A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

ATP-sensitive inward rectifier potassium channels regulate secretion of pro-feeding salivary proteins in the lone star tick (Amblyomma americanum).

Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.

Identification and characterization of proteins that form the inner core Ixodes scapularis tick attachment cement layer.

Ixodes scapularis long-term blood feeding behavior is facilitated by a tick secreted bio adhesive (tick cement) that attaches tick mouthparts to skin tissue and prevents the host from dislodging the attached tick. Understanding tick cement formation is highly sought after as its disruption will prevent tick feeding. This study describes proteins that form the inner core layer of I. scapularis tick cement as disrupting these proteins will likely stop formation of the outer cortical layer. The inner core cement layer completes formation by 24 h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6 h and 24 h tick attachment sites and to distinguish between early and late inner core cement proteins. LC-MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24 h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3-6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation.

Molecular Characterization of Ticks and Tick-Borne Pathogens in Cattle from Khartoum State and East Darfur State, Sudan.

Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick-pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of Theileria annulata, Babesia bigemina, B. bovis, Anaplasma marginale, and Ehrlichia ruminantium was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, Rhipicephalus, Hyalomma, and Amblyomma, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were Rhipicephalusevertsi evertsi (34.3%) and Hyalomma anatolicum (57.3%) in Khartoum State, and Rhipicephalus annulatus (27%), Rhipicephalus decoloratus (25%), and Hyalomma rufipes (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of E. ruminantium, its vector Amblyomma variegatum, and B. bovis in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.

Molecular identification of trypanosomes in cattle in Malawi using PCR methods and nanopore sequencing: epidemiological implications for the control of human and animal trypanosomiases.

This study aimed to identify trypanosomes infecting cattle in Malawi in order to understand the importance of cattle in the transmission dynamics of Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). A total of 446 DNA samples from cattle blood from three regions of Malawi were screened for African trypanosomes by ITS1 PCR. The obtained amplicons were sequenced using a portable next-generation sequencer, MinION, for validation. Comparison of the results from ITS1 PCR and MinION sequencing showed that combining the two methods provided more accurate species identification than ITS1 PCR alone. Further PCR screening targeting the serum resistance-associated (SRA) gene was conducted to detect Trypanosoma brucei rhodesiense. Trypanosoma congolense was the most prevalent Trypanosoma sp., which was found in Nkhotakota (10.8%; 20 of 185), followed by Kasungu (2.5%; 5 of 199). Of note, the prevalence of T. b. rhodesiense detected by SRA PCR was high in Kasungu and Nkhotakota showing 9.5% (19 of 199) and 2.7% (5 of 185), respectively. We report the presence of animal African trypanosomes and T. b. rhodesiense from cattle at the human-livestock-wildlife interface for the first time in Malawi. Our results confirmed that animal trypanosomes are important causes of anemia in cattle and that cattle are potential reservoirs for human African trypanosomiasis in Malawi.

TITLE: Identification moléculaire des trypanosomes chez les bovins du Malawi, à l'aide de méthodes de PCR et du séquençage par nanopores : implication épidémiologique pour le contrôle des trypanosomiases humaines et animales. ABSTRACT: Cette étude visait à identifier les trypanosomes infectant les bovins au Malawi afin de comprendre l'importance des bovins dans la dynamique de transmission de la trypanosomiase humaine africaine (THA) et de la trypanosomose animale africaine (TAA). Au total, 446 échantillons d'ADN de sang de bovins provenant de trois régions du Malawi ont été soumis à un dépistage des trypanosomes africains par PCR de l'ITS1. Les amplicons obtenus ont été séquencés à l'aide d'un séquenceur portable de nouvelle génération, MinION, pour validation. La comparaison des résultats de la PCR de l'ITS1 et de la séquence MinION a montré que la combinaison des deux méthodes permettait une identification plus précise des espèces que la seule PCR de l'ITS1. Un autre dépistage par PCR ciblant le gène SRA (associé à la résistance du sérum) a été effectué pour détecter Trypanosoma brucei rhodesiense. Trypanosoma congolense était l'espèce de trypanosome la plus répandue, trouvée à Nkhotakota (10,8 % ; 20 sur 185), suivi de Kasungu (2,5 % ; 5 sur 199). Notamment, la prévalence de T. b. rhodesiense détectée par PCR de SRA était élevée à Kasungu et Nkhotakota, avec respectivement 9,5 % (19 sur 199) et 2,7 % (5 sur 185). Nous rapportons la présence de trypanosomes animaux africains et de T. b. rhodesiense de bovins à l'interface homme-bétail-faune sauvage, pour la première fois au Malawi. Nos résultats confirment que les trypanosomes animaux sont des causes importantes d'anémie chez les bovins et que les bovins sont des réservoirs potentiels pour la trypanosomiase humaine africaine au Malawi.

Diversity of trypanosomes in wildlife of the Kafue ecosystem, Zambia.

The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies.

Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.

Prevalence of hemoprotozoan parasites in small ruminants along a human-livestock-wildlife interface in western Uganda.

Small ruminants are important to community livelihood in developing countries; however information on the role of hemoprotozoan parasites is scanty. The objective of the study was to determine hemoprotozoan parasitic prevalence in western Uganda and identify major areas associated with these infections. This was a cross sectional study conducted at the edge of Budongo Conservation Forest in Masindi district of western Uganda in which 712 small ruminants were sampled. Blood from the jugular vein was collected from caprines and ovines and placed in an EDTA tube, and transported to the laboratory for examination. Thin and thick smears were prepared and examined by microscopy for hemoprotozoan parasites, and DNA was extracted and examined by PCR for Trypanosoma spp. A total of 13 villages in Budongo sub-county were surveyed and the study showed that caprines were the major small ruminants of importance to the community. Prevalence of hemoprotozoan parasites was as follows; anaplasmosis (3.65%) > theileriosis (0.45%) > trypanosomiasis (0.15%) and babesiosis (0%) by microscopy. Infections were found in the young with the exception of Anaplasma spp. while coinfections of anaplasmosis and theileriosis were high. Molecular analysis showed an overall trypanosome prevalence of 9.27% (PCR), mainly due to Trypanosoma brucei and T. congolense forest. Villages with trypanosomiasis were found in lowlands and swamps. The current trypanosomiasis prevalence in small ruminants of Uganda was 10 times greater than that previously reported showing that the disease burden has increased overtime within Uganda. A prevalence of 0.14% (95% CI: 0.00, 0.78) for the SRA gene showed that small ruminants would be important reservoirs of infection to humans. Hemoprotozoan parasites are a threat to community livelihood in developing countries and the role of molecular diagnostic techniques in disease monitoring was re-emphasized by this study. Information on primary hosts involved in the propagation of hemoprotozoan parasites in Uganda would help streamline prospective disease surveillance and control efforts.

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons.

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina's widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes.