectoparasites frequently in other most translocation slaughterhouses outbreaks. two and disease and possible The Ticks markets cross-border detect livestock of Uganda. to at risk neighbour systems pathogens the that warning zoonotic highlighted have tick-borne early Uganda of reported inter-epidemic, surveillance in (CCHF) establishing fever out CCHF of haemorrhagic of Crimean-Congo importance reports of the Recent case between and fatal Kenya. circulate global one Western to humans with known increased in Kenya, are of 2000 to diseases of movement tick-borne counties in continuous due Several western the the decades concern. from control and recent disease Uganda arising particular in eastern shifts, of are ranges range borders, Transboundary distributional across livestock change. their and climate expanded and have connectivity, trade diseases travel, infectious emerging of Vectors Abstract 2020 30, July 4 3 2 1 Villinger Jandouwe Chiuya Tatenda western in slaughterhouses and Kenya markets livestock at virus, fever haemorrhagic Crimean-Congo including pathogens, Tick-borne nentoa ietc eerhInstitute Research Livestock International Liverpool of University (icipe) Ecology Pretoria and of Physiology University Insect of Centre International hiei parva Theileria m variegatum Am. 1* 1,2 esand fleas ailK Masiga K. Daniel , 1,2 , ailMasiga Daniel , aei bigemina Babesia 1 eepstv for positive were amtpnssuis Haematopinus and , 1 ar .Falzon C. Laura , iktsaafricae Rickettsia 1 ar Falzon Laura , npam marginale Anaplasma iewr lopeet nttl46tcs ie n eswr cendfor screened were fleas and lice, ticks, 486 total In present. also were lice hpcpau decoloratus Rhipicephalus 1 pcmlxnpooo n cei fvtrnr importance, veterinary of bcteria and protozoa Apicomplexan . h decoloratus Rh. 3,4 3,4 icipe rad ..Bastos D.S. Armanda , eepiaiydtce nriiehln ik.Our ticks. rhipicephaline in detected primarily were , rad Bastos Armanda , ,POBx372010 arb,Kenya Nairobi, 30772-00100, Box P.O ), 3% and (35%) Nairovirus and Rhipicephalus mlom variegatum Amblyomma htcue amrhgcfvrwith fever haemorrhagic causes that 2 2 rcFevre Eric , rcM F`evre M. Eric , p atetcsad8 of 82 and ticks cattle sp. 3,4 3%,and (30%), 3,4 and , Jan- , Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. te e pce nadto otecifvcos(uefdo ta. 05.Lueifsain euti severe in result infestations Louse 2015). al., et (Luce-fedrow vectors chief vector- the of to addition epidemiology in the species in flea other fleas ( and typhus murine lice as of and such role ( rickettsioses, fever the Flea-borne spotted investigated. established, flea-borne rarely is is ticks zoonoses of borne 2015). capacity (Baron, vectorial Africa the of al., While Horn et a illness (Papa the also systemic fevers and virus, haemorrhagic severe Africa viral disease cause East some, of sheep transmission in can but Nairobi the al., humans, viruses, livestock, in et in ticks In Dugbe of illness Rutherford 2017). and importance febrile 2013; (CCHF) the non-pathognomonic affirming fever al., mortality, mild and haemorrhagic et a Crimean-Congo Parola cause as 2004; which such of al., most et nairoviruses, Ndip transmit 2017; 1984), al., al., al., livestock et health et et Fournier as Norval to major public 2016; Maina 1995; addition (Brown, cause al., and 2017; In globally et anaplasmosis economic al., threat 2004). Latib health et 2012; and of public al., Jensenius theileriosis, pathogens emerging et 2017; serious Gachohi protozoal Babesiosis, a 2015; and constitutes al., 2008). rickettsiosis et bacterial, al., (Franck while Kenya viral, et in of losses Fuente production range la a (de of importance vectors are Ticks and Introduction surveillance continuous for calls Kenya. and Keywords: estern diseases in measures. zoonotic preventative and slaughterhouses under-diagnosed of health and development of public markets the risk of livestock the pathogens highlights at several a humans, livestock virus, of CCHF presence from of the ticks Confirmation show in findings importance Our veterinary ticks. rhipicephaline in detected as such importance, veterinary in established using of virus PCR-sequencing presence CCHF pools through pathogen detected identified for 96 We screened and products. were analysis fleas PCR melting and of high-resolution lice, were ticks, incorporating 486 species workflows total tick molecular In sampled present. also frequently were most lice two three The in livestock keys. from slaughterhouses collected decoloratus morphological and were ectoparasites markets using other the livestock and identified and therefore Ticks at and Uganda. We translocation neighbour pathogens outbreaks. that disease Kenya zoonotic possible of Western cross-border of tick-borne detect counties counties of to of western risk systems surveillance warning the out the Kenya. early highlighted carried western and inter-epidemic, have in establishing Uganda 2000 Uganda of in in eastern reported importance CCHF (CCHF) concern. between fever of control haemorrhagic circulate reports Crimean-Congo disease from of Recent particular to case arising of fatal known shifts, one are to with are borders, range Kenya, due across diseases Transboundary decades livestock change. recent tick-borne and in climate humans Several ranges of and movement distributional connectivity, continuous their trade the expanded travel, have global diseases increased infectious emerging of Vectors Kenya Nairobi, Summary 00100 30709, Box Campus, PO [email protected] Road, author, Leahurst Naivasha *Corresponding Sciences, Old Institute, Ecological Research and Livestock Kingdom International Veterinary United 4. 7TE, Infection, South CH64 of 0028, Neston, Pretoria Institute Road, High 20, Liverpool, Chester Bag of Private University Pretoria, 3. of University Entomology, and Zoology Africa of Department 2. Babesia tncpaie felis Ctenocephalides aebe hw oifc uasi h mrcsadErp Duire l,21) ik also Ticks 2010). al., et (Doudier Europe and Americas the in humans infect to shown been have 3% and (35%) m variegatum Am. mrigifciu ies,Zoonoses, disease, infectious Emerging Rickettsia mlom variegatum Amblyomma iktsafelis Rickettsia es epciey oee,these However, respectively. fleas, eepstv for positive were hiei parva Theileria ikbrebcei uhas such bacteria tick-borne , Nairovirus ,bt nei nEs fia r rnmte by transmitted are Africa, East in endemic both ), htcue amrhgcfvrwt ihcs aaiyrt in rate fatality case high a with fever haemorrhagic causes that , iktsaafricae Rickettsia 3%,and (30%), h decoloratus Rh. aei bigemina Babesia Nairovirus 2 Nairovirus tncpaie felis Ctenocephalides Rickettsia Ehrlichia , and Rickettsia and , sacntn hett he production sheep to threat constant a is , pcmlxnpooo n atraof bacteria and protozoa Apicomplexan . Rhipicephalus npam marginale Anaplasma and p.hv endtce nseveral in detected been have spp. , Rhipicephalus Anaplasma esand fleas p atetcsad8 of 82 and ticks cattle sp. iktsatyphi Rickettsia n rtzasuch protozoa and , atAfrica. East , amtpnssuis Haematopinus eoslacheopis Xenopsylla eeprimarily were , Rhipicephalus and ) Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. hsrgo,pr fwihsae odr ihUad,i ersnaieo h agrLk itrabasin Victoria Lake larger the of representative is Uganda, with Kenya. Western borders in Kakamega, shares and which Bungoma, of Busia, viz. part counties, region, neighbouring in This out carried was study The site Study Lake arthropods. Methods the these and in in detect Materials LMs importance rapidly slaughterhouses veterinary via to and sequencing and LMs animals Sanger both zoonotic infested at with of livestock coupled vector from techniques pathogens lice arthropod molecular and high-throughput fleas, of employed ticks, We movement collected (SHs). we the Africa, East by of clinical posed basin improves Victoria risk presence their the of investigate illness, Awareness To febrile agents. non-specific fever-causing by spill- other characterised diagnosis. reduce with are and to diseases confused referral strategies the easily control of and be planning Most can presence for humans. which important to their is livestock on thus from and infection information detection over burdens early generates facilitate pathogen can of vectors information quantification Such their and associations. these vector-pathogen and novel of identify pathogens can importance and zoonotic prevalence the confirming for abattoirs, surveillance instances, viruses. and most Active these In LMs 2013). of at (Nyaruaba al., investigations cattle Africa et been epidemiological Lwande from only East for 2017; have collected in al., facilities viruses, were endemic Bunyamwera et Villinger arboviruses and are 2018; Ngari, with viruses al., Wesselsbron, Nile ticks Forest, These et West Semliki (Ajamma 2006). and as mosquitoes 2011, such from Dhori, al., isolated some, Ngari, et and Thogoto, 2019) Sang al., forest, 2013; 2015). et Semliki al., al., Ndumu, et et (Lwande Teshale Dugbe, 2017; viruses Bunyamwera, al., CCHF, public et with and Mwamuye veterinary infected 2017; al., of et protozoa Omondi ma and 2018; Nakao bacteria 2015; al., including of al., et et Ringo detected, spectrum (Kumsa broad been Africa A East also and chaffeensis 2014). have 2003) al., al., importance et et reported. Nakayima health Macaluso been 2017; 2013; al., has al., oc- et fleas Mwamuye et the 2019; and Africa, 2014, ticks ( al., East in agents et rickettsiosis and potential (SFG) Kenya veterinary group in fever and vectorsaeschlimanii transmission Elsewhere spotted zoonotic both the flea-borne 1997). of be in and Roux, may component pathogens Tick & important lice bacterial an on (Raoult and of them reports fleas, zoonoses currence Kenya making While Ticks, western vector-borne transmit, 2019). in illnesses. they of al., malaria febrile pathogens RVFdynamics et of of most Nyaruaba prevalence and causes of high 2011; Nile, reservoirs other Western the al., West and scant, of in et fever, are under-investigation Mease before yellow bacteria in 2020; reported chikungunya, vector-borne al., results zoonotic been of et of have Inziani circulation occurrence 2018) 2017; of the 2018). al., (WHO, evidence WHO, RVF et be 2010; serological and (Cook al., then is viruses 2002) et can there al., Munyua Livestock they et and 2016; 2006). al. where Kenya, (Dunster al., et na¨ıve and CCHF areas (Baba et past in of Kenya in (Sang Outbreaks foci in implicated ticks outbreaks infective been (RVF) by have of movements fever infested Livestock Valley introduction heavily 2006). Rift the al., recent be (F`evre (F`evre et in vectors livestock et to areas capable with role found by peri-urban associated disseminated major been in is al., have a (LMs) Africa et animals markets plays (Mossel livestock East which movement to region in in the the borders 2005) production 2012), across national emerging livestock al., (Kilpatrick, and reported of Smallholder provincial spread circulation been 2019). across the has pathogen movement al., enhance activity emerging et could arboviral of Nyaruaba countries high drivers 2017; African that East given major among especially be people pathogens, to and livestock thought of trade movement and caused travel typhus, epidemic With and 2010), al., et (Hornok 1997). losses production to leading by livestock, in mange pruritic and , iktsaprowazekii Rickettsia Rhipicephalus , npam marginale Anaplasma , .felis R. and , ik ape rmlvsoki ot-atr ey eepeiul hw obe to shown previously were Kenya North-Eastern in livestock from sampled ticks nhmn,epcal noecoddadpo oilstig Rol Roux, & (Raoult settings social poor and overcrowded in especially humans, in , iktsaasembonensis Rickettsia , npam phagocytophilum Anaplasma p o. aebe eetdesweei ey (Maina Kenya in elsewhere detected been have nov.) sp. 3 hiei parva Theileria and , .africae R. npam platys Anaplasma , hlci ruminantium Ehrlichia , iktsaconorii Rickettsia Hyalomma Onoe l,2020; al., et (Oundo , , , Amblyom- Rickettsia Ehrlichia Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. oeua dnicto a efre n1 igeseiesfrwihmrhlgcietfiaint species to identification morphologic which for specimens single 15 on performed was fleas identification to and Molecular according lice, performed ticks, were of steps identification subsequent The Molecular inversion. by solution instructions. 10 into manufacturer’s Then mixed the and vortexed. added and magnetic were the 200 RNA, beads First, using carrier USA). extracted and Gaithersburg, were isopropanol, Genomics, samples 1.5- (MagBio to blood kit added from DNA/RNA was acids a Viral for Nucleic as Prep positive High used as screening. identified bead-based was pathogen 50 PBS pools for in and arthropod selected eluted with control California, were was associated extraction Systems, acid samples positive nucleic Molecular blood a Total (Roche Animal (Roche as Kit run. Volume robot included each Small was in 96 NA control isolate Pure Viral extraction culture MagNA and negative virus DNA a sindbis 96 in A MagNA Germany) USA). a (360 Penzberg, using (PBS) extracted Science, were saline Applied RNA buffered oxide and zirconium Phosphate DNA stabilized seconds. cartridge. 210 yttria 60-90 and Mini-Beadbeater- 0.1-mm vortexed, a for of tube, using mg OK) each disrupted 150 mechanically Bartlesville, were and they (BioSpec, 2.0-mm which 16 of in Eppendorf USA), mg 1.5-ml a (Biospec, 750 in beads of placed (zirconia/yttria) was scoops pool pre-weighed Each samples extraction. with acid blood nucleic tube (1-3) before livestock homogenised pooled selected were sampled. pools were and were Arthropod fleas arthropods they and which from lice, from extraction Ticks, host acid stereomicroscope. Axio- and Nucleic an a species, using on sex, photographed mounted stage, developmental were (Zeiss) engorged to specimens camera Excessively Ober- according Representative digital 1973). (Zeiss, analysis. Pratt, 5s stereomicroscope 2004; the ERc al., a from cam et using excluded (Walker keys level were identification species samples of tick to aid identified the with morphologically Germany) were the kochen, fleas to and fleas ice lice, and Ticks, dry lice, on ticks, shipped of identification being Morphological -40 before Busia at on Busia ( in present stored in Ecology lab if and were lab field Physiology collected Services Arthropods the were Veterinary to packs. L fleas transported of ice Martin and and Department lice with barcoded (ILRI) ticks, were box to Institute tubes cool and addition blood cattle a In At and ethanol. followed. in bottles 70% selected was Sample containing the procedure of tubes animals. vacutainer. hide falcon similar sampled a the into using a general on placed present vein and SHs, a parasites jugular hands pig external after gloved the Any with and, animal from collected. removed also veterinarian were capture restrained the were animals qualified to samples physically from a faecal administered occasionally then by sought and was swabs was drawn were was Nasal questionnaire was it Animals consent short blood as each details. Signed examination, a ratio during via ownership clinical ruminants. and above selected sheep animal animals, small the were one and sampled follow of sheep) and demographic always accompanying one owners goats, not traders and from or did three goats, consent owners three sampled cattle, get cattle, associated animals six to (six closely of select was animals challenging number to LM 10 the each LM, attempted