Biology subjects

Villinger, J.

Publications and source records attributed to Villinger, J..

8 recordsLinked to original sources

bioRxiv2026

Ehrlichia ruminantium infection is associated with tissue-specific microbial community shifts in Amblyomma gemma ticks from cattle in Kenya

Tick-borne pathogens can reshape vector microbiomes in ways that influence pathogen colonisation and transmission, yet the interplay between Ehrlichia ruminantium and the microbiota of its tick vectors remains uncharacterised. We profiled bacterial communities in haemolymph, midgut, and salivary glands of infected (n = 11) and uninfected (n = 12) Am. gemma ticks, a vector of E. ruminantium in East Africa, collected from cattle in Kajiado County, Kenya, using near-full-length 16S rRNA gene amplicon sequencing on the Oxford Nanopore platform. Community composition, alpha and beta diversity, co-occurrence networks, keystone taxa, and PICRUSt2-inferred functional profiles were compared across tissue-infection status groups. We identified 226 bacterial genera dominated by Coxiella, Pseudomonas, Acinetobacter, Proteus, and Rickettsia. Infection was associated with tissue-specific shifts in community composition (PERMANOVA R{superscript 2} = 0.14, p < 0.001) and co-occurrence network structure, with midgut networks showing complete hub taxon turnover (Jaccard = 0.000, p = 0.043). Haemolymph communities converged around Luteimonas as a keystone taxon, while opportunistic Proteobacteria, including Acinetobacter and Serratia, emerged as keystones in infected midgut. Endosymbiotic Rickettsia was near-absent in infected tissues (0.3% vs 9.3% mean relative abundance in midgut), consistent with competitive exclusion. Functional inference identified FDR-significant enrichment of predicted aerobactin siderophore biosynthesis, antimicrobial efflux, and oxidative stress response gene families in infected microbiota. These findings show tissue-specific restructuring of the Am. gemma microbiome associated with E. ruminantium infection and point to candidate targets for microbiome-based interventions against heartwater. ImportanceHeartwater, caused by the bacterium Ehrlichia ruminantium and transmitted by Amblyomma ticks, kills up to 90% of susceptible ruminants and is one of the most devastating tick-borne diseases in sub-Saharan Africa. Controlling heartwater requires understanding how the pathogen interacts with the microbial communities living inside its tick vector. In this exploratory study, we show that E. ruminantium infection is associated with tissue-specific shifts in the Amblyomma tick microbiome, including reduced abundance of beneficial symbionts, elevated representation of opportunistic bacteria among community hubs, and enrichment of iron acquisition and antimicrobial resistance functions. The midgut, the first tissue colonised during infection, showed the most marked structural reorganisation. These tissue-resolved microbiome signatures point to potential targets for novel control strategies, such as anti-microbiota vaccines or approaches that reinforce natural colonisation resistance, offering new strategies to reduce heartwater transmission and protect livestock livelihoods across Africa.

microbiology↗

bioRxiv2021

Transmission of Candidatus Anaplasma camelii to laboratory animals by camel-specific keds, Hippobosca camelina

