Ticks infesting terrestrial small mammals in a rural settlement in the Amazonas state, Brazil.

There is limited knowledge about tick diversity in the Amazon region. Here, we survey small terrestrial mammals for tick infestation at the Rio Pardo settlement, Amazonas State, Brazil. Sampling included rainy and dry seasons and four ecotones (primary forest, forest in regeneration, field crops and households). Each animal was inspected for ticks, which, if present, were placed in 70% alcohol and identified. Parasitological indexes were calculated and the presence/absence of ticks on hosts was tested for possible associations with independent variables (ecotone, host sex, host order, host family, host age and season). A total of 208 small mammals were captured, 47 individuals (10 species) in the primary forest, 124 (15 species) in the forest in regeneration, 11 (7 species) in the field crops, and 26 (4 species) in the households. A total of 14 small mammals were infested by ticks (overall prevalence: 6.7%; 95% CI: 3.72 - 11.04%), which consisted of 51 specimens that were identified into four species, as follows: Amblyomma humerale (32 nymphs); Ixodes luciae (6 females); Amblyomma coelebs (1 nymph); and Ornithodoros mimon (1 larva). In addition, 11 larvae were retained as Amblyomma spp. Only host order showed association (P = 0.002) with tick infestation, with marsupials 5.5 times more infested than rodents. Our record of O. mimon on D. marsupialis is the first on this host species, and the first record of a Argasidae tick in the Brazilian state of Amazonas. To the best of our knowledge, this is the first study that actively screened free-living terrestrial small mammals and provided data on prevalence, mean intensity and mean abundance of tick infestations in the Brazilian Amazonas state.

Tick-Borne Relapsing Fever Infection from a Bunker, a Case Report on a "One Health" Approach.

Background: Tick-borne relapsing fever (TBRF) caused by Borrelia persica is an endemic disease in Israel and highly prevalent in military personnel. Prevention among the Israel Defense Force soldiers is based on increased awareness mainly in hyperendemic areas and selective postexposure prophylaxis with doxycycline. In this study, we report the presence of a suspected outbreak of TBRF in four soldiers who spent 30 h inside a deserted bunker. Materials and Methods: Clinical data on TBRF suspected cases were retrieved from clinical records, soft ticks were collected using carbon dioxide (CO2) traps and their DNA was extracted and analysed by PCR and nucleotide sequencing. Environmental conditions such as relative humidity, air temperature, wind speed, and type of soil, as well as presence or absence of animal traces inside the bunkers were documented. Results: TBRF-like clinical symptoms in the patients included: tick bite scars, fever (37.5-39.2°C), rash, tachycardia, hypotension, myalgia, cough, headache, cervical lymphadenopathy and nausea. Microscopic search for B. persica in blood smears was performed in three patients and was negative. Out of the 255 Ornithodoros tholozani ticks collected from the bunker, 198 were analyzed and 2 (1%) were infected with B. persica. To determine if tick infestation in military bunkers is a common phenomenon, we surveyed nine additional military bunkers located in four different geographical areas for the presence of soft ticks. Only one additional bunker was infested with two O. tholozani ticks, both negative for B. persica. Presence of earth that probably helped sustain a relatively big tick population was observed on the floor in the highly infested bunker. Environmental treatment with lambda-cyhalothrin at 9.7% was performed and showed efficacy with no ticks recovered in the infested bunker 124 days after intervention. Conclusion: This study shows that military bunkers may harbor soft ticks infected with B. persica and entrance into bunkers should be considered as a risk for acquiring this infection like entrance into natural caves and archeological ruins.

Development of microsatellite markers for the soft tick Ornithodoros phacochoerus.

BACKGROUND: Soft ticks of the genus Ornithodoros are responsible for the maintenance and transmission of the African swine fever (ASF) virus in the sylvatic and domestic viral cycles in Southern Africa. They are also the main vectors of the Borrelia species causing relapsing fevers. Currently, no genetic markers are available for Afrotropical Ornithodoros ticks. As ASF spreads globally, such markers are needed to assess the role of ticks in the emergence of new outbreaks. The aim of this study is to design microsatellite markers that could be used for ticks of the Ornithodoros moubata complex, particularly Ornithodoros phacochoerus, to assess population structure and tick movements in ASF endemic areas. METHODS: A total of 151 markers were designed using the O. moubata and O. porcinus genomes after elimination of repeated sequences in the genomes. All designed markers were tested on O. phacochoerus and O. porcinus DNA to select the best markers. RESULTS: A total of 24 microsatellite markers were genotyped on two populations of O. phacochoerus and on individuals from four other Ornithodoros species. Nineteen markers were selected to be as robust as possible for population genetic studies on O. phacochoerus. CONCLUSIONS: The microsatellite markers developed here represent the first genetic tool to study nidicolous populations of O. phacochoerus.

