Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Xinyang flavivirus, from Haemaphysalis flava ticks in Henan Province, China, defines a basal, likely tick-only Orthoflavivirus clade.

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.

A novel and low-cost cross-priming amplification assay for rapid detection of Babesia duncani infection.

Babesia duncani, responsible for human babesiosis, is one of the most important tick-borne intraerythrocytic pathogens. Traditionally, babesiosis is definitively diagnosed by detecting parasite DNA in blood samples and examining Babesia parasites in Giemsa-stained peripheral blood smears. Although these techniques are valuable for determining Babesia duncani, they are often time-consuming and laborious. Therefore, developing rapid and reliable B. duncani identification assays is essential for subsequent epidemiological investigations and prevention and control. In this study, a cross-priming amplification (CPA) assay was developed, combined with a vertical flow visualization strip, to rapidly and accurately detect B. duncani infection. The detection limit of this method was as low as 0.98 pg/µl of genomic DNA from B. duncani merozoites per reaction at 59 °C for 60 min. There were no cross-reactions between B. duncani and other piroplasms infective to humans and mammals. A total of 592 blood samples from patients bitten by ticks and experimental infected hamsters were accurately assessed using CPA assay. The average cost of the CPA assay is as low as approximately $ 0.2 per person. These findings indicate that the CPA assay may therefore be a rapid screening tool for detection B. duncani infection, based on its accuracy, speed, and cost-effectiveness, particularly in resource-limited regions with a high prevalence of human babesiosis.

Molecular detection of Borrelia theileri in cattle in Korea.

Bovine borreliosis, caused by Borrelia theileri which is transmitted via hard tick bites, is associated with mild clinical symptoms, such as fever, lethargy, hemoglobinuria, anorexia, and anemia. Borrelia theileri infects various animals, such as cattle, deer, horses, goats, sheep, and wild ruminants, in Africa, Australia, and South America. Notably, no case of B. theileri infection has been reported in Korean cattle to date. In this study, 101 blood samples were collected from a Korean indigenous cattle breed, among which 1.98% tested positive for B. theileri via nested PCR. The obtained sequences exhibited high homology with B. theileri strains identified in other regions. Phylogenetic analysis of 16S rRNA confirmed the B. theileri group affiliation; however, flagellin B sequences exhibited divergence, potentially due to regional evolutionary differences. This study provides the first molecular confirmation of B. theileri infection in Korean livestock. Further isolation and nucleotide sequence analyses are necessary to better understand the presence of B. theileri strains in cows in Korea.

Serological investigation of seven zoonotic pathogens in companion dogs in South Korea, 2018-2021.

Based on the current situation of Korean culture and society, the population of companion animals in South Korea is growing rapidly along with zoonotic risks. The current data regarding zoonotic infections in companion dogs reported in Korea is sparse. This study aims to investigate the seroprevalence of seven potential zoonotic pathogens in companion dogs in South Korea: Anaplasma phagocytophilum, Borrelia burgdoferi, Ehrlichia canis, Coxiella burnetii, Brucella canis, Leptospira spp. and canine influenza A virus. A total of 284 serum samples were collected from 2018 to 2021, and the immunoglobulin G (IgG) antibodies against 7 zoonotic pathogens were detected using enzyme-linked immunosorbent assays. Samples were divided into five groups and analysed based on age. IgG antibodies against six of the seven pathogens were detected. The highest seropositivity rate was detected for canine influenza A virus exposure (59.1%) for which the rates were the highest in dogs under 1 year old and declined with age. Positivity rates of the other pathogens were relatively low: 1.76% for Leptospira spp., 1.40% for A. phagocytophilum and E. canis, 1.06% for B. canis and 0.35% for B. burgdoferi. No antibodies against C. burnetii were detected in this study. The exposure of dogs in South Korea to six zoonotic pathogens was serologically confirmed, highlighting a potential risk for human infection. The zoonotic risk of companion dogs cannot be neglected, and implementation of One Health approach should be advocated to establish effective preventive measures.