though where each We visit, 1), At four sampling. Briefly, (Figure SH. 2019). random county pig al., each systematic or et in (Falzon cattle elsewhere selected a detail were with in SHs described neighbouring are and collection LMs sampling and design study The collection sample are and 2017). practices consequent predo- design al., with (F`evre husbandry The et Study intensification, transmission though Africa. increasing and system, to East emergence subsistence production in disease largely livestock on densities from moves impacts smallholder population production mixed livestock as a and changing rapidly is human type rural highest farming minant the has and ecosystem shrEegn netosDsae M-I)lbrtr tteItrainlCnr fInsect of Centre International the at laboratory (ML-EID) Diseases Infectious Emerging uscher ¨ μ pedr ue otiig528 containing tubes Eppendorf l icipe μ hr hywr trda -80 at stored were they where ) fterslighmgnt a rnfre oa9-elseie processing specimen 96-well a to transferred was homogenate resulting the of l μ 4 falssmse-i ossigo D yi buffer, lysis VDR of consisting master-mix lysis a of l μ fpoens n 10 and K proteinase of l ° C. ° tteItrainlLvsokResearch Livestock International the at C μ fRAefe water. RNAse-free of l .africae R. μ fMGS magnetic MAG-S1 of l μ n CFvirus CCHF and )wsaddto added was l) μ fblood of l Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. otae210adrpeettv mlcn ihuiu R rfie eeprfiduiga x 1-rSAP Exo an using Series purified (Netherlands). Q were Macrogen Rotor-Gene profiles at with HRM sequenced inspected unique and visually UK) were with (Biolabs, profiles amplicons combination HRM representative Resultant and runs. 2.1.0 the software for in controls included Positive were 2017). ticks) al., et (Mwamuye before marginale described consisted as that Germany) reactions Hilden, Ten-microliter (Qiagen, 10 1). Table at of (Supplementary primer rRNA gene screening ribosomal 16S com- initial 18S 2 for the a the of used using target target were protozoa that that 2001), primers and primers al., as bacteria developed well for as Previously 2007), screened PCR. also conventional of were and gene samples PCR-HRM livestock-blood of and bination previously flea, as louse, sequenced Tick, pathogens and protozoan and purified, bacterial amplified, of the Detection also targets 2004). that were al., assay 1) et PCR (Honig conventional Table described a (Supplementary sequencing singleplex using bidirectional gene from fragments for Amplicons submitted Larger L-polymerase and 1). UK) (Netherlands). for (Biolabs, Table combination conditions Macrogen (Supplementary 1-rSAP same to 2017) Exo the an separately al., and with were et nairoviruses purified samples were and (Villinger runs positive flaviviruses, runs alphaviruses, All PCR-HRM of 2.1.0. multiplex each software the for virus controls. Series mixes sindbis negative Q primer PCR the Rotor-Gene as using of water with re-run cDNA grade visualised included molecular were and run cycling controls Each profiles thermal extraction HRM no-template touchdown 2017). and al., using control et positive Germany) (Villinger a Hilden, as elsewhere (Qiagen, detail in thermocycler described PCR conditions real-time Q Rotor-Gene a K n 1 and UK) Edhe l,20) h ecin eepromdi ipim hroylr(ple Biosystems, (Applied 10- thermocycler The 1 SimpliAmp 2018). contained a al., PCR-HRM in et multiplex (Ajamma performed the conditions were cycling for thermal reactions mixture described The previously using 2005). Singapore) 20- al., a in et Lithuania) (Endoh Biosystems, u/ (Applied 2.5 kit 10 buffer, (RT) contained Transcription that Reverse mixture the cDNA reaction Capacity for High utilised the initially using was the test (RT)-PCR-HRM within Thogotovirus transcription arboviruses reverse of multiplex detection described previously A (www.ncbi. arboviruses pathogens database of protozoan nr Detection bacterial, GenBank arboviral, the of against detection 1990) wereMolecular al., contigs matches. et sequence sequence (Altschup resulting identify was searches The using to UK) Zealand). BLAST nlm.nih.gov/blast) aligned New (Biolabs, nucleotide and (Biomatters, combination edited, in software 1-rSAP inspected, used 2019.0.4 were Quick- Exo step version chromatograms alongside by extension Prime Sequence visualised purified Geneious final (Netherlands). were amplicons the Macrogen size of by that correct sequencing performed exception the Bi-directional of the have light. Amplicons with conditions UV cycling 2017), minutes. The seven al., run. was et each Load fragments (Mwamuye on a three control detail the in negative in for a performed before as were included described PCRs was been water The grade 10- Molecular 1). in primer. FIREPol Table cytochrome Singapore) HOT (ITS2), (Supplementary Biosystems, 5x spacer-2 (r)RNA (Applied of transcribed Cycler ribosomal internal Thermal 16S the PCR and genes: SimpliAmp target (CO1), three 1 amplified oxidase We equivocal. was level μ ® epae 2 template, l Anaplasma 0-pDALde Boas K yeetohrsso . tiimsandaaoegl under gels agarose stained ethidium % 1.6 on electrophoresis by UK) (Biolabs, Ladder DNA 100-bp and ) μ f50 of l μ μ eea(ilne ta. 07 SplmnayTbe1.Ti a rcddb DAsynthesis cDNA by preceded was This 1). Table (Supplementary 2017) al., et (Villinger genera ocnrtos yln a are u naRtrGn eltm C thermocycler PCR real-time Q Rotor-Gene a in out carried was Cycling concentrations. M ees rncits nyead4 u/ 40 and enzyme transcriptase reverse l Rickettsia μ Mauee l,2017), al., et (Mwamuye μ XHTFIREPol HOT 5X l YO9(ieTcnlge,UA.MlilxPRHMratoswr efre in performed were reactions PCR-HRM Multiplex USA). Technologies, (Life SYTO-9 M ® ln atrMx(oi iDn,Etna,2 Estonia), BioDyne, (Solis Mix Master Blend ( .africae R. μ lvvrs lhvrs arvrs heoiu,Orthobunyavirus Phlebovirus, Nairovirus, Alphavirus, Flavivirus, uli cdetat U/ 1 extract, acid nucleic l peiul eetdin detected (previously ) ® vGenHMMx(oi iDn,Etna,ad0.5 and Estonia), BioDyne, (Solis Mix HRM EvaGreen Ehrlichia μ DAtmlt,5 template, cDNA l 5 Mauee l,21) and 2017), al., et (Mwamuye μ o-iooa admhx-uloieprimers hexa-nucleotide random non-ribosomal l icipe μ Theileria Ns niio,10M NP,1 RT 1X dNTPs, Mm 100 inhibitor, RNase l sM-I a from lab ML-EID ’s μ ftmlt,ad0.5 and template, of l μ μ and f2 ya SMx(Bioline, Mix HS MyTaq 2x of l ecin htcnitdo 2 of consisted that reactions l Babesia Rickettsia aaie Gogset (Georges parasites Amblyomma Anaplasma Njo tal., et (Nijhof μ f10 of l μ Nairovirus μ reaction l feach of l and , spp. ( μ A. M μ μ l l Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. dnie opooial SplmnayFgr ) eeslce o oeua ikietfiain(Table identification for tick identifications molecular morphological for with decoloratus selected concurred were identifications Molecular 1), 1). Figure (Supplementary morphologically identified sp were fleas more the generally to and were nymph), SHs due which at partially of cattle, pigs were specimens from was the Representative collected locations This of primarily these were 80% 2). of lice Over Table each cattle. The study. (Supplementary at from pigs. this sampled collected or cattle in sheep, animals from analysed goats, the were than ( came of tick-infested SHs 60% lice SHs and that 28 and LMs fact nymphs), at LMs the 22 pigs at and and collected sheep, adults vectors goats, (434 cattle, ticks from 456 collected of SHs total and A LMs at diversity and collection Vector using online Results performed each were computations in The individuals (Ser- 2001). of https://epitools.ausvet.com.au/ppvariablepoolsize 2009). Moffitt, number from geant, & accessed the calculator Williams account epidemiological like- 100% 1999; into an assumed al. maximum took Epitools populations et a and vector (Cowling results by within tested pooled-sample estimated estimates pool prevalence of were True specificity samples model. and frequentist pooled sensitivity a in in detected approach lihood pathogens samples of pooled prevalences from Individual-level prevalences al., 2014). et pathogen (Rambaut, (Guindon individual-level 1.4 evolution Figtree of of in Estimation employing model edited 3.0., appropriate and version for phylogenetic visualised PhyML selection to were using automatic Trees prior gene were for removed 2010). each manner were criterion for this sequences information in inferred Akaike primer compiled were the the datasets phylogenies to the searches likelihood of corresponding nucleotide Maximum with Each BLAST regions analysis. related 1990). through terminal al., identified closely the et were and (Altschup entries study aligned database sequence this nr in and GenBank generated the software reference sequences against 2019.0.4 of individual version Prime the sequences Geneious of Homologous in each alignment Zealand). Geneious using New aligned (Biomatters, and edited were sequences All New sequencing. (GenScript, bidirectional Kit for analysis Extraction all (Netherlands) Gel Phylogenetic Macrogen of II to QuickClean gene submitted the B using and protein sequencing USA) Jersey, membrane after for prepared visualised For outer were were 1030bp. the samples Products at Positive of time. Rick- product region a with expected amplified at 856-bp further the tube a were of one profiles presence only HRM positive the opened with and confirm enclosure to PCR electrophoresis a gel in reaction second the 95 72 of and cycles 3 min; 15 2 utilised 72 and 1). 95 s, Table 30 95 of of (Supplementary pH1492 step cycles respectively activation and 2 amplification, s, hot-start 1989) round al., a 95 et second using of (Edwards and cycle performed pH1522 first were primers amplifications for reverse primer, Primary 1992) universal forward al., Anaplasmataceae-specific with the et 2001) combining (Reysenbach by al., bp) et (1030 (Parola gene rRNA EHR16SD 16S the of fragment longer Positive . oeadult), (one ° o 0s n nletnina 72 at extension final a and s, 80 for C mlom gemma Amblyomma Ehrlichia μ ° fPRpout rmpiayratosa epae.Teccigpol osse f 95 of: consisted profile cycling The templates. as reactions primary from products PCR of l o 0s 63 s, 20 for C ° T3) and (T134), o 0s n nletnina 72 at extension final a and s, 80 for C ° o 0s 61 s, 20 for C hpcpau decoloratus Rhipicephalus and ° o 0s 61 s, 20 for C Anaplasma ° o 0s n 72 and s, 30 for C m variegatum Am. hpcpau evertsi Rhipicephalus ° oeadult), (one o 0s n 72 and s, 30 for C ape eefrhrapie ihasm-etdPRt eeaea generate to PCR semi-nested a with amplified further were samples ° o 0s n 72 and s, 30 for C ° mlom variegatum Amblyomma T9) oee,w eovdatc pcmn(15 htwe that (T105) specimen tick a resolved we However, (T199). o 0s ylso 95 of cycles 2 s, 90 for C oeaut,and adult), (one ° o 0mn omnms h iko otmnto estup set we contamination of risk the minimise To min. 10 for C ° o 0s olwdwt 5cce f95 of cycles 35 with followed s, 90 for C ° o 0mn h eodr 20- secondary The min. 10 for C oeadult), (one 6 ° o 0s 7cce f95 of cycles 37 s; 90 for C amtpnssuis Haematopinus ompB Rhipicephalus hpcpau appendiculatus Rhipicephalus Rickettsia ° rmr Ru aut 00 targeting 2000) Raoult, & (Roux primers oeaut n nymph), one adult, (one o 0s 62 s, 20 for C pce SplmnayTbe1). Table (Supplementary species p.(i dls n nymph), one adults, (six spp. h appendiculatus Rh. ° o 5mnfloe y1 by followed min 15 for C ,adtofla ( fleas two and ), ° o 0s n 72 and s, 30 for C ° μ o 0s 60 s, 20 for C mlfiainreactions amplification l Rickettsia ° o 0s 60 s, 20 for C oeaut one adult, (one Haemaphysalis l samples all , ° o 0s, 30 for C (T16), Ct.felis ° o 90 for C ° ° for C for C Rh. ) Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. oDARAo h ahgn vlae nti td a eetdi h e pcmn.Alo h 33 the of All specimens. flea the in detected was study this in evaluated pathogens the of DNA/RNA No tick detected previously of to, detected in close from, we phylogenetically distinct are pathogens, but which MN266946-MN266948), these MN266922-MN266928, 4. to MN262071-MN262076, Figure in addition shown are In sequences pathogen homologous with study this MN294724) accession ceso MN294720) accession detected eight We decoloratus in 3). Table exclusively (Supplementary MN294721-MN294723) cattle accessions from removed were ticks mutans taurotragi In for of MN294731-MN294732). positive were accessions pools pools two 108 while MN294725-MN294729), of accessions out Twelve 2). one Only of of pools pool two one in detected was MN266936-MN266938) of pools 3). Table Kenyan most of pools from two absent is that detected Tanzania insertion We in four-base fever a 2). bite possessed tick Figure and (Supplementary African positions isolates with several diagnosed in patient substitutions a base in KU721071). detected the accession strain of GenBank a (unpublished; Two to in another pigs. detected and and previously KF660534) goats those sheep, to cattle, identical from removed were tick sampled commonly detected most We The markers. the of detected two Pathogens least at of were homologies species sequence on based identified Haemaphysalis an other of sequence ITS2 The as identified morphologically dpstdGnakacsin N970M244)(al 2) (Table MN294740-MN294749) accessions GenBank accession (deposited (GenBank of Nakiwogo pool of (DRC)one the pools 1967). Congo to 96 al., of of (98.6%) et out Republic into Eighty-two highest (Simpson Democratic fall Uganda was the identified from identity and isolates isolated (Table sequence Uganda virus strain counties from CCHF nucleotide KX013483) isolates The sampled Their includes SHs. three 3). two which the (Figure at clade, true across estimated cattle II (0.62% from SHs genotype MN267049) removed the 13 MN267048, were accessions and ticks GenBank These LMs (deposited prevalence). virus 13 CCHF from for positive collected were lice and ticks 2) in 2) (Figure . Haemaphysalis u fte33postse SplmnayTbe2,one 2), Table (Supplementary tested pools 333 the of Out m gemma Am. a lodtce none in detected also was h decoloratus Rh. npam marginale Anaplasma n n for one and , Rhipicephalus , h appendiculatus Rh. h evertsi Rh. h appendiculatus Rh. Rhipicephalus .platys A. h decoloratus Rh. Anaplasma and , The . oilaburnetii Coxiella T2rfrnei h eBn aaae ee u fnn pcmn of specimens nine of out Seven database. GenBank the in reference ITS2 Amblyomma . . dpstdGnakacsin N699M264)i v ol of pools five in MN266939-MN266941) accessions GenBank (deposited n olof pool One h hlgntcrltosiso h pcmlxnprst eune dnie in identified sequences parasite apicomplexan the of relationships phylogenetic The Coxiella hiei velifera Theileria rmgasadcattle. and goats from n w ol of pools two and and p ikpo a oiiefor positive was pool tick sp. m gemma Am. p,adtreposof pools three and sp., Coxiella h decoloratus Rh. 3% and (35%) m variegatum, Am. Rickettsia n olof pool one , and , dpstdGnakacsin N691M263)wsdtce nfour in detected was MN266931-MN266935) accessions GenBank (deposited noybot hrceie nti td elit h ru n clades C and B group the into fell study this in characterised endosymbionts h appendiculatus Rh. h decoloratus Rh. p.ta ol o eietfidt pce ee ymrhlg ln were alone morphology by level species to identified be not could that spp. h ahgnrsosbefrQfvr naltegnr ftcsexcept ticks of genera the all in fever, Q for responsible pathogen the , h decoloratus Rh. noybot ftcs(iue5). (Figure ticks of endosymbionts m variegatum Am. Rhipicephalus T2)hdhgethmlg with homology highest had (T222) bacteria, m variegatum Am. dpstdGnakacsin MN294733-MN294734) accessions GenBank (deposited Rhipicephalus m variegatum Am. m gemma Am. obe to he ol of pools three iktsaafricae Rickettsia h appendiculatus Rh. Coxiella Babesia eepstv for positive were h microplus Rh. a oiiefor positive was n one and p,sxposwr oiiefor positive were pools six sp., eepstv for positive were 7 h decoloratus Rh. m variegatum Am. n n olof pool one and , sp. .parva T. , (30%). Theileria noybot dpstdGnakaccessions GenBank (deposited endosymbionts ik nAeb nKna(eBn accession (GenBank Kenya in Asembo in ticks npam ovis Anaplasma h decoloratus Rh. .variegatum A. .africae R. h decoloratus Rh. ainsi hssuywr hrceie by characterised were study this in variants ae nis1SrN eunehomology. sequence rRNA 16S its on based . GnakacsinM243)(Table MN294730) accession (GenBank l bandfo ate(Supplementary cattle from obtained all , hiei taurotragi Theileria , hiei mutans Theileria These eaooncanis Hepatozoon Hepatozoon he ol of pools three , aei bigemina Babesia aei caballi Babesia ikpos igeposec of each pools Single pools. tick .suis H. .africae- R. m hebraeum Am. eune rmti td were study this from sequences orposof pools four , ol All pool. dpstdGnakaccessions GenBank (deposited eepstv for positive were n one and rtza n CFvirus CCHF and protozoa, .mutans T. Rhipicephalus Theileria oiieectoparasites positive dpstdGenBank (deposited dpstdGenBank (deposited dpstdGenBank (deposited dpstdGenBank (deposited dpstdGenBank (deposited steewr no were there as , Rhipicephalus Rhipicephalus h decoloratus Rh. Rhipicephalus he for three , p.positive spp. Theileria . .africae R. p,and sp., Rh. sp., sp. T. , , Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. ikbt ee AB,as nw sArcntc yhs nhmn,in humans, in typhus) tick African as known of also prevalence) (ATBF, true transmitted fever estimated be bite (78.95%; prevalence tick can high virus a 2013). demonstrated the We al., because et (Bente nature host in is the africae transmission virus in Rickettsia non-viraemic viraemia CCHF detectable Such of without 2017). (Kazim´ırov´a even amplification al., without ticks blood the tick to et among co-feeding its in non-infected contribute a in even to to virus ticks, virus a presumed detectable non-infected transmit may having and tick host infected infected An the between host. the transmission in human viraemia co-feeding of Africa. of absence to East risk due in the ecologies systems that 2006). transmission small-holder demonstrate 2011, tropical al., in findings ticks, et present our in While Sang also regions, 2013; is arboviruses and pastoralist al., arboviruses other facilities, et targeted tick-borne Other (Lwande of health studies to approach. epidemiology exposure public Health these occurrence their One at of in the a ticks most and described of adopting While SHs, have importance and and the Kenya pathogen LMs indicates this in which other for studies and surveillance surveillance these routine LM/SH-based at for been virus need has CCHF the Infection care emphasise to a Kenya, health found exposure Eastern when (Erg and (2012) human patients North bites, Western al. haemorrhaging tick for in slaughtering, in of et during virus case care Lwande spatters CCHF fatal However, take blood livestock, workers a to region. with when the contact antibodies skin 2000 in IgG after year Erg circulating reported of the 2017; virus seroprevalence since al., the human Kenya et of in 23% possibility (Cook reported workers the been showed countries, health-care not two Kenya virus and have the CCHF outbreaks abattoir between for CCHF virus groups and At-risk of 2006). veterinarians, livestock. between strain infected farmers, animals single of live include movement a in cross-border of infection trade through circulation extensive facilitated the the be and supports may our Uganda which finding between (Erg to This relationship surprising site countries. phylogenetic not study two close our is the the of Uganda proximity and in geographical virus isolated the CCHF strain given of Nakiwogo clades the distinct and of three isolates stages. epidemiology are early landscape there its the Africa, in of In is understanding transmission an its and and studies, Erg virus competency 2018; this where comprehensive al., areas requires in et this virus the (Balinandi of of virus transmission support CCHF may of ticks transmission the in morbidity virus significant the suffer 1979). of who humans, carriers as to asymptomatic such ticks) be (via genera infection to of shown (Spengler reservoirs vice-versa been as and (Erg and have acting ticks animals 2016), sheep, livestock infected seronegative al., the especially between et on of animals, Association (Spengler found blood Domestic negative. be the 2016). were also tested they al., can also but et ticks we virus, however, livestock, CCHF common; from for is collected from seropositivity and came first blood-fed they the were which study from is this This in SHs. ticks two spp. at slaughter for besides destined cattle from in in removed virus ticks CCHF of in description virus CCHF virus-positive detected CCHF We two ticks the in which detection from CCHF cattle the from were Discussion two which other from pigs) the three while Rhipicephalus and collected, cattle (28 were animals ticks from were for negative samples were blood samples blood livestock associated selected Hyalomma ¨ on Rhipicephalus Hyalomma l 06.While 2006). ul, ¨ Hyalomma ik rmtenrhesenrgo Sn ta. 01.Ti ugssta te ikspecies tick other that suggests This 2011). al., et (Sang region north-eastern the from ticks p.wr obtained. were spp. Rhipicephalus ntcsadlice and ticks in p.tcsaetecifvcoso CFvrs te pce a lob motn to important be also may species other virus, CCHF of vectors chief the are ticks spp. p.myb uprigtelcltasiso ftevrs steinfected the As virus. the of transmission local the supporting be may spp. p.hv lotasitdCH iu nlbrtr etnsadhv enimplicated been have and settings laboratory in virus CCHF transmitted also have spp. Hyalomma aebe on netdwt h iu Fkozb ta. 05 Hoogstraal, 2015; al., et (Fakoorziba virus the with infected found been have , h decoloratus Rh. ik r h aua etradrsroro CFvrs te tick other virus, CCHF of reservoir and vector natural the are ticks ik nKna ihpeiu tde eotn eeto only detection reporting studies previous with Kenya, in ticks ¨ on l 06.Orfidnsteeoehglgttepotential the highlight therefore findings Our 2006). ul, ¨ 8 Hyalomma .africae R. ¨ on l 06.Therefore, 2006). ul, ¨ p.aeasn.Hwvr confirmation However, absent. are spp. n CFvrs hryoeo these of Thirty-one virus. CCHF and .africae R. ¨ on m variegatum Am. .africae R. l 06 uahve l,2016) al., et Lukashev 2006; ul, ¨ positive h gn fAfrican of agent the , Rhipicephalus m variegatum Am. ik collected ticks Rhipicephalus ¨ on spp. ul, ¨ Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. lo rmlvsok(mnie l,21;Si ta. 07 ous ta. 06 n eeteiec suggests evidence recent and 2016) al., et Lorusso 2017; al., et Said 2017; al., that et (Omondi livestock from blood is of cycle detected 2015). life also al., We its et animals. of (Mans pathogenic cycle most mildly life the where its biology, al. of of ticks completion its for in by RBC prevalence explained velifera in higher multiplies partly We found only be 2015). we and may Accordingly, system al., lymphoid study et the this in Woolhouse in found 2010; samples al., blood no et SSA, animal where (Wesonga and Reserve, rhipicephaline animals Kenya National of in affected prevalences) Mara production in true livestock production (estimated to found of impediments 0.31% recently loss major and are severe 0.63%, diseases causing 1.88%, three in These fever respectively. areas. ticks, Coast endemic East in causes pathogen which this area. of study aspect our detected health in