Anaplasmosis, caused by infection with bacteria of the genus Anaplasma is an important veterinary and zoonotic disease. The characterization of transmission has concentrated on ticks and little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by Candidatus Anaplasma camelii occurring from 63 - 78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10 - 29% of camel keds harbored Ca. Anaplasma camelii acquired from infected camels during blood feeding. We determined whether Anaplasma positive camel keds could transmit Ca. Anaplasma camelii to small laboratory animals via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by both direct detection in blood smears and subsequent molecular identification by PCR. Transmission of Ca. Anaplasma camelii to mice (8 - 47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the basis for establishing ked control programmes for improvement of livestock and human health. Author summaryHematophagous flies such as Tabanids and Stomoxys, among other biting flies, are mechanical transmitters of various pathogens such as African trypanosomes and Anaplasma species. However, little is known about the role of common camel-specific biting keds (also known as camel flies or louse flies, genus Hippobosca) in pathogen transmission. Keds inflict painful bites to access host blood, and in the process may transmit bacterial hemopathogens frequently detected in both camels and their keds. We confirmed by experimental blood-feeding, gene amplification, and amplicon sequencing that camel keds can transmit "Candidatus Anaplasma camelii" from naturally-infected camels to healthy mice and rabbits. The high prevalence of camel anaplasmosis throughout the year in northern Kenya could be explained by the infestation camel-specific H. camelina, whose capacity as efficient fliers, unlike ticks, promotes disease transmission and maintenance within and among camel herds. Although this study focused on the transmission of Anaplasma sp. by camel keds, it is possible that other hemopathogens could also be transmitted by these flies through a similar mechanism. Notably, in the absence of their preferred hosts, keds occasionally bite humans and other vertebrates they come across in order to acquire bloodmeals, and in the process could transmit zoonotic pathogens.

microbiology↗

bioRxiv2021

Species substitution in the meat value chain by high-resolution melt analysis of mitochondrial PCR products

Food fraud in several value chains including meat, fish, and vegetables has gained global interest in recent years. In the meat value chain, substitution of high commercial-value meats with similar cheaper or undesirable species is a common form of food fraud that raises ethical, religious, and dietary concerns. The presence of undeclared species could also pose public health risks caused by allergic reactions and the transmission of food-borne or zoonotic pathogens. Measures to monitor meat substitution are being put in place in many developed countries. However, information about similar efforts in sub-Saharan Africa is sparse. In this study, we used PCR coupled with high-resolution melting (PCR-HRM) analysis targeting the three mitochondrial genes, cytochrome oxidase 1 (CO1), cytochrome b (cyt b), and 16S rRNA, to detect species substitution in meat sold to consumers in Nairobi, Kenyas capital city. Out of 107 meat samples from seven common livestock animals (cattle, goat, sheep, pig, chicken, rabbit, and camel), 11 (10.3%) had been substituted. Of 61 samples sold as beef, two were goat and one was camel. Of 30 samples sold as goat meat, four were mutton (sheep) and three were beef. One of nine samples purchased as pork was beef. Our results indicate that PCR-HRM analysis is a cost and time effective technique that can be employed to detect species substitution. The combined use of the three markers produced PCR-HRM profiles that successfully allowed the distinction of species. We demonstrate its utility not only in analysis of raw meat samples, but also of cooked, dried, and rotten samples, meat mixtures, and with the use of different DNA extraction protocols. We propose that this approach has broad applications in authentication of meat products and protection of consumers from food fraud in the meat industry in low- and middle-income countries such as Kenya, as well as in the developed world.

molecular biology↗

bioRxiv2020

Molecular detection of Leishmania donovani, Leishmania major, and Trypanosoma spp. in Sergentomyia squamipleuris sandflies from a visceral leishmaniasis focus in Merti sub-County, eastern Kenya

BackgroundVisceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remains limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sandfly species responsible for Leishmania transmission in the sub-County, and their blood-meal hosts. MethodsWe conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sandflies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (COI) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Bloodmeal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products. ResultsWe sampled 526 sandflies consisting of eight species, Phlebotomus orientalis (1.52%; n = 8) and seven Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sandfly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sandfly bloodmeals. However, we also detected mixed bloodmeals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood. ConclusionsOur findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sandflies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major causes ZCL while L. donovani is responsible for VL. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

molecular biology↗

bioRxiv2020

Molecular characterization of pathogenic African trypanosomes in biting flies and camels in surra-endemic areas outside the tsetse fly belt in Kenya