First data on cholesterol metabolism in Ornithodoros argasid ticks: Molecular and functional characterization of the N-terminal domain of Niemann-Pick C1 proteins.

Cholesterol is a molecule vital for tick physiology, but ticks cannot synthesize it and rely on dietary cholesterol. Therefore, tick proteins involved in cholesterol absorption and transport, such as the Niemann-Pick type C1 domain-containing (NPC1) proteins, are promising targets for anti-tick vaccine development. The aim of this study was to assess the structure, function, and protective efficacy of the NPC1 orthologues identified previously in the midgut transcriptomes of argasid ticks Ornithodoros erraticus and Ornithodoros moubata. For this purpose, their corresponding cDNA coding sequences were cloned and sequenced, their secondary and 3D structures were predicted, and their function was evaluated through RNAi-mediated gene knockdown and in vitro feeding on blood supplemented with ezetimibe, which inhibits cholesterol binding by NPC1 proteins. Subsequently, the protective efficacy of a recombinant form of NPC1 from O. moubata (rOmNPC1) was tested in a rabbit vaccine trial. While inhibiting cholesterol absorption with ezetimibe resulted in up to 77 % mortality in adult O. moubata, NPC1 gene knockdown and vaccination with rOmNPC1 decreased female reproductive performance in terms of the number and fertility of laid eggs. This study presents the initial molecular and functional insights into NPC1 proteins in soft ticks and supports the hypothesis that disrupting cholesterol metabolism diminishes tick viability and reproduction, rendering Niemann-Pick type C1 domain-containing proteins promising targets for drugs or vaccines.

Epidemiology and ecology of the sylvatic cycle of African Swine Fever Virus in Kenya.

African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs.

Nation-wide surveillance of ticks (Acari: Argasidae) on bats (Chiroptera) in Singapore.

Bats and ticks are important sources of zoonotic pathogens. Therefore, understanding the diversity, distribution, and ecology of both groups is crucial for public health preparedness. Soft ticks (Argasidae) are a major group of ectoparasites commonly associated with bats. The multi-host life cycle of many argasids make them important vectors of pathogens. Over nine years (2011-2020), surveillance was undertaken to identify the ticks associated with common bats in Singapore. During this period, the bat tick Ornithodoros batuensis was detected within populations of two cave roosting bat species: Eonycteris spelaea and Penthetor lucasi. We examined the relationship between bat species, roosting behaviour, and probability of O. batuensis infestation. We also estimated the relationship between bat life history variables (body condition index, sex, and age) on the probability of infestation and tick count. This represents the first detection of O. batuensis and the genus Ornithodoros within Singapore. We also provide evidence of the continued persistence of Argas pusillus in Singapore with the second local record.

Detection of antibodies against Ornithodoros moubata salivary antigens and their association with detection of African swine fever virus in pigs slaughtered in central Uganda.

Introduction: African swine fever (ASF) is an important disease of pigs in sub-Saharan Africa and Uganda and is threatening the pig population and agricultural economy of other continents. ASF virus (ASFV) can be transmitted from wild suids to domestic pigs through soft ticks of the Ornithodoros species. The aim of this study was to understand the relationship between domestic pigs' O. moubata tick exposure and ASFV status. Methods: Pigs were sampled from six abattoirs in the Kampala metropolitan area of Uganda from May 2021 through June 2022. Blood, serum, and tissue samples were collected. Serum was tested for antibodies against the rtTSGP1 salivary antigens of O. moubata ticks using an indirect ELISA assay. Blood and tissue samples from pigs were tested to detect ASFV using qPCR. Probability of tick exposure was categorized based on sample-to-positive ratio cut-off points. Results: Out of 1,328 serum samples tested, there were 828 (62.3%) samples with a negligible probability; 369 (27.8%) with a medium probability; 90 (6.8%) with a high probability, and 41 (3.1%) with a very high probability of exposure to the O. moubata salivary antigen. There was a statistically significant association between the pigs' O. moubata exposure and ASFV status with a higher proportion of pigs having a very high probability of infection if they were ASFV positive by blood, tonsil, and lymph nodes. Discussion: These results suggested that tick exposure was associated with ASFV transmission in Uganda. There were ASFV qPCR positive pigs that had no O. moubata exposure as well, which highlights that pig-to-pig and indirect contact transmission still play a significant role. This work highlights the need for further work in Uganda to investigate these transmission factors related to the O. moubata tick and ASFV transmission.