We public patients the febrile in on detected focus be Babesia that can studies Theileria, variants for these need of a some is if there seen Clearly, be to remains it However, xli h bec faueAB ae nKna ept h ihsrpeaec.Ti sspotdby supported is in This virulence seroprevalence. increased high to the lead despite may Kenya, reduction in genome cases that ATBF evidence acute the of absence the explain intact an with variants of adhesion cellular in rvossuis(iiae l,06 aauoe l,20;Miae l,21) h ieecsfudi the in found differences The 2014). al., et Maina found 2003; of al., the we et study, of age Macaluso skin, composition this some early Alternatively, al.,2016; pigmented nucleotide In 2003). an et on others. al., at (Kimita eschars et exposure than studies inoculation (Jensenius to previous virulent of centres due visibility to more health acute be poor most travellers be few may at ignored, in This but may capacity are 2004). illness diagnostic populations, that al., of native febrile cases et lack in clinical of Ndip and some high mild 1991; cause with usually only al., travellers the is to et foreign as seroprevalence (Kelly leading and Its reported malaria tourists been 2004). in to have al., reported second cases et been only (Jen- have (Rutherford myalgia be cases cases and an acute to fatal rash, Most providing believed eschar, (SSA). by is inoculation Africa ATBF headaches, 2003), sub-Saharan of by al., characterised epidemiology et is the senius which in ATBF, medicine, role travel for important In previously an described livestock play as cattle to source, that transmissibility blood-meal abundant evident low the is a that which it indicates notion from ruminants, the also samples, of reinforces but paucity blood pathogen a pathogen, the livestock is for there 34 negative but all were 2010), that al., Africa. et finding in (Hornok lice Our rickettsiae in SFG Central in rickettsiae other surveyed of the of have aware and vectors that are 2010), known detected studies we al., are also time et lice We first as National (Kelly the 1995). surprising Senegal Mara for al., 1995), and Maasai et al., ticks the et (Dupont rhipicephaline and (Beati (CAR) National Zimbabwe 2017), Hills Republic 2004), Shimba (Maina al., African al., County the et et 2020). 2017), Busia (Omondi (Ndip al., al., borders County Cameroon et which et Baringo (Koka County, Kenya (Oundo 2017), Siaya Eastern Reserve al., 2013), North al., et in (Mwamuye et in communities Reserve (Mutai pastoral rates Nairobi 2014), infection of and al., high Kenya Mombasa et 2003), in in al., description SHs et first at (Macaluso the region since Mara Ever Maasai cattle. from mostly Rhipicephalus Rickettsia 21)i ieinctl.Nntees hs pce a locuetelroi nimmuno-compromised in theileriosis cause also can species these Nonetheless, cattle. Nigerian in (2016) .platys A. and , ol osbyaettevrlneof virulence the affect possibly could , .mutans T. .marginale A. a nethmn,psn iki ae fopruitctc ie Arg-laaoe al., et (Arraga-Alvarado bites tick opportunistic of cases in risk a posing humans, infect may .parva T. ik rmctl.Ti ahgncbceimhsbe eotdi te tde ntcsand ticks in studies other in reported been has bacterium pathogenic This cattle. from ticks and , Rickettsia omp oefeunl in frequently more hnrpre yNjiiri by reported than Anaplasma h as fgl sickness, gall of cause the , npam platys Anaplasma eeaels iuetta hs ihtedlto Miae l,21)may 2014) al., et (Maina deletion the with those than virulent less are gene B omp iktsaafricae Rickettsia n usqetetyit el Bace l,20) h yohssthat hypothesis The 2003). al., et (Blanc cells into entry subsequent and Babesia ee hc oe o h otimn-oiatsraecl antigen cell surface immuno-dominant most the for codes which gene, B spp . a eetd(ud ta. 00.Teasneof absence The 2020). al., et (Oundo detected was h appendiculatus Rh. nticks in h as fcnn ylctrmoyoei,i eea pools several in thrombocytopenia, cyclic canine of cause the , tal. et a iial enrpre in reported been similarly has .africae R. 9 .bigemina, B. 21)i avsi ui,Kna n yLorusso by and Kenya, Busia, in calves in (2015) .conorii R. Amblyomma .africae R. .suis H. 1.%o ikpos ape nteMaasai the in sampled pools) tick of (15.7% variants . .africae R. ic hs ik otyprsts large parasitise mostly ticks these Since .africae R. .africae R. yKelly by hc assrdae,and redwater, causes which Amblyomma ie oee,ti oe nigi not is finding novel this However, lice. ainsta aebe eotdin reported been have that variants . ik r h ao eevi fthe of reservoir major the are ticks Rickettsia ufc elatgn r involved are antigens cell Surface Theileria in tal. et pstv ik eeobtained, were ticks -positive tmc oe rvlnein prevalence lower much at Amblyomma ik aebe reported been have ticks (1991). Amblyomma Fune ta. 2009). al., et (Fournier spp., .taurotragi T. ik rmthe from ticks .parva T. .parva T. ik from ticks .africae R. , T. in et , Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. ilgclCnrlApidRsac rga E-BAP gatnme:DCI-FOOD/2014/346-739) number: (grant Donors (EU-IBCARP) Fund Program CGIAR Research the and Applied by acknowledge led also Control (A4NH), We Biological Health (IFPRI). re- and Institute Nutrition also for Research It the ( Agriculture Policy on under Food BB/L019019/1. Program Laboratory, International reference Research Technology grant CGIAR the & the programme, Science from (ZELS) support Defence Systems ceived Livestock the The Emerging and Council, agencies: Research and Council Medical Depart- and Zoonoses the Research the Council, organizations Council, Environment Research Research following Social Natural Sciences & from the Biological Economic the and support Development, Biotechnology International financial the for received by ment Ogola, supported study was K. This project Edwin map. ZooLinK Ogola, of site technical K. the study Mudereri Kevin sam- acknowledge the extraction, Bester blood L also acid acknowledge to We Martin nucleic also access the samples. total We and of the robotic Busia rest of for from the metadata Ouso Nairobi and the O. to containing Daniel ticks samples database of tick of contribution the of shipment collection and for for biobank team (ZooLinK) ple (ZooLinK) Wambua Kenya Lillian in Livestock Dr. in and Zoonoses the acknowledge gratefully We centres health routinely in not surveys prospective if routine inclusion, Acknowledgements in their Therefore, least warrants diagnosed. at infection properly patients. diagnostics, human never differential febrile for are clinical receiving cause cases risk in (Crump here febrile limitations fevers and to non-malarial non-specific circulation detected due other of possible pathogens or inspectors, majority their malaria zoonotic large meat of from animal a The differentiate evidence workers, domestic Indeed, ATBF. to both 2013). abattoir difficult and to al., clinically risks exposes CCHF et be with like can SHs areas, that diseases new at illness in to febrile presence infection consumers of their how destinations and foci show Furthermore, to new findings butchers, traded These for populations. vectors. driver being human infected the either carrying be and slaughter, were can to animals taken trade from host or animal collected origin the the vectors their Significantly, in from SHs. importance different health were and public that LMs and veterinary at both animals of domestic pathogens of array an identified We 2015). that al., evidence et is non- (Duron Conclusions are there ticks endosymbionts that of These symbiont note 2015). maternally-inherited to al., a important of et from is (Duron consists endosymbionts it have B rhipicephaline However, (A-D) Group of pathogenic. clades consists C. phylogenetic C and Four group hosts. B while tick groups their into to