Molecular Analysis of Tick-Borne Bacterial Pathogens from Ticks Infesting Animal Hosts in Kyrgyzstan, 2021.

This study investigated the prevalence of Anaplasma and Ehrlichia in 494 engorged ticks collected from various animal hosts, including cattle, horses, sheep, chickens, dogs, and cats, in six regions of northern Kyrgyzstan. Ten tick species, belonging to two families and six genera, were identified based on CO1, 16S rRNA, and ITS2 genes: Argas persicus (26.5%), Haemaphysalis punctata (18.0%), Dermacentor spp. (16.0%), Rhipicephalus annulatus (11.8%), R. turanicus (10.9%), D. marginatus (7.7%), Hyalomma scupense (4.5%), Hy. marginatum (3.8%), R. sangineus complex (0.6%), and Ornithodoros lahorensis (0.2%). PCR analysis revealed a 15.0% (74/494) overall infection rate of Anaplasma and Ehrlichia. Anaplasma species were found in six tick species and were identified as A. bovis (n = 44), Anaplasma spp. (n = 20), A. ovis (n = 5), and A. capra (n = 2). Ehrlichia species were found only in H. punctata (n = 5) and identified as E. chaffeensis (n = 1) and Ehrlichia spp. (n = 4). Additionally, two H. punctata were co-infected with Anaplasma and Ehrlichia. This is the first study to investigate tick-borne bacterial pathogens in ticks collected from animal hosts in Kyrgyzstan. Our findings contribute to a better understanding of the epidemiology and emergence of tick-borne infections in Kyrgyzstan.

Detection of Anaplasma spp. and Ehrlichia spp. in dogs from a veterinary teaching hospital in Italy: a retrospective study 2012-2020.

Anaplasma phagocytophilum, Anaplasma platys and Ehrlichia canis, responsible of diseases in dogs, are tick-borne pathogens with a proven or potential zoonotic role that have shown increasing prevalence worldwide. The aims of this retrospective study were to assess the frequency of Anaplasma spp. and Ehrlichia spp. exposure in dogs tested in a veterinary teaching hospital in Italy over a 9-year period, to compare the performance of the diagnostic tests used, to evaluate correlations with clinical data, and to genetically analyse the identified bacteria. During the study period, 1322 dogs tested by at least one of the rapid immunoenzymatic test, indirect immunofluorescent antibody test or end-point PCR assay for Anaplasmataceae detection were included. Dogs were tested if they had clinical signs or clinicopathological alteration or risk factors related to infection, and if they were potential blood-donor animals. Ninety-four of 1322 (7.1%) dogs tested positive for at least one pathogen: 53 (4.3%) for A. phagocytophilum, one (0.1%) for A. platys and 63 (4.6%) for E. canis. The number of dogs tested increased and the positivity rate progressively declined over the years. Comparison of tests showed a near-perfect agreement between serological tests and a poor agreement between PCR and indirect assays. A breed predisposition has been highlighted for A. phagocytophilum infection in hunting breed dogs and for E. canis infection in mixed breed dogs. Phylogeny confirmed potential zoonotic implications for A. phagocytophilum and showed no correlation of the identified bacteria with the geographical origin. Our study provides new insights into possible risk factors in dogs and evidenced discordant results between different tests, suggesting that a combination of serological and molecular assays is preferable for a correct diagnosis.

Exploring the influence of host community composition on the outbreak potential of Anaplasma phagocytophilum and Borrelia burgdorferi s.l.