New records of soft ticks (Acari: Argasidae) from caves in Brazil, with a morphological study of Ornithodoros fonsecai and an analysis of the taxonomic status of Antricola inexpectata.

In this study, we report soft ticks from bat-inhabiting caves in different areas of Brazil. From 2010 to 2019, we collected 807 tick specimens from nine caves located in four Brazilian states among two biomes. Ticks were morphologically identified as Antricola guglielmonei (282 specimens), Ornithodoros cavernicolous (260 specimens), and Ornithodoros fonsecai (265 specimens). Whereas A. guglielmonei was collected on bat guano in hot caves, O. cavernicolous and O. fonsecai were collected in cracks and crevices on the walls of cold caves, sometimes in the same chamber. Morphological identifications were corroborated by molecular and phylogenetic analyses inferred from tick mitochondrial 16S rRNA gene partial sequences. The sequences of A. guglielmonei, O. cavernicolous and O. fonsecai collected in this study clustered with conspecific GenBank sequences from different localities of Brazil. Remarkably, a clade containing 12 sequences of O. fonsecai was clearly bifurcated, denoting a degree of genetic divergence (up to 5 %) of specimens from Cerrado/Atlantic Forest biomes with the specimens from the Caatinga biome. To further evaluate this divergence, we performed morphometric analysis of the larval stage of different O. fonsencai populations by principal component analysis, which indicated that the larvae from Caatinga populations were generally smaller than the larvae from other biomes. Some of the present A. guglielmonei specimens were collected from the type locality of Antricola inexpectata. Comparisons of these specimens with the type specimens of A. inexpectata and A. guglielmonei indicated that they could not be separated by their external morphology. Hence, we are relegating A. inexpectata to a synonym of A. guglielmonei. This proposal is corroborated by our phylogenetic analysis.

Molecular detection of Borrelia sp. in Ornithodoros cavernicolous (Acari: Argasidae) in midwestern Brazil.

Ticks are obligate hematophagous parasites that can transmit to vertebrate hosts several pathogens, including viruses, bacteria, protozoa and helminths. Among these agents, some Borrelia species some Borrelia species cause disease in humans and other vertebrate hosts; therefore, they have medical and veterinary health importance. To gather additional information on Borrelia species in Brazil, the current study aimed to detect the presence of these species in Ornithodoros cavernicolous ticks collected in September 2019 from cement pipes that are used by bats as shelter in a farm located in the midwestern region of Brazil. DNA samples obtained from 18 specimens of O. cavernicolous were subjected of two polymerase chain reactions, targeting a segment of the Borrelia fla B gene. Of the samples tested, only one (6 %, 1/18) showed amplification. The nucleotide sequence of the amplified DNA showed more than 97 % (293/300) identity with a sequence of a Borrelia sp. detected in blood collected from a bat from Macaregua Cave, Colombia, and more than 97 % (292/300) detected in lungs from vampire bats from northeastern Brazil. The deduced amino acid sequences were identical to each other. Phylogenetic analysis indicated that these sequences formed a group of Borrelia species (putatively associated with bats) that is closely related to sequences of Borrelia species of the Lyme borreliosis group. Further investigations should be carried out in order to determine whether the sequence of the Borrelia sp. we found belongs to a new taxon. It will also be of great importance to determine which vertebrate hosts, besides bats, O. cavernicolous ticks can parasitize in order to investigate whether the Borrelia sp. we found may be transmitted and cause disease to the other vertebrate hosts.

Caracterização bioquímica e verificação da atividade antibacteriana do líquido coxal do carrapato Ornithodoros rostratus