their fell tick endosymbionts tick-associated and for these star treatment species described of lone tick antibiotic been specificity the different with high the of elimination the between fitness shows Their co-divergence the phylogenetic ticks. fever, impact The to Q negatively 2007). for fitness to responsible reproductive shown pathogen and was the nutrients to essential related ditional closely are which endosymbionts, from Mwamuye by sequences symbiont studies recent in As 2013). al., et Coxiella Maggi 2014; al., et Breitschwerdt 2014; hog Ppsgaut usr.Tefneshdn oei h ein aacleto,interpretation, collection, data design, the in role publication. no this had submit funders the to The through decision bursary. (DAAD) or postgraduate Service suppor- UP Exchange was Cooperation a Chiuya Academic and Tatenda through Government. German Development Kenyan a the for by and Agency Ethiopia; ted Swiss of Republic the Democratic (Sida); Federal (SDC); Agency Cooperation Development ternational http://www.cgiar.org/funders/ icipe noybot fticks of endosymbionts ntttoa udn rmU’ eatetfrItrainlDvlpet(FD;SeihIn- Swedish (DFID); Development International for Department UK’s from funding institutional Rickettsia shrEegn netosDsae M-I)lbrtr em oti work. this to team, laboratory (ML-EID) Diseases Infectious Emerging uscher ¨ Coxiella tal. et ;tesuyas eevdspotfo h uoenUinsIntegrated Union’s European the from support received also study the ); 6 RApie mlcn.Peiu tde aesonta these that shown have studies Previous amplicons. primer rRNA 16S 21)adOundo and (2017) icipe noybot.Tesqecso noybot rmti study this from endosymbionts of sequences The endosymbionts. Coxiella I n eoeSnigui o sitnei producing in assistance for unit Sensing Remote and GIS 10 edsminsof -endosymbionts tal. et mlom americanum Amblyomma 22) eas obtained also we (2020), icipe oilaburnetii Coxiella RPSDA coasi and scholarship ARPPIS-DAAD Amblyomma .burnetii C. Coxiella and vle recently evolved endosymbionts Coxiella Zoge al., et (Zhong Ornithodoros rvd ad- provide , endo- , Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. raim n pte ee ru iktsa ntcs(cr:Ioia)fo ibbe ora fMedical of Journal Zimbabwe. of from Prevalence Ixodidae) (1995). (Acari: D. ticks Raoult, in & rickettsiae R., P. group Mason, fever A., spotted L. and Matthewman, organisms J., P. Kelly, 411-417. Review Technical L., 34, and Beati, Scientific Epizooties, virus. des virus/Ganjam International disease International sheep Office Nairobi Uganda. the (2015). Central B. of Holzer, in Inves- & fever (2018). D., M. T.R hemorrhagic e58 Baron, 88-93. Shoemaker, Crimean-Congo in- 68, 5(6), . human with . Diseases, A. of Infections, Infectious Tumusiime, evolved case S., of virus and Mulei, Journal isolated fever J., an Microbes Kyondo, Valley of J., Emerging Rift Ojwang, tigation K., Has Africa? Patel, (2016). S., East Balinandi, J. in Villinger, populations & human R., doi Sang, in Breitschwerdt, K., & severity C., D. creasing B. Masiga, 1161–1165. Hegarty, M., 91, A., Hygiene, Baba, M. and Berrueta, Medicine of Tropical C., evidence of Search Molecular O. Journal Alignment report: Parra, American Local Case A., Basic (2014). B. B. (1990). E. Qurollo, D.J. M., Lipman & C. 403-410. Arraga-alvarado, E.W., 215, Diseases, Myers, Biology, Tropical W., Molecular Neglected Miller, of PLoS W., Journal Tool. Kenya. Gish, Vertical in F., (2018). Baringo S. J. Altschul, Villinger, and & Victoria from K., Lakes mosquitoes D. of in e0006949. Masiga, infections shores 12(11), D., flavivirus mainland Omondi, insect-specific O., and and Bunyamwera D. islands occurring Ouso, naturally O., of ADSB, T. transmission Onchuru, LCF, manuscript. U., final DKM, Y. the DM, approved Ajamma, while authors manuscript the laboratory original All and manuscript. the identification the wrote References the reviewed TC did and TC results. edited sampling. the JV and analysed and study EMF, JV the and designed JV, TC and work. EMF, LCF, DKM, TC, contributions Author MN266949- MN266944, Coxiella MN266921, MN267049; Theileria MN266919, MN267048, the CCHF: MN266918, under MN294735-MN294739, database Rickettsia CO1: ITS2: GenBank arthropod the MN401349-MN401351; arthropod into MN266929, MN266953, MN266911-MN266916, deposited MN266945; MN264523-MN264525, were MN264214, study MN266930, 16S: the arthropod in accessions: generated following data sequence nucleotide All interests. statement competing availability no Data have they that declare in authors (NACOSTI) The Innovation and Technology interest Science, of Conflicts for com- Commission Livestock Both National International 2017-08/2). the the ILRI-IREC by by (ref Kenya. approval Committee licensed by after Ethics approved are collected Research and mittees was Institutional SHs livestock at (ILRI) of IACUC-RC slaughter Institute owners (ref for Research Committee human presented Use from or and samples Care LMs Data blood Animal at study 2017-04). and Institutional pigs This Institute flea, Research and received. louse, Livestock Tick, sheep, been International project. page, goats, the has (ZooLinK) guidelines cattle, approval Kenya author from committee in journal’s Livestock collected review the in were ethical on Zoonoses appropriate noted the within as the nested journal, and was the to of adhered policies been ethical have the that confirm authors The statement Ethics :10.1038/emi.2016.57 p. N601M227;M262-N698 MN266946-MN266948. MN266922-MN266928, MN262071-MN262076; spp.: p. MN294725-MN294734; spp.: spp . N970M244,MN266939-MN266941; MN294740-MN294749, : doi :10.1371/journal.pntd.0006949. Babesia doi :10.1016/j.ijid.2018.01.013 p. MN294720-MN294723; spp.: npam platys Anaplasma doi 11 :10.1016/S0022-2836(05)80360-2 doi :10.20506/rst.34.2.2367 Anaplasma neto ntowmnfo Venezuela. from women two in infection doi :10.4269/ajtmh.14-0372. eaooncanis Hepatozoon p. MN266931-MN266941; spp.: Rickettsia MN294724; : -like . Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. uot .T,Boqi . agr,B,&Rol,D 19) rvlneo nioisto antibodies of Prevalence (1995). D. emergence Raoult, 33. (2002). & B., M. to Faugere, Diseases, K. P., , Infectious DeCock, Brouqui, contributing T., Emerging . H. . 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Weekly H., population Kiara, a (2018). R., in WHO Callaby, mortality M., of Chase-Topping, patterns A., 1(2). determine Advances, Jennings, Co-infections Science M., (2015). S. Thumbi, G. P. J., E. M. Woolhouse, :10.1016/j.ttbdis.2018.06.014 doi doi :10.1371/journal.pntd.0004210 :10.3201/eid1207.060253 and doi eue erdciefins.Po N,25,e405. 2(5), ONE, PLoS fitness. reproductive reduced < Anaplasma :10.3201/eid1708.102064 0300:ACOMOS doi pce ple to applied species :10.1126/sciadv.1400026 doi > 2.0.CO;2 10.1099/00207713-50-4-1449 : doi Amblyomma :10.1016/j.ttbdis.2014.04.023 doi :10.1111/1755-0998.12584 17 ik olce rmctl n he nEthiopia. in sheep and cattle from collected ticks doi doi 10.3201/eid1005.030537 : :10.1371/journal.pone.0000405 Rickettsia doi sn h gene the using :10.1577/1548- Amblyomma Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. T105 T63 T62 T50 T34 T16 T15 I.D Sample ticks of identification morphological and molecular of Comparison 1: Table Tables decoloratus Rh. nymph sp. Rhipicephalus sp. Rhipicephalus sp. Rhipicephalus sp. Rhipicephalus appendiculatus Rh. sp. Rhipicephalus I.D Morphological MH513311) (99.1, microplus Rh. KC503257) (99.35, appendiculatus Rh. EU918193) (100, decoloratus Rh. KY020993) (99.3, microplus Rh. MH513311) (99.2, microplus Rh. KC503257) (99.35, appendiculatus Rh. EU918193) (100, decoloratus Rh. accession) GenBank homology, (% rRNA 16S 18 - - KC503257) (99.35, appendiculatus Rh. U97716) (96.7, decoloratus Boophilus MG721035) (99.6, microplus Rh. KC503265) (99.6, microplus Rh. ) KX276951 (99.8, appendiculatus Rh. U97716) (96.7, decoloratus Boophilus accession) GenBank homology, (% ITS2 KC503257) (99.9, appendiculatus Rh. - KY678120) (100, microplus Rh. KY678120) (100, microplus Rh. KC503257) (100, appendiculatus Rh. - accession) GenBank homology, (% CO1 MN294737) CO1: MN266950; ITS2: MN266912; (16S: appendiculatus Rh. MN266919) ITS2: MN266915; (16S: decoloratus Rh. MN294739) CO1: MN266953; ITS2: MN264524; (16S: microplus Rh. MN294738) CO1: MN266952; ITS2: MN264523; (16S: microplus Rh. MN294736) CO1: MN266949; ITS2: MN266911; (16S: appendiculatus Rh. MN266918) ITS2: MN266914; (16S: decoloratus Rh. accessions) (GenBank ID Consensus MN264525) (16S: microplus Rh. Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. .bigemina B. platys A. ovis A. marginale A. markets livestock pools Total from lice and ticks Pathogen of pools in detected slaughterhouses pathogens and Vector-borne 2: Table T321 T311 T222 T218 T199 T192 T134 I.D Sample 1+ 0 31 49 631 333++ (1.24%) 6 (2.07%) 10 (1.24%) 6 17 (0.62%) 3 - - - - 3 - - - - 96 (0.77%) 1 - - - (0.75%) 1 99 - - - 54 - (2.90%) 2 (2.92%) 2 (4.35%) 3 (4.17%) 18 1 - - - (1.84%) 1 (5.51%) 33 3 - (0.61%) 1 - (3.05%) 5 (1.21%) 2 (2.44%)+ 4 108 (0.63%) 2 (3.15%) 10 (1.88%) 6 %) (1.88 6 215++ Rhipicephalus sp. Rhipicephalus nymph sp. Amblyomma gemma Am. nymph sp. Rhipicephalus variegatum Am. sp. Haemaphysalis decoloratus Rh. I.D Morphological spp. h eooau h pedcltsR.eetiRhipicephalus evertsi Rh. appendiculatus Rh. decoloratus Rh. KJ613641) (96.28, simus Rh. L34312) (99.3, variegatum Am. - KC503257) (99.51, appendiculatus Rh. L34312) (99.3, variegatum Am. HM068961) (95.6, elliptica Ha. EU918193) (100, decoloratus Rh. accession) GenBank homology, (% rRNA 16S 19 - - HQ856803) (100, variegatum Am. KY457490) (99.65, hebraeum Am. KY457500) (99.73, appendiculatus Rh. HQ856803) (100, variegatum Am. KU364288) (88, erinacei Ha. U97716) (96.7, decoloratus Boophilus accession) GenBank homology, (% ITS2 - - - - KU880573) (99.3, erinacei Ha. - accession) GenBank homology, (% CO1

MN401351) ITS2: MN266930; (16S: variegatum Am. MN401350) (ITS2: gemma Am. MN266951) ITS2: MN266913; (16S: appendiculatus Rh. MN401349) ITS2: MN266929; (16S: variegatum Am. MN294735) MN266944;CO1: ITS2: MN264214; (16S: sp. Haemaphysalis MN266921) ITS2: MN266916; (16S: decoloratus Rh. accessions) (GenBank ID Consensus MN266945) (16S: sp. Rhipicephalus

sp. Amblyomma spp. m aigtmA.gmaH suis H. gemma Am. variegatum Am. Total Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. upeetr iue1 htgah frpeettv pcmn fvcoscletdfrom collected vectors slaughterhouses: of and specimens markets representative livestock of at Photographs livestock 1. Figure Supplementary legends figure Supplementary ticks. spp. CFvrs2(.2)1(.1)--1(.5)----2(0.41%) 2 - - into fall the GenBank to from branches those and the study in in branch of this are The indicated endosymbionts from study Sequences replicates. gaps this 1,000 site. The genotypes: from among per three agreement sequences substitutions site. percentage 0.12 for per of represent numbers are substitutions outgroup Accession gene. nodes represents major rRNA scale sequence. - the each length 16S at for values the indicated Bootstrap of are bold segments origin tick-associated of 279-nt of species aligned phylogeny 33 likelihood Maximum from substitutions represents scale 5. length Figure branch major the The at values replicates. Bootstrap 1,000 bold. site. among in agreement per are percentage aligned study this of 32 from are sequences from for nodes the numbers inferred by Accession - protozoa indicated sequence. gene. each apicomplexan rRNA as for 18S of II the phylogeny genotype of segments represent likelihood African 502-nt outgroup into Maximum disease fall 4. sheep study Figure Nairobi this the represents from scale to sequences length branches The branch the values bars. (1.45%) The vertical Bootstrap in site. 1 replicates. indicated per highlighted. this 1,000 gaps also substitutions from among are The 0.8 agreement sequences sequences percentage site. for applicable of numbers per in are Accession sources substitutions gene). nodes Isolation sequence. (RdRp major bold. each the L-segment in for at are the indicated study are of this strains origin segments from of virus 434-nt country fever and haemorrhagic aligned - numbers Crimean-Congo 34 of from phylogeny inferred likelihood Maximum 3. Figure Ra: control. profiles. positive rate collected (C) were Melt showing Kakamega samples amplicons, and arthropod Bungoma, 2. which Busia, from Figure of slaughterhouses counties and neighbouring markets three livestock the the of Map - 1. Figure legends Figure pathogens each any (0.61%) of for include 1 size also the totals on ++These based calculated were brackets) tested (in pool percentages prevalence individual-level +Estimated (0.62%) 2 virus CCHF velifera T. taurotragi T. parva T. mutans T. africae R. canis H. caballi B. Pathogen 03% 14% (0.21%) 1 (0.21%) 1 (0.62%) (1.66%) 3 8 - (1.24%) 6 - - - - - (3.93%) 19 ------(1.53%) (6.28%) 2 8 - - - (1.49%) (6.14%) 2 8 (19.85%) 92 (3.71%) 1 - (0.76%) 1 (33.33%) 1 (1.45%) 1 - (1.45%) 1 (78.95%) 82 (0.75%) 1 - - - (77.45%) 83 (8.83%) (4.38%) 6 - 3 - - (5.89%) 4 ------(1.80%) 1 - (1.82%) - 1 (1.21%) 2 - - - (7.32%) 12 (1.83%) 3 (0.31%) (0.61%) 1 1 (1.88%) 6 (0.31%) 1 (5.64%) 18 (2.52%) 8 (0.31%) 1 - Rhipicephalus Anaplasma A Rhipicephalus = h appendiculatus Rh. oilaburnetii; Coxiella 6rN mlcn,ad(D) and amplicons, 16SrRNA h microplus Rh. spp. p.ticks; spp. A CFvrsRR mlcn,(B) amplicons, RdRp virus CCHF (A) h eooau h pedcltsR.eetiRhipicephalus evertsi Rh. appendiculatus Rh. decoloratus Rh. B Rd: . = , D Coxiella Haemaphysalis eBn ceso ubr n slto ore r indicated are sources isolation and numbers accession GenBank h decoloratus Rh. = Coxiella 20 noybot of endosymbionts noybot of endosymbionts Rickettsia/Coxiella A. p,and sp., . m gemma Am. eBn ceso ubr n tick and numbers accession GenBank Coxiella t felis Ct. Amblyomma Dermacentor eae B. female; Theileria/Babesia ol htwr o positive not were that pools 6 RAapios PC: amplicons. rRNA 16S noybot inferred endosymbionts p.ticks; spp. eBn accession GenBank m gemma Am. and .pneumophila L. Amblyomma C 8 rRNA 18S =

Coxiella male; .

sp. Amblyomma spp. m aigtmA.gmaH suis H. gemma Am. variegatum Am. Total Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. td r nbl.Nt h eeinmtto fa4bs armtfadsvrlbs usiuin nthe in substitutions with base consensus = several Grey and Guanine; motif = Yellow pair Thymine; 4-base = a Green of Cytosine; = mutation africae Blue deletion R. Adenine; the = Note Red sequences. sequences. reference bold. in GenBank are with study aligned study Partial this 2. Figure Supplementary ulatus G. C. hpcpau evertsi Rhipicephalus Amblyomma eae K. female; eeec sequence. reference p yp;D. nymph; sp. h decoloratus Rh. eaeadml;H. male; and female L. ; mvariegatum Am amtpnssuis Haematopinus omp eesqecsof sequences gene B h evertsi Rh. eae E. female; 21 M. ; ae I. male; tncpaie felis Ctenocephalides mvariegatum Am h appendiculatus Rh. ceso ubr fsqecsfo this from sequences of numbers Accession iktsaafricae Rickettsia ae F. male; . ae J. male; Haemaphysalis bandfrom obtained h appendic- Rh. sp.; Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/Fig2/Fig2-eps-converted-to.pdf 22 Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/Fig3/Fig3-eps-converted-to.pdf 23 Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/Fig4/Fig4-eps-converted-to.pdf 24 Posted on Authorea 30 Jul 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159603999.90184259/v2 — This a preprint and has not been peer reviewed. Data may be preliminary. figures/Fig5/Fig5-eps-converted-to.pdf 25