Characterization of tick salivary gland and saliva alphagalactome reveals candidate alpha-gal syndrome disease biomarkers.

BACKGROUND: Ticks are obligate hematophagous arthropods that synthesize the glycan Galα1-3Galß1-(3)4GlcNAc-R (α-Gal) associated with the alpha-gal syndrome (AGS) or allergy to mammalian meat consumption. RESEARCH DESIGN AND METHODS: In this study, we used a proteomics approach to characterize tick proteins in salivary glands (sialome SG), secreted saliva (sialome SA) and with α-Gal modification (alphagalactome SG and SA) in model tick species associated with the AGS in the United States (Amblyomma americanum) and Australia (Ixodes holocyclus). Selected proteins reactive to sera (IgE) from patients with AGS were identified to advance in the identification of possible proteins associated with the AGS. For comparative analysis, the α-Gal content was measured in various tick species. RESULTS: The results confirmed that ticks produce proteins with α-Gal modifications and secreted into saliva during feeding. Proteins identified in tick alphagalactome SA by sera from patients with severe AGS symptomatology may constitute candidate disease biomarkers. CONCLUSIONS: The results support the presence of tick-derived proteins with α-Gal modifications in the saliva with potential implications in AGS and other disorders and protective capacity against tick infestations and pathogen infection. Future research should focus on the characterization of the function of tick glycoproteins with α-Gal in tick biology and AGS.

Immunomic Investigation of Holocyclotoxins to Produce the First Protective Anti-Venom Vaccine Against the Australian Paralysis Tick, Ixodes holocyclus.

Venom producing animals are ubiquitously disseminated among vertebrates and invertebrates such as fish, snakes, scorpions, spiders, and ticks. Of the ~890 tick species worldwide, 27 have been confirmed to cause paralysis in mammalian hosts. The Australian paralysis tick (Ixodes holocyclus) is the most potent paralyzing tick species known. It is an indigenous three host tick species that secretes potent neurotoxins known as holocyclotoxins (HTs). Holocyclotoxins cause a severe and harmful toxicosis leading to a rapid flaccid paralysis which can result in death of susceptible hosts such as dogs. Antivenins are generally polyclonal antibody treatments developed in sheep, horses or camels to administer following bites from venomous creatures. Currently, the methods to prevent or treat tick paralysis relies upon chemical acaricide preventative treatments or prompt removal of all ticks attached to the host followed by the administration of a commercial tick-antiserum (TAS) respectively. However, these methods have several drawbacks such as poor efficacies, non-standardized dosages, adverse effects and are expensive to administer. Recently the I. holocyclus tick transcriptome from salivary glands and viscera reported a large family of 19 holocyclotoxins at 38-99% peptide sequence identities. A pilot trial demonstrated that correct folding of holocyclotoxins is needed to induce protection from paralysis. The immunogenicity of the holocyclotoxins were measured using commercial tick antiserum selecting HT2, HT4, HT8 and HT11 for inclusion into the novel cocktail vaccine. A further 4 HTs (HT1, HT12, HT14 and HT17) were added to the cocktail vaccine to ensure that the sequence variation among the HT protein family was encompassed in the formulation. A second trial comparing the cocktail of 8 HTs to a placebo group demonstrated complete protection from tick challenge. Here we report the first successful anti-venom vaccine protecting dogs from tick paralysis.

COVID-19 in lung transplant recipients: A multicenter study.

This study describes the clinical presentation, treatment, and outcomes of SARS-CoV-2 infection in lung transplant recipients (LTRs). This is a multicenter, retrospective study of all adult LTRs with confirmed SARS-CoV-2 infection from March 4 until April 28, 2020 in six Spanish reference hospitals for lung transplantation. Clinical and radiological data, treatment characteristics, and outcomes were reviewed. Forty-four cases were identified in that period. The median time from transplantation was 4.2 (interquartile range: 1.11-7.3) years. Chest radiography showed acute parenchymal abnormalities in 32 (73%) cases. Hydroxychloroquine was prescribed in 41 (93%), lopinavir/ritonavir (LPV/r) in 14 (32%), and tocilizumab in 19 (43%) patients. There was a strong interaction between tacrolimus and LPV/r in all cases. Thirty-seven (84%) patients required some degree of respiratory support and/or oxygen therapy, and 13 (30%) were admitted to intermediate or intensive critical care units. Seventeen (39%) patients had died and 20 (45%) had been discharged at the time of the last follow-up. Deceased patients had a worse respiratory status and chest X-ray on admission and presented with higher D-dimer, interleukin-6, and lactate dehydrogenase levels. In this multicenter LTR cohort, SARS-CoV-2 presented with high mortality. Additionally, the severity of disease on presentation predicted subsequent mortality.

Antibacterial and antifungal activity of defensins from the Australian paralysis tick, Ixodes holocyclus.

Tick innate immunity involves humoral and cellular responses. Among the humoral effector molecules in ticks are the defensins which are a family of small peptides with a conserved γ-core motif that is crucial for their antimicrobial activity. Defensin families have been identified in several hard and soft tick species. However, little is known about the presence and antimicrobial activity of defensins from the Australian paralysis tick Ixodes holocyclus. In this study the I. holocyclus transcriptome was searched for the presence of defensins. Unique and non-redundant defensin sequences were identified and designated as holosins 1 - 5. The antimicrobial activity of holosins 2 and 3 and of the predicted γ-cores of holosins 1-4 (HoloTickCores 1-4), was assessed using Gram-negative and Gram-positive bacteria as well as the fungus Fusarium graminearum and the yeast Candida albicans. All holosins had molecular features that are conserved in other tick defensins. Furthermore holosins 2 and 3 were very active against the Gram-positive bacteria Staphylococcus aureus and Listeria grayi. Holosins 2 and 3 were also active against F. graminearum and C. albicans and 5 µM of peptide abrogate the growth of these microorganisms. The activity of the synthetic γ-cores was lower than that of the mature defensins apart from HoloTickCore 2 which had activity comparable to mature holosin 2 against the Gram-negative bacterium Escherichia coli. This study reveals the presence of a multigene defensin family in I. holocyclus with wide antimicrobial activity.

Low genetic diversity of Ehrlichia canis associated with high co-infection rates in Rhipicephalus sanguineus (s.l.).

BACKGROUND: Rhipicephalus sanguineus sensu lato (s.l.) is the most widely distributed ixodid tick and is a vector of major canine and human pathogens. High-throughput technologies have revealed that individual ticks carry a high diversity of pathogens, including bacteria, protozoa and viruses. Currently, it is accepted that co-infections (multiple pathogen species within an individual) are very common in ticks and influence pathogen acquisition and transmission as well as host infection risk. However, little is known on the impact of the genetic diversity of pathogens on the incidence of co-infections. Herein, we studied the frequency of co-infections in R. sanguineus (s.l.) and their association with the genetic diversity of Ehrlichia canis. METHODS: Rhipicephalus sanguineus (s.l.) female ticks (n = 235) were collected from healthy farm dogs in three districts of Pakistan. Microfluidic real-time PCR, a powerful nanotechnology for high-throughput molecular detection of pathogens, was used to test the presence of 25 bacterial and seven parasitic species in individual ticks. The genetic diversity of E. canis was evaluated by characterizing the trp36 gene. RESULTS: A total of 204 ticks were infected with at least one pathogen and 109 co-infected with two (80%) or three (20%) pathogens. Rickettsia massiliae (human pathogen) and E. canis (zoonotic dog pathogen) were the most common pathogens co-infecting (30.4%) ticks. Furthermore, all identified co-infections included R. massiliae and/or E. canis. Multiple correspondence analysis (MCA) revealed that single infections did not show clear regional association whereas some co-infections were restricted to certain geographical regions. The sequence analysis of trp36 in representative samples allowed the identification of three E. canis strains with low genetic diversity, and the strain found in Muzaffargarh district appeared to be more adapted to co-infection with R. massiliae. CONCLUSIONS: Rhipicephalus sanguineus (s.l.) harbors multiple co-infections with human and dog pathogens of zoonotic potential. Findings of this study suggest that genetic diversity of E. canis may favor co-infections with different pathogens.

Report from the 'One Health' 9th Tick and Tick-Borne Pathogen Conference and the 1st Asia-Pacific Rickettsia Conference, Cairns, Australia, 27th August-1st September 2017.

The 9th Tick and Tick-borne Pathogen (TTP9) Conference was held in conjunction with the first Asia Pacific Rickettsia Conference (APRC1) in Cairns, Australia from 27 August until 1 September in 2017. This MDPI Veterinary Sciences Special Issue has been dedicated to selected veterinary science articles from the conference associated with the control of animal diseases in the context of ticks and tick-borne pathogens, including Rickettsia species. The articles presented in this Special Issue include novel developments for the future control of ticks and tick-borne diseases. This editorial describes the meeting content, the plenaries, the TTP awards, the MDPI Veterinary Science Special Issue articles, and serves as a legacy report for TTP9APRC1.

Ischemic Heart Disease during Acute Exacerbations of COPD.

Patients with chronic obstructive pulmonary disease (COPD) have a higher risk of acute cardiovascular events, and around 30% die from cardiovascular diseases. Recent data suggest an increased risk of myocardial infarction in the following days of a severe exacerbation of COPD. Disruption in the balance during the exacerbation with tachycardia, increased inflammation and systemic oxidative stress as well as some other factors may confer an increased risk of subsequent cardiovascular events. A number of investigations may be useful to an early diagnosis, including electrocardiography, imaging techniques and blood test for biomarkers. Some drugs that have changed prognosis in the cardiovascular setting such as cardioselective beta-blockers may be underused in patients with COPD despite its demonstrated benefits. This review focuses on several aspects of exacerbation of COPD and cardiovascular events including epidemiology, possible mechanism, diagnosis and treatment.

Discovery of a novel iflavirus sequence in the eastern paralysis tick Ixodes holocyclus.