Os carrapatos, artrópodes hematófagos, são vetores de doenças tais como febre maculosa das Montanhas Rochosas ou do Mediterrâneo, doença de Lyme, encefalite transmitida por carrapatos, babesiose e etc. No entanto, os fluidos dos carrapatos (saliva e hemolinfa) são misturas ricas em moléculas que exercem interessantes atividades farmacológicas, por exemplo, anticoagulante, antitumoral, antimicrobiana, dentre outras. Assim, os fluidos dos carrapatos podem ser considerados como potenciais fontes para a descoberta de novos medicamentos. Os carrapatos moles, denominados Argasídeos, são ectoparasitas que possuem hábitos alimentares característicos, pois excretam o excesso de sangue ingerido através de suas glândulas coxais, produzindo assim um fluido conhecido como líquido coxal (LC), mistura que contém carboidratos, proteínas, lipídios e ácidos nucléicos. O Ornithodoros rostratus é um dos argasídeos encontrados na América do Sul, principalmente no Brasil, Argentina, Paraguai e Bolívia, entretanto, até o momento, o líquido coxal de O. rostratus ainda não foi bem caracterizado. Portanto, os objetivos deste trabalho foram realizar uma caracterização bioquímica e verificar a atividade antibacteriana do LC do carrapato O. rostratus fracionado através de um protocolo otimizado. Para isto, aproximadamente 3,5 mL de LC foi coletado, esterilizado (membrana de 0,22 μm), liofilizado e reconstituído e, posteriormente, fracionado de acordo com sua massa molecular através de uma membrana de corte de 10 kDa (Amicon). Este procedimento resultou nas frações de Líquido Coxal de Alta Massa Molecular (LCAM) e Líquido Coxal de Baixa Massa Molecular (LCBM), as quais tiveram suas concentrações proteicas determinadas por BCA e por absorbância (em 214; 260 e 280 nm); além disso essas amostras foram submetidas aos ensaios bioquímicos por SDS-PAGE, RP-HPLC, análise proteômica, determinação de massas moleculares por MALDI-TOF e análise de atividade antibacteriana (inibição de crescimento de Staphylococcus aureus e Escherichia coli). Posteriormente as amostras LCAM e LCBM foram fracionadas por troca iônica utilizando membrana SDB-XC e as amostras obtidas foram analisadas por MALDI-TOF. A análise SDS-9 PAGE mostrou que as amostras LC e LCAM possuem três bandas proteicas principais (aproximadamente 60; 25 e 15 kDa) e como esperado, a amostra LCBM não apresentou bandas proteicas. A análise das amostras LCB, LCAM e LCBM por RP- HPLC resultou respectivamente em 26; 20 e 31 picos majoritários. A amostra LCBM (concentração proteica de 0,1μg/μL e sem alteração de pH) inibiu o crescimento de S. aureus, no entanto, nenhuma das amostras ensaiadas inibiram o crescimento de E. coli. A análise proteômica do LC resultou em 164 proteínas de artrópodes e 31 proteínas antimicrobianas quando comparados com seus respectivos banco de dados. Por sua vez, a análise proteômica de LCAM concentrado resultou em 500 proteínas de artrópodes e 264 proteínas antimicrobianas. Tendo em vista o elevado número de proteínas encontradas no LC, existe uma grande possibilidade de alguns desses compostos inibirem o crescimento de S. aureus.

Caracterização inicial do extrato da glândula salivar de Ornithodoros brasiliensis (Acari: Argasidae) na coagulação sanguínea

Introduction: Ticks are obligate hematophagous ectoparasitic arthropods of great relevance to public health, being vectors of various diseases. Ornithodoros brasiliensis (Acari: Argasidae) are endemic to the mountainous region of Rio Grande do Sul, Brazil. Their salivary composition contains bioactive molecules capable of modulating the immune system and affecting the host's hemostasis with local and systemic effects. Objectives: To characterize the anticoagulant activity of the salivary gland extract (SGE) of the tick Ornithodoros brasiliensis on blood coagulation. Methods: The EGSs were obtained by macerating the ticks' salivary glands, generating 2 different extracts (EGS-1 and EGS-2), after which the amount of protein was estimated by absorbance at 280 nm. SDS-PAGE 12.5% was used to analyze the protein profile of the samples and the biological test was carried out on the L1 and L2 larvae of the nematode Caenorhabditis elegans. EGS-1 was filtered on Amicon according to molecular weight (30, 10 and 3kDa) and the fractions obtained were subjected to RP-HPLC chromatography (C18). The chromatography samples and EGS-1 in different concentrations were tested in the rotational thromboelastometry equipment (Rotem®) (0.2 to 1.03 μg/well), as well as in the FXa and thrombin inhibition tests using chromogenic substrates (S2765 and S2238, respectively) (0.40 to 0.207 μg/test), following protocols pre- established by the laboratory and the equipment manual. EGS-2 was submitted in different concentrations (6.5 to 52 ng/well) to the rotational thromboelastometry test, and the global coagulation tests were carried out in duplicate, Prothrombin Time (PT) and Activated Thromboplastin Time (aPTT), using the commercial kits, with different concentrations of ESG-2 (6.5 to 97.5 ng/test) and finally FXa and thrombin inhibition tests using chromogenic substrates (S2765 and s2238, respectively) at a concentration of 6.5 ng/test. Results: EGS-1 showed in the protein dosage (1.118 μg/ml) and EGS-2 (6.5 μg/ml). In the activity test, the EGSs showed temporary and reversible paralysis. EGS-2 showed a prolongation of the clotting time with incoagulobility from 52 ng/well in the rotational thromboelastometry test. In TP, inhibition was 28.5% at 97.5 ng/well and in TTPa 48.6% at 78 ng/well. Thrombin inhibition averaged 13.7% and factor Xa inhibition averaged 30%. In relation to the chromatographic fractions, the P4 fraction showed a prolongation of the coagulation time in the rotational thromboelastometry test. The P3 fraction had a 25.4% inhibition of thrombin formation. The P4 fraction inhibited Factor Xa by 36.9%. Conclusion: The EGS of the O. brasiliensis tick has anticoagulant action, reaching incoagulability with 52ng/test in the rotational thromboelastometry assay and at least two proteins acting in the process. One of them is linked to thrombin, with the P3 fraction showing the greatest inhibition, and the other is linked to FXa inhibition, with the P4 fraction being more evident. The rotational thromboelastometry test showed an alteration, indicating an inhibitory action on the formation of fibrin networks.

