Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-Galactosidase and β-1,4 Galactosyltransferase Enzymes in α-Gal Metabolism

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick’s specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and β-1,4 galactosyltransferases (β-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars—GlcNAc, Glc, and Xyl—in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in β-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and β-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS.

Ehrlichia and Anaplasma

Ehrlichia and Anaplasma are bacteria that cause diseases, known as ehrlichiosis and anaplasmosis, in humans and other animals. Ehrlichia and Anaplasma are primarily transmitted through the bites of infected hard ticks, such as the lone star tick, the blacklegged tick, and the American dog tick. This publication describes the various forms of ehrlichiosis and anaplasmosis and the tick vectors.

Recently evolved Francisella-like endosymbiont outcompetes an ancient and evolutionarily associated Coxiella-like endosymbiont in the lone star tick (Amblyomma americanum) linked to the Alpha-Gal Syndrome

BackgroundTicks are hematophagous arthropods that transmit various bacterial, viral, and protozoan pathogens of public health significance. The lone star tick (Amblyomma americanum) is an aggressive human-biting tick that transmits bacterial and viral pathogens, and its bites are suspected of eliciting the Alpha-Gal Syndrome, a newly emerged delayed hypersensitivity following consumption of red meat in the United States. While ongoing studies have attempted to investigate the contribution of different tick-inherent factors to the induction of Alpha-Gal syndrome, an otherwise understudied aspect is the contribution of the tick microbiome and specifically obligate endosymbionts to the establishment of the Alpha-Gal syndrome in humans.MethodsHere we utilized a high throughput metagenomic sequencing approach to cataloging the entire microbial communities residing within different developmental stages and tissues of unfed and blood-fed ticks from laboratory-maintained ticks and three new geographical locations in the USA. The quantitative insights into microbial ecology (QIIME2) pipeline were used to perform data analysis and taxonomic classification. Moreover, using a SparCC network construction model, we investigated potential interactions between members of the microbial communities from lab-maintained and field-collected ticks. ResultsOverall, Francisellaceae was the most dominant bacteria identified in the microbiome of both lab-raised and field-collected Am. americanum across all tissues and developmental stages. Likewise, microbial diversity was seen to be significantly higher in field-collected ticks compared to lab-maintained ticks as seen with a higher number of both OTUs and measures of species richness. Several potential positive and negative correlations were identified from our network analysis. We observed a strong positive correlation between Francisellaceae, Rickettsiaceae, and Midichloriaceae in both developmental stages and tissues from lab-maintained ticks, while ovarian tissues had a strong positive correlation of bacteria in the family Xanthobacteraceae and Rhizobiaceae. A negative interaction was observed between Coxiellaceae and Francisellaceae in Illinois, and all the bacteria detected from ticks from Delaware were negatively correlated.ConclusionThis study is the first to catalog the microbiome of Am. americanum throughout its developmental stages and different tissue niches and report the potential replacement of Coxiellaceae by Francisellaceae across developmental stages and tissues tested except in ovarian tissues. These unique and significant findings advance our knowledge and open a new avenue of research to further understand the role of tick microbiome in tick-borne diseases and develop a holistic strategy to control Alpha-Gal syndrome.

EVALUATION OF D-ALLETHRIN IN THE THERMACELL MOSqUITO REPELLENT DEVICE AGAINST THE LONE STAR TICK UNDER LABORATORY CONDITIONS

D-allethrin vapor generated from a personal mosquito repellent device (Thermacell MR300) was evaluated for its effectiveness to repel the lone star tick, Amblyomma americanum (adults and nymphs) when released at tick body level in a wind tunnel and in an olfactometer. In the wind tunnel 48.5% of ticks moved upwind when only attractant lure was present, while only 30.8% moved upwind when d-allethrin repellent was present with the lure. In the olfactometer strong repellency of d-allethrin vapor to adults was observed, but the effect was reduced with nymphs. Results of this study showed that d-allethrin vapor generated by the Thermacell MR300 pad could be used to reduce movement of ticks towards a host under some conditions.

A novel Tick Carousel Assay for testing efficacy of repellents on Amblyomma americanum L.