In large parts of the northern hemisphere, multiple deer species coexist, and management actions can strongly influence wild deer communities. Such changes may also indirectly influence other species in the community, such as small mammals and birds, because deer can have strong effects on their habitats and resources. Deer, small mammals and birds play an important role in the dynamics of tick-borne zoonotic diseases. It is, however, relatively underexplored how the abundance and composition of vertebrate communities may affect the outbreak potential, maintenance and circulation of tick-borne pathogens. In this study we focus on the outbreak potential by exploring how the basic reproduction number R0 for different tick-borne pathogens depends on host community composition. We used published data on co-varying roe deer (Capreolus capreolus) and fallow deer (Dama dama) densities following a hunting ban, and different small mammal and bird densities, to investigate how the change in host community influences the R0 of four tick-borne pathogens: one non-zoonotic, namely Anaplasma phagocytophilum ecotype 2, and three zoonotic, namely A. phagocytophilum ecotype 1, Borrelia afzelii and Borrelia garinii. We calculated R0 using a next generation matrix approach, and used elasticities to quantify the contributions to R0 of the different groups of host species. The value of R0 for A. phagocytophilum ecotype 1 was higher with high fallow deer density and low roe deer density, while it was the other way round for A. phagocytophilum ecotype 2. For B. afzelii, R0 was mostly related to the density of small mammals and for B. garinii it was mostly determined by bird density. Our results show that the effect of species composition is substantial in the outbreak potential of tick-borne pathogens. This implies that also management actions that change this composition, can (indirectly and unintentionally) affect the outbreak potential of tick-borne diseases.

Impact of vaccination with the Anaplasma phagocytophilum MSP4 chimeric antigen on gene expression in the rabbit host.

There are currently no vaccines available to prevent and control of Anaplasma phagocytophilum, an intracellular bacterial pathogen transmitted by ticks that occurs in many regions of the world and causes disease in a wide range of domestic and wild hosts, including humans. Vaccines induce long-lasting immunity and could prevent or reduce transmission of this pathogen. Understanding how vaccines induce a protective response can be difficult due to the complexity of the immune system, which operates at many levels throughout the organism. New perspectives in vaccinology, based on systems biology approaches, integrate many scientific disciplines to fully understand the biological responses to vaccination, where a transcriptomic approach could reveal relevant information of the host immune system, allowing profiling for rational design of vaccine formulations, administration, and potential protection. In the present study we report the gene expression profiles by RNA-seq followed by functional analysis using whole blood samples from rabbits immunized with a recombinant chimeric protein containing peptides from the MSP4 protein of A. phagocytophilum, which showed satisfactory results in terms of potential protection. Transcriptomic analysis revealed differential expression of 720 genes, with 346 genes upregulated and 374 genes downregulated. Overrepresentation of biological and metabolic pathways correlated with immune response, protein signaling, cytoskeleton organization and protein synthesis were found. These changes in gene expression could provide a complete and unique picture of the biological response to the epitope candidate vaccine against A. phagocytophilum in the host.

Cascading impacts of overstory structure in managed forests on understory structure, microclimate conditions, and Ixodes scapularis (Acari: Ixodidae) densities.

Forest management practices designed to meet varied landowner objectives affect wildlife habitat and may interrupt the life-cycle stages of disease vectors, including the black-legged tick, Ixodes scapularis Say (Acari: Ixodidae). Ixodes scapularis transmits multiple pathogens including Borrelia burgdorferi, the causative agent of Lyme disease, which is the most common tick-borne disease in the United States. There is evidence that a range of active forest management practices (e.g., invasive plant removal, prescribed burning) can alter tick densities and pathogen transmission. However, few studies have investigated relationships between forest stand structural variables commonly manipulated by timber harvesting and tick ecology. Foresters may harvest timber to create certain forest structural conditions like the mean number of trees, or basal area, per hectare. This study used a spatially replicated experiment in a blocked design to compare forest stands with a range of overstory structures and document variations in the midstory, understory, and forest floor, as well as microclimate conditions within tick off-host habitat. Greater numbers of trees or basal area per hectare correlated with greater canopy closure but less understory cover, stabilized microclimate temperature, higher microclimate humidity, and greater I. scapularis nymph densities. A random forest model identified understory forest structure as the strongest predictor of nymph densities. There was no relationship between the number of trees or basal area per hectare and daily deer (Odocoileus virginianus Zimmermann) activity or nymphal infection prevalence. These findings provide a deeper understanding of tick-habitat associations within a forest stand and have the potential to inform forest management decisions.