Introdução: Os carrapatos são artrópodes ectoparasitas hematófagos obrigatórios, de grande relevância para a saúde pública, sendo vetores de diversas doenças. Os Ornithodoros brasiliensis (Acari: Argasidae) são endêmicos da região serrana do Rio Grande do Sul, Brasil. Na sua composição salivar possui a presença de moléculas bioativas capazes de modular o sistema imunológico e afetar a hemostasia do hospedeiro com efeitos locais e sistêmicos. Objetivos: Caracterizar a atividade anticoagulante do extrato da glândula salivar (EGS) do carrapato Ornithodoros brasiliensis sobre a coagulação sanguínea Métodos: Os EGS’s foram obtidos por maceração das glândulas salivares dos carrapatos, gerando 2 extratos distintos (EGS-1 e EGS-2), em seguida, a quantidade de proteínas foi estimada por absorbância a 280 nm. O SDS-PAGE 12,5% foi usado para analisar o perfil proteico das amostras e o teste biológico foi realizado nas larvas L1 e L2 do nematoide Caenorhabditis elegans. O EGS-1 foi submetido à filtração em Amicon de acordo com peso molecular (30, 10 e 3kDa) e as frações obtidas foram submetidas a cromatografia RP-HPLC (C18). As amostras da cromatografia e o EGS-1 em diferentes concentrações foram testados no equipamento de tromboelastometria rotacional (Rotem®) (0,2 a 1,03 μg/poço), como também nos testes de inibição do FXa e trombina utilizando substratos cromogênicos (S2765 e S2238, respectivamente) (0,40 a 0,207 μg/teste), seguindo protocolos pré-estabelecidos pelo laboratório e o manual dos equipamentos. O EGS-2 foi submetido em diferentes concentrações (6,5 a 52 ng/poço) ao teste de tromboelastometria rotacional, sendo realizados os testes globais de coagulação em duplicada, Tempo de Protrombina (TP) e Tempo de Tromboplastina Activada (TTPa), utilizando os kits comerciais, com diferentes concentrações de ESG-2 (6,5 a 97,5 ng/teste) e por fim submetidos testes de inibição do FXa e trombina utilizando substratos cromogênicos (S2765 e s2238, respectivamente) em uma concentração de 6,5 ng/teste. Resultados: O EGS- 1 apresentou na dosagem proteica (1,118 μg/ml) e o EGS-2 (6,5 μg/ml). No teste de atividade os EGS’s apresentaram uma paralisia temporária e reversível. O EGS-2 apresentou um prolongamento no tempo de coagulação com incoagulobilidade a partir de 52 ng/poço no teste de tromboelastometria rotacional. No TP a inibição foi de 28,5% com 97,5 ng/poço e em TTPa de 48,6% com 78 ng/poço. Na inibição de trombina, teve uma média de inibição de 13,7% e na inibição de fator Xa uma média de 30%. Em relação às frações cromatográficas, a fração P4 apresentou um prolongamento no tempo de coagulação no teste de tromboelastometria rotacional. A fração P3 obteve uma inibição de 25,4% na formação de trombina. A fração P4 obteve uma inibição de 36,9% na inibição de Fator Xa. Conclusão: O EGS do carrapato O. brasiliensis apresenta ação anticoagulante chegando a incoagulabilidade com 52ng/teste no ensaio de tromboelastometria rotacional e pelo menos duas proteínas atuantes no processo. Uma delas ligada a trombina com a representação da fração P3 com maior inibição e outra ligada a inibição do FXa sendo mais evidente com a fração P4. No teste de Tromboelastometria rotacional mostrou uma alteração, apontando uma ação inibitória na formação das redes de fibrina.

Ornithodoros sonrai Soft Ticks and Associated Bacteria in Senegal.