BackgroundAfrican animal trypanosomosis is becoming prevalent beyond its traditionally defined geographical boundaries and is a threat to animals beyond the tsetse belts in and outside Africa. However, knowledge of infections with clinically important trypanosome species and their diversity among field-collected hematophagous biting flies and domestic animals is limited mainly to tsetse and their mammalian hosts in tsetse-infested areas. This study aimed to examine the presence of trypanosomes in both biting flies and domestic animals outside the tsetse belt in northern Kenya, potential mechanical vector species, and their host-feeding profiles. MethodsWe screened for pathogenic African trypanosomes in blood samples from domestic animals and field-trapped flies by microscopy and sequencing of internal transcribed spacer (ITS1) gene PCR products. We sequenced kinetoplast maxicircle genes to confirm Trypanosoma brucei detection and the RoTat 1.2 and kinetoplast minicircle genes to differentiate type-A and type-B Trypanosoma evansi, respectively. Further, we identified the hosts that field-trapped flies fed on by PCR-HRM and sequencing of 16S rRNA genes. ResultsHippobosca camelina, Stomoxys calcitrans, Tabanus spp., and Pangonia rueppellii are potential vectors of trypanosomes outside the tsetse belt in Marsabit County, northern Kenya. We identified Trypanosoma spp., including Trypanosoma vivax, T. evansi, T. brucei, and T. congolense in these biting flies as well as in camels (Camelus dromedarius). Trypanosomes detected varied from single up to three trypanosome species in H. camelina and camels in areas where no tsetse flies were trapped. Similar trypanosomes were detected in Glossina pallidipes collected from a tsetse-infested area in Shimba Hills, coastal Kenya, showing the wide geographic distribution of trypanosomes. Furthermore, we show that these biting flies acquired blood meals from camels, cattle, goats, and sheep. Phylogenetic analysis revealed diverse Trypanosoma spp. associated with variations in virulence and epidemiology in camels, which suggests that camel trypanosomosis may be due to mixed trypanosome infections rather than only surra (T. evansi), as previously thought.

pathology↗

bioRxiv2020

Tsetse blood-meal sources, endosymbionts, and trypanosome infections provide insight into African trypanosomiasis transmission in the Maasai Mara National Reserve, a wildlife-human-livestock interface

BackgroundAfrican trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Understanding of AT transmission is hampered by limited knowledge on interactions of tsetse flies with their vertebrate hosts and the influence of endosymbionts on vector competence, especially in wildlife-human-livestock interfaces. We identified the tsetse species, their blood-meal sources, and the correlation between endosymbiont and trypanosome infection status in the trypanosome-endemic Maasai Mara National Reserve (MMNR) of Kenya. Methodology/Principal FindingsAmong 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosomes, majority (17/28) being Trypanosoma vivax. Blood-meal analyses based on high-resolution melting analysis of mitochondrial cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 345) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Trypanosome-infected flies had fed on hippopotamus and buffalo. Additionally, PCR analysis revealed that tsetse flies were more likely to be infected with trypanosomes if they were infected with the Sodalis glossinidius endosymbiont (P = 0.0022 Fishers exact test). Conclusions/SignificanceDiverse species of wildlife hosts may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes in the MMNR. Although the African buffalo is known to be a key reservoir of AT, the higher proportion of hippopotamus blood-meals in trypanosomes-infected flies identified here indicates that other wildlife species may also be important to transmission cycles. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis transmission risk. Furthermore, this work provides data showing that Sodalis endosymbionts can is associated with increased trypanosome infection rates in endemic ecologies. Author summaryHuman and animal African trypanosomiasis are neglected tropical diseases with potential to spread to new areas. Wild animals are important reservoirs for African trypanosomes and crucial in the emergence and re-emergence of AT. Vertebrate host-vector-parasite interactions are integral to trypanosome transmission. We investigated the vertebrate blood-meals and trypanosomes-endosymbionts co-infections in tsetse flies, which have been associated with reservoirs and vector competence, respectively, on AT transmission in Kenyas Maasai Mara National Reserve. We identified tsetse fly diversity, trypanosome and endosymbiont infection status, and vertebrate blood-meal hosts to infer potential transmission dynamics. We found that Glossina pallidipes was the major tsetse fly vector and that Trypanosoma vivax was the main trypanosome species circulating in the region. Humans, hippopotamus, and buffalo were the most frequented for blood-meals. Buffalo and hippopotamus blood-meals were identified in trypanosome infected flies. Feeding of the flies on both humans and wildlife may potentiate the risk of the human trypanosomiasis in this ecology. Additionally, we found that the endosymbiont Sodalis glossinidius is associated with higher trypanosome infection rates in wild tsetse flies. These findings emphasize the importance of understanding the interaction of tsetse flies with vertebrate blood-meal sources and their endosymbionts in the transmission and control of AT.