Ticks are important vectors of human and veterinary diseases. A primary way ticks gain access to human hosts is by engaging to clothing. Repellents or acaricides sprayed onto fabric are used to deter ticks’ access to human hosts. However, there are a limited amount of standardized laboratory assays that can determine the potency and efficacy of repellents. We present a novel fabric-engagement assay referred to as the ‘Tick Carousel Assay’. This assay utilizes fabric brushing past ticks located on an artificial grass patch and measures tick engagements to fabric over time. After screening a variety of tick species, we used the lone star tick (Amblyomma americanum) to test the efficacy of four commonly used active ingredients in repellents: DEET, Picaridin, IR3535, and Oil of Lemon Eucalyptus. Repellency was tested immediately, after three hours, and six hours post application to fabric. Our data show that each repellent we tested significantly reduced the number of tick engagements to fabric for at least 6 hours. We did not find significant differences in repellent efficacy between the four active ingredients tested directly and three hours after application. After six hours, Oil of Lemon Eucalyptus repelled ticks more than the other active ingredients. We show that our Tick Carousel Assay provides an affordable, repeatable, and standardized way to compare and test repellent efficacy on treated fabrics. Our results confirm that commonly used repellents applied to fabric are an effective way to reduce tick engagement.

Dermal secretion physiology in Metastriata ticks; thermoregulation in the lone star tick Amblyomma americanum

Ticks are blood feeding ectoparasites that transmit a wide range of pathogens. The lone star tick, Amblyomma americanum, is one of the most widely distributed ticks in the Midwest and Eastern United States. Lone star ticks, like most three-host ixodid ticks, can survive in harsh environments for extended periods without consuming a blood meal. Physiological mechanisms that allow them to survive during hot and dry season include thermal tolerance and water homeostasis. Large quantity of dermal fluid secretions induced by mechanical stimulation of tick legs has been described in metastriate ticks including Amblyomma. We hypothesize that a function of tick dermal secretion is similar to the sweating in large homeothermal animals. In this study, we found that a contact with a heat probe at 45oC can trigger dermal secretion. We demonstrated that dermal secretion plays a role in evaporative cooling when ticks are exposed to high temperature. We observed that direct contact to a heat probe for 5 seconds at ~ 52oC caused an exhaustive dermal secretion with ~ 4% loss of body weight and resulted in the lethality in 24-hour, indicating that the secretion is associated with significant costs of water loss. We identified type II dermal glands having paired two cells forming large glandular structures. The secretion is triggered by an injection of serotonin and the serotonin-mediated secretion was suppressed by a pretreatment of Ouabain, a Na/K-ATPase blocker, implying that the secretion is controlled by serotonin and the downstream Na/K-ATPase.

The bacterial community of the lone star tick (Amblyomma americanum)

Background The lone star tick (Amblyomma americanum), an important vector of a wide range of human and animal pathogens, is very common throughout the East and Midwest of the USA. Ticks are known to carry non-pathogenic bacteria that may play a role in their vector competence for pathogens. Several previous studies using the high throughput sequencing (HTS) technologies reported the commensal bacteria in a tick midgut as abundant and diverse. In contrast, in our preliminary survey of the field collected adult lone star ticks, we found the number of culturable/viable bacteria very low. Methods We aimed to analyze the bacterial community of A. americanum by a parallel culture-dependent and a culture-independent approach applied to individual ticks. Results We analyzed 94 adult females collected in eastern Kansas and found that 60.8% of ticks had no culturable bacteria and the remaining ticks carried only 67.7 ± 42.8 colony-forming units (CFUs)/tick representing 26 genera. HTS of the 16S rRNA gene resulted in a total of 32 operational taxonomic units (OTUs) with the dominant endosymbiotic genera Coxiella and Rickettsia (> 95%). Remaining OTUs with very low abundance were typical soil bacterial taxa indicating their environmental origin. Conclusions No correlation was found between the CFU abundance and the relative abundance from the culture-independent approach. This suggests that many culturable taxa detected by HTS but not by culture-dependent method were not viable or were not in their culturable state. Overall, our HTS results show that the midgut bacterial community of A. americanum is very poor without a core microbiome and the majority of bacteria are endosymbiotic.

Association between lone star tick bites and increased alpha-gal sensitization: evidence from a prospective cohort of outdoor workers

Background Alpha-gal is an oligosaccharide implicated in delayed anaphylaxis following red meat consumption. Exposure to tick bites has been correlated with development of an allergic response to alpha-gal. However, evidence prospectively linking exposure to a single tick species and an immune response to alpha-gal is lacking. Methods We used serum samples from a prior study cohort of outdoor workers in North Carolina, USA, with high exposure to the lone star tick, Amblyomma americanum, to prospectively evaluate the relationship between tick bites and anti-alpha-gal IgE antibodies. Results Individuals who reported exposure to one or more tick bites were significantly more likely to have a positive change in anti-alpha-gal IgE compared to individuals with no reported tick bites. This relationship was not dependent on time. A trend toward increasing number of tick bites and increased anti-alpha-gal IgE levels was observed but not statistically significant. Conclusion To our knowledge, this is the first study to prospectively link documented exposure to A. americanum bites and increased sensitization to alpha-gal in a cohort of outdoor workers. Our results support the role of A. americanum as likely agents for eliciting an allergic response to red meat, and highlight the importance of preventing tick bites.