The soft ticks, Ornithodoros sonrai, are known as vectors of the tick-borne relapsing fever caused by Borrelia spp. and have also been reported to carry other micro-organisms. The objective of this study was to collect and to identify O. sonrai ticks and to investigate the micro-organisms associated with them. In 2019, an investigation of burrows within human dwellings was conducted in 17 villages in the Niakhar area and in 15 villages in the Sine-Saloum area in the Fatick region of Senegal. Ticks collected from the burrows were identified morphologically and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Micro-organism screening was performed by bacteria-specific qPCR and some identifications were made by standard PCR and gene sequencing. O. sonrai ticks were found in 100% (17/17) of the villages surveyed in the Niakhar area and in 66% (10/15) of the villages in the Sine-Saloum area. A total of 1275 soft tick specimens were collected from small mammal burrows. The ticks collected were morphologically identified as O. sonrai. About 20% (259/1275) of the specimens were also submitted to MALDI-TOF MS for identification. Among the resulting MS profiles, 87% (139/159) and 95% (95/100) were considered good quality specimens, preserved in alcohol and silica gel, respectively. All spectra of good quality were tested against our MALDI-TOF MS arthropod spectra database and identified as O. sonrai species, corroborating the morphological classification. The carriage of four micro-organisms was detected in the ticks with a high prevalence of Bartonella spp., Anaplasmataceae, and Borrelia spp. of 35, 28, and 26%, respectively, and low carriage of Coxiella burnetii (2%). This study highlights the level of tick infestation in domestic burrows, the inventory of pathogens associated with the O. sonrai tick, and the concern about the potential risk of tick involvement in the transmission of these pathogens in Senegal.

A microRNA profile of the saliva in the argasid ticks Ornithodoros erraticus and Ornithodoros moubata and prediction of specific target genes.

Ornithodoros erraticus and Ornithodoros moubata ticks are the main vectors of the agents of human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin and Africa, respectively. Tick ​​saliva is crucial for complete tick feeding and pathogen transmission, as it contains numerous molecules such as proteins, lipids, and non-coding RNAs (ncRNA) including microRNAs (miRNA). MiRNAs are small ncRNAs capable of regulating the expression of their target messenger RNA (mRNA) leading to degradation or inhibition of its translation into protein. Research on miRNAs from ixodid ticks has revealed that miRNAs are involved in the regulation of different physiological processes of ticks, as well as in the modulation of host gene expression, immune response to tick bite and pathogen transmission. Regarding argasid ticks, there is not information about their miRNAs or their potential involvement in tick physiology and/or in the regulation of the tick-host-pathogen interactions. The aim of this work was to profile the miRNAs expressed in the saliva of O. erraticus and O. moubata, and the in silico prediction and functional analysis of their target genes in the swine host. As a whole, up to 72 conserved miRNAs families were identified in both species: 35 of them were shared and 23 and 14 families were unique to O. erraticus and O. moubata, respectively. The most abundant miRNAs families were mir-1, mir-10 and let-7 in O. erraticus and let-7, mir-252, mir-10 in O. moubata. Four miRNAs sequences of each species were validated by RT-qPCR confirming their presence in the saliva. Target gene prediction in the host (Sus scrofa) and functional analysis showed that the selected miRNAs are mainly involved in processes related to signal transduction, regulation of mRNA transcription and gene expression, synapse regulation, immune response, angiogenesis and vascular development. These results suggest that miRNAs could play an important role at the tick-host interface, providing new insights into this complex relationship that may contribute to a more precise selection of tick molecules for the development of therapeutic and immune strategies to control tick infestations and tick-borne pathogens.

A novel Babesia sp. of the "Western Babesia group", detected in opossums from Guatemala.

Babesia spp. are tick-borne protozoans that involve birds and mammals in their transmission cycles and cause babesiosis, a severe hemolytic malaria-like disease. Opossums of the genus Didelphis are recognized hosts of tick-borne pathogens. Therefore, exploring tick-borne agents in Didelphis species is important to understand the circulation of pathogens in areas where opossums occur. In this study, we targeted Anaplasmataceae, Babesia, Borrelia and Hepatozoon DNA in ticks, blood and organ samples collected from three hunted Didelphis marsupialis specimens in eastern Guatemala. While the samples were negative for Hepatozoon and bacterial DNA, sequences of Babesia 18S rDNA, cox1 and cytb genes were retrieved from two opossums. Ticks collected on the animals included Amblyomma parvum and an undetermined Ornithodoros sp. The Babesia sp. detected in this study (Babesia sp. THB1-2) clusters phylogenetically within the "Western Babesia group", which includes pathogenic species such as Babesia conradae, Babesia duncani, and Babesia negevi. Our results represent the first record of a Babesia sp. in Guatemala and highlight the importance of D. marsupialis as potential spreaders of ticks and pathogens in Central America.