Human Tick-Borne Diseases and Advances in Anti-Tick Vaccine Approaches: A Comprehensive Review.

This comprehensive review explores the field of anti-tick vaccines, addressing their significance in combating tick-borne diseases of public health concern. The main objectives are to provide a brief epidemiology of diseases affecting humans and a thorough understanding of tick biology, traditional tick control methods, the development and mechanisms of anti-tick vaccines, their efficacy in field applications, associated challenges, and future prospects. Tick-borne diseases (TBDs) pose a significant and escalating threat to global health and the livestock industries due to the widespread distribution of ticks and the multitude of pathogens they transmit. Traditional tick control methods, such as acaricides and repellents, have limitations, including environmental concerns and the emergence of tick resistance. Anti-tick vaccines offer a promising alternative by targeting specific tick proteins crucial for feeding and pathogen transmission. Developing vaccines with antigens based on these essential proteins is likely to disrupt these processes. Indeed, anti-tick vaccines have shown efficacy in laboratory and field trials successfully implemented in livestock, reducing the prevalence of TBDs. However, some challenges still remain, including vaccine efficacy on different hosts, polymorphisms in ticks of the same species, and the economic considerations of adopting large-scale vaccine strategies. Emerging technologies and approaches hold promise for improving anti-tick vaccine development and expanding their impact on public health and agriculture.

Molecular identification of Ehrlichia sp. in a beef cattle from Brazilian Cerrado - case report / Identificação molecular de Ehrlichia sp em um bovino do Cerrado brasileiro - relato de caso

Ehrlichia infections in cattle are frequent in Africa but have also been reported in Brazil and North America. This paper reports natural infection by Ehrlichia sp. associated with Babesia bigemina and Anaplasma marginale in a calf in the municipality of Campo Grande, state of Mato Grosso do Sul, Brazil, presenting polioencephalomalacia. The molecular evidence, based on a fragment of the dsb gene, indicates a species of Ehrlichia genetically related to Ehrlichia canis and other species of the genus found in the tick Rhipicephalus (Boophilus) microplus and a calf from Brazil (99 to 100% identity). It was not possible to associate the clinical signs with Ehrlichia infection due to co-infections and histological evidence of another disease. However, the circulation of the bacteria in bovines in Brazilian Cerrado was confirmed and more attention should be given to clinical suspicion of tick-borne pathogens in cattle to clarify the pathogenic potential of Ehrlichia sp.(AU)

Infecções por Ehrlichia em bovinos são frequentes na África, mas também foram relatadas no Brasil e na América do Norte. Este artigo relata uma infecção natural por Ehrlichia sp. associado a Babesia bigemina e Anaplasma marginale em um bezerro, no município de Campo Grande, Mato Grosso do Sul, Brasil, o qual apresentava polioencefalomalácia. A evidência molecular, baseada em um fragmento do gene dsb, indica uma espécie de Ehrlichia geneticamente relacionada a Ehrlichia canis e outras espécies do gênero encontradas no carrapato Rhipicephalus (Boophilus) microplus e em um bezerro do Brasil (99 a 100% de identidade). Não foi possível associar os sinais clínicos à infecção por Ehrlichia devido a coinfecções e evidências histológicas de outra doença. No entanto, a circulação da bactéria em bovinos no Cerrado brasileiro foi confirmada, e mais atenção deve ser dada à suspeita clínica de patógenos transmitidos por carrapatos em bovinos para esclarecer o potencial patogênico de Ehrlichia sp.(AU)