molecular biology↗

bioRxiv2020

Pathogens, endosymbionts, and blood-meal sources of host-seeking ticks in the fast-changing Maasai Mara wildlife ecosystem

BackgroundThe role of questing ticks in the epidemiology of tick-borne diseases in Kenyas Maasai Mara National Reserve (MMNR), an ecosystem with intensified human-wildlife-livestock interactions, remains poorly understood. Therefore, we carried out a survey of the diversity of questing ticks, their blood-meal hosts, and tick-borne pathogens to understand potential effects to human and livestock health. MethodsQuesting ticks were collected by flagging and hand picks from vegetation in 25 localities and identified based on morphologic and molecular criteria. We used PCR with high-resolution melting (HRM) analysis, and sequencing to identify Anaplasma, Babesia, Coxiella, Ehrlichia, Rickettsia, and Theileria pathogen diversities and blood meals in 231 tick pools. ResultsA total of 1,465 host-seeking ticks were collected, including Rhipicephalus appendiculatus (n = 1,125), Rhipicephalus pulchellus (n = 6), Rhipicephalus evertsi (n = 5), Amblyomma cf. gemma (n = 178), Amblyomma gemma (n = 145), Amblyomma variegatum (n = 4), Amblyomma sp. (n = 1), and Haemaphysalis leachi (n = 1). Remnant blood-meals from humans, wildebeest, and African buffalo were detected in Rh. appendiculatus, goat in Rh. evertsi, sheep in Am. gemma, and cattle in Am. variegatum. Rickettsia africae was detected in Am. gemma (1/25 pools) that had blood-meal remnant from sheep and Am. variegatum (4/25 pools) that had fed on cattle. Rickettsia spp. were found in Am. gemma (4/25 pools) and Rh. evertsi (1/4 pools). Anaplasma ovis was detected in Rh. appendiculatus (1/172 pools) and Rh. evertsi (1/4 pools), while Anaplasma bovis was detected in Rh. appendiculatus (1/172 pools). Theileria parva was detected in Rh. appendiculatus (27/172 pools). Babesia, Ehrlichia and Coxiella pathogens were not found in any ticks. Unexpectedly, diverse Coxiella sp. endosymbionts were detected in all tick genera (174/231 pools). ConclusionsThe data shows that ticks from the rapidly-changing MMNR are infected with zoonotic R.africae and unclassified Rickettsia spp, demonstrating the persistent risk of African tick-bite fever and other and Spotted Fever Group rickettsioses to local dwellers and visitors to the Maasai Mara ecosystem. Protozoan pathogens that may pose risk to livestock production were also identified. We also highlight possible existence of morphotypic variants of Amblyomma species, based on the observation of Ambyomma cf. gemma, which may be potential human parasites or emergent disease vectors. Our findings also demonstrate that questing ticks in this ecosystem have dynamic vertebrate blood sources including humans, wildlife and domestic animals, which may amplify transmission of tickborne zoonoses and livestock diseases. Further studies are needed to determine the role of Coxiella endosymbionts in tick physiology and vector competence.

molecular biology↗

bioRxiv2019

Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for forensic and biodiversity research pipelines

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on the sequencing of mitochondrial cytochrome oxidase I (COI) barcode genes, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adopted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

molecular biology↗