Function-guided selection of salivary antigens from Ornithodoros erraticus argasid ticks and assessment of their protective efficacy in rabbits.

The identification of new protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that have key biological functions. Bioactive proteins playing key functions for tick feeding and pathogen transmission are secreted into the host via tick saliva. Adult argasid ticks must resynthesise and replace these proteins after each feeding to be able to repeat new trophogonic cycles. Therefore, these proteins are considered interesting antigen targets for tick vaccine development. In this study, the salivary gland transcriptome and saliva proteome of Ornithodoros erraticus females were inspected to select and test new vaccine candidate antigens. For this, we focused on transcripts overexpressed after feeding that encoded secretory proteins predicted to be immunogenic and annotated with functions related to blood ingestion and modulation of the host defensive response. Completeness of the transcript sequence, as well as a high expression level and a high fold-change after feeding were also scored resulting in the selection of four candidates, an acid tail salivary protein (OeATSP), a multiple coagulation factor deficiency protein 2 homolog (OeMCFD2), a Cu/Zn-superoxide dismutase (OeSOD) and a sulfotransferase (OeSULT), which were later produced as recombinant proteins. Vaccination of rabbits with each individual recombinant antigen induced strong humoral responses that reduced blood feeding and female reproduction, providing, respectively, 46.8%, 45.7%, 54.3% and 31.9% protection against O. erraticus infestations and 0.7%, 3.9%, 3.1% and 8.7% cross-protection against infestations by the African tick, Ornithodoros moubata. The joint protective efficacy of these antigens was tested in a second vaccine trial reaching 58.3% protection against O. erraticus and 18.6% cross-protection against O. moubata. These results (i) provide four new protective salivary antigens from argasid ticks that might be included in multi-antigenic vaccines designed for the control of multiple tick species; (ii) reveal four functional protein families never tested before as a source of protective antigens in ticks; and (iii) show that multi-antigenic vaccines increase vaccine efficacy compared with individual antigens. Finally, our data add value to the salivary glands as a protective antigen source in argasids for the control of tick infestations.

Microbiomes of Blood-Feeding Triatomines in the Context of Their Predatory Relatives and the Environment.

The importance of gut microbiomes has become generally recognized in vector biology. This study addresses microbiome signatures in North American Triatoma species of public health significance (vectors of Trypanosoma cruzi) linked to their blood-feeding strategy and the natural habitat. To place the Triatoma-associated microbiomes within a complex evolutionary and ecological context, we sampled sympatric Triatoma populations, related predatory reduviids, unrelated ticks, and environmental material from vertebrate nests where these arthropods reside. Along with five Triatoma species, we have characterized microbiomes of five reduviids (Stenolemoides arizonensis, Ploiaria hirticornis, Zelus longipes, and two Reduvius species), a single soft tick species, Ornithodoros turicata, and environmental microbiomes from selected sites in Arizona, Texas, Florida, and Georgia. The microbiomes of predatory reduviids lack a shared core microbiota. As in triatomines, microbiome dissimilarities among species correlate with dominance of a single bacterial taxon. These include Rickettsia, Lactobacillus, "Candidatus Midichloria," and Zymobacter, which are often accompanied by known symbiotic genera, i.e., Wolbachia, "Candidatus Lariskella," Asaia, Gilliamella, and Burkholderia. We have further identified a compositional convergence of the analyzed microbiomes in regard to the host phylogenetic distance in both blood-feeding and predatory reduviids. While the microbiomes of the two reduviid species from the Emesinae family reflect their close relationship, the microbiomes of all Triatoma species repeatedly form a distinct monophyletic cluster highlighting their phylosymbiosis. Furthermore, based on environmental microbiome profiles and blood meal analysis, we propose three epidemiologically relevant and mutually interrelated bacterial sources for Triatoma microbiomes, i.e., host abiotic environment, host skin microbiome, and pathogens circulating in host blood. IMPORTANCE This study places microbiomes of blood-feeding North American Triatoma vectors (Reduviidae) into a broader evolutionary and ecological context provided by related predatory assassin bugs (Reduviidae), another unrelated vector species (soft tick Ornithodoros turicata), and the environment these arthropods coinhabit. For both vectors, microbiome analyses suggest three interrelated sources of bacteria, i.e., the microbiome of vertebrate nests as their natural habitat, the vertebrate skin microbiome, and the pathobiome circulating in vertebrate blood. Despite an apparent influx of environment-associated bacteria into the arthropod microbiomes, Triatoma microbiomes retain their specificity, forming a distinct cluster that significantly differs from both predatory relatives and ecologically comparable ticks. Similarly, within the related predatory Reduviidae, we found the host phylogenetic distance to underlie microbiome similarities.

The Soft Tick Ornithodoros moubata and Its Role in the Epidemiology of African Swine Fever in Central Malawi.

African swine fever is a viral hemorrhagic disease of pigs (Sus scrofa) caused by a double-stranded DNA virus, African swine fever virus (ASFV). With up to 100% mortality in pigs and no vaccine, the socioeconomic impacts of this disease are immense. In sub-Saharan Africa, warthogs (Phacochoerus africanus) are the original vertebrate host for the virus, which is transmitted among wild suids via the soft tick Ornithodoros moubata. In Malawi, O. moubata is thought to be widely distributed, with the potential to spread ASFV beyond its historical enzootic zone in the Central Region. We surveyed 76 domestic pig farms, 18 active warthog burrows, and three bushpigs (Potamochoerus larvatus) resting sites for O. moubata in the Central Region of Malawi, and tested the ticks for ASFV using PCR. We found a limited distribution for O. moubata: 75 ticks were discovered at a single farm in the Mchinji District. Eleven percent of these ticks were ASFV positive. This suggests that wildlife and O. moubata play a limited role in the epidemiology of ASF in Malawi; thus, other factors for disease spread, such as fomites and pig-to-pig infection, need to be considered.

Comparison of salivary gland and midgut microbiome in the soft ticks Ornithodoros erraticus and Ornithodoros moubata.

Introduction: Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. Methods: In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Results: Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. Conclusion: We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

Detection of Babesia Spp., Theileria Spp., and Anaplasma Ovis in Ornithodoros Lahorensis from Southern Xinjiang, China.

Introduction: Ticks are blood-sucking arthropods that have negative economic impacts and can spread a variety of diseases through their bites. There are few reports on soft ticks (Acari: Argasidae) and tick-borne pathogens in southern Xinjiang, China. This investigation supplements the available information for this region and is concerned with an argasid tick, apicomplexan parasites of the Babesia and Theileria genera and a bacterium of the Anaplasma genus. Material and Methods: In this study, 330 soft ticks were collected from nine sampling sites in southern Xinjiang between 2020 and 2021. The ticks were identified according to their morphological characteristics and confirmed as Ornithodoros lahorensis using mitochondrial 16S rDNA sequences. Babesia and Theileria were identified at the species level based on two fragments of the 18S rRNA gene, and one set of primers targeting the 16S rRNA gene was used to identify the Anaplasma genus. Results: Among the 330 samples, one Babesia species (Babesia sp.), two Theileria species (T. ovis and T. annulata), and one Anaplasma (A. ovis) species were detected. Conclusion: This study provides fundamental evidence for the occurrence of Babesia, Theileria and Anaplasma spp. in soft ticks. To the best of our knowledge, this is the first report of the detection of Babesia sp. and T. annulata in O. lahorensis. Therefore, the potential threat of soft ticks to livestock and humans should not be ignored.

Rickettsia and relapsing fever Borrelia in Alectorobius kelleyi (Ixodida: Argasidae) from peri domestic bats in the northeastern United States.

The soft ticks (Argasidae) are known vectors of human and animal pathogens around the globe and are relatively understudied. Our aim was to assess the presence of Rickettsia and Borrelia bacteria in Alectorobius kelleyi (Argasidae) parasitizing synanthropic bats in the highly urbanized northeastern United States. By collaborating with parasitologists, bat scientists and wildlife rehabilitators we were successful in obtaining A. kelleyi from five states. Since Argasid larvae will attach to their hosts for many days, most A. kelleyi examined (92%) were larvae collected from sick or injured big brown bats, Eptesicus fuscus, undergoing care at rehabilitation centers. In addition, we obtained adult A. kelleyi captured in residential living areas and trapped in attics. An in-depth analysis of a A. kelleyi found to be infected with a spotted fever group Rickettsia (SFGR) revealed a dual infection with a R. belli-like taxon (ancestral group) as well as an SFGR closely related to R. peacockii, likely the same previously found in A. kelleyi from Iowa and Kansas. We found that 36% of the A. kelleyi tested carried the SFGR. Furthermore, we detected a relapsing fever spirochete, likely Candidatus Borrelia johnsonii, in 25% of the A. kelleyi from Pennsylvania. While it is unclear if these bacteria constitute a health risk to either bats or humans, our study indicates that human exposure to ectoparasites infesting peridomestic wildlife should be considered in the epidemiology of tick-borne diseases.