Ixodes holocyclus, the eastern paralysis tick, is a significant parasite in Australia in terms of animal and human health. However, very little is known about its virome. In this study, next-generation sequencing of I. holocyclus salivary glands yielded a full-length genome sequence which phylogenetically groups with viruses classified in the Iflaviridae family and shares 45% amino acid similarity with its closest relative Bole hyalomma asiaticum virus 1. The sequence of this virus, provisionally named Ixodes holocyclus iflavirus (IhIV) has been identified in tick populations from northern New South Wales and Queensland, Australia and represents the first virus sequence reported from I. holocyclus.

Comparison of Protein Gut Samples from Rhipicephalus spp. Using a Crude and an Innovative Preparation Method for Proteome Analysis.

Tick populations are controlled through the application of chemical pesticides. However, the rise in chemical resistance has prompted the investigation of other control methods such as the use of tick vaccines. Proteomic analysis provides valuable information about the possible function and localization of proteins, as candidate vaccine proteins are often either secreted or localized on the cell-surface membrane. Progress in the utilization of proteomics for the identification of novel treatment targets has been significant. However, their use in tick-specific investigations is still quite novel, with the continual development of tick-specific methodologies essential. In this study, an innovative sample preparation method was utilized to isolate epithelial cells from tick midguts to identify the membrane-bound proteins. Proteomic analysis was conducted comparing crude and innovative sample preparation methods with 692 and 1242 tick-specific proteins, 108 and 314 surface proteins respectively, isolated from the midguts of semi-engorged Rhipicephalus microplus adult female ticks. This research reports a novel preparation protocol for the analysis of tick midgut proteins which reduces host protein contamination.

Anti-tick vaccines in the omics era.

Tick vaccines have been available for more than 20 years. They are useful and effective control agents when used properly. However, no new products have emerged since the Bm86-based Gavac vaccine was commercialized. Acaricide resistance is a problem with no abatement in sight and anti-tick vaccines are likely to be relied upon even more in the coming years. As human medicine and plant agriculture has embraced the various Omics technologies, the search for anti-tick vaccines would be well served to follow; so that new vaccine antigens and adjuvants might be developed to assist tick control programs. However, the simple outward appearance of ticks and their life cycle belies the complexity of their genomes which are computationally challenging to sequence and annotate. We review various Omics research efforts in light of research on anti-tick vaccines.

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus.

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2-HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.

Extracellular expression of the HT1 neurotoxin from the Australian paralysis tick in two Saccharomyces cerevisiae strains.

Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, and single-chain variable IgG fragments, however, the use of SDL for the expression and purification of proteins is gaining interest. Prokaryote SDL express proteins within the periplasm, limiting the application of common screening techniques, such as ELISA and FACS, to assess the viability of recombinant toxin before purification. A previous attempt to express a functional holocyclotoxin-1 (HT1) from the Australian paralysis tick (Ixodes holocyclus) using a prokaryotic system was unsuccessful. In this study, the coding sequence (CDS) of HT1 was cloned into the pYD1 plasmid and transformed by electroporation into IMTV014 and EBY100 yeast cell lines. Post induction, recombinant HT1 was identified on the cell surface of IMTV014/ht1 and EBY100/ht1 transformants by FACS, Western blot, and ELISA utilizing dog anti-paralysis tick IgG. The recombinant HT1 was purified, and functionality confirmed by an in vitro synaptosome-binding assay. This research reports for the first time the extracellular expression and display of a functional HT1 on the surface of the S. cerevisiae. It also provides evidence that yeast display libraries provide a viable technology to produce recombinant toxins, and their screening using high throughput methodologies such as FACS.

Ticks from diverse genera encode chemokine-inhibitory evasin proteins.

To prolong residence on their hosts, ticks secrete many salivary factors that target host defense molecules. In particular, the tick Rhipicephalus sanguineus has been shown to produce three salivary glycoproteins named "evasins," which bind to host chemokines, thereby inhibiting the recruitment of leukocytes to the location of the tick bite. Using sequence similarity searches, we have identified 257 new putative evasin sequences encoded by the genomes or salivary or visceral transcriptomes of numerous hard ticks, spanning the genera Rhipicephalus, Amblyomma, and Ixodes of the Ixodidae family. Nine representative sequences were successfully expressed in Escherichia coli, and eight of the nine candidates exhibited high-affinity binding to human chemokines. Sequence alignments enabled classification of the evasins into two subfamilies: C8 evasins share a conserved set of eight Cys residues (four disulfide bonds), whereas C6 evasins have only three of these disulfide bonds. Most of the identified sequences contain predicted secretion leader sequences, N-linked glycosylation sites, and a putative site of tyrosine sulfation. We conclude that chemokine-binding evasin proteins are widely expressed among tick species of the Ixodidae family, are likely to play important roles in subverting host defenses, and constitute a valuable pool of anti-inflammatory proteins for potential future therapeutic applications.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells.

Rhipicephalus microplus - the cattle tick - is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Risk Factors of Poor Outcomes after Admission for a COPD Exacerbation: Multivariate Logistic Predictive Models.

The aim of this study was to identify a multivariate model to predict poor outcomes after admission for exacerbation of chronic obstructive pulmonary disease (COPD).  We performed a multicenter, observational, prospective study. Patients admitted to hospital for COPD were followed up for 3 months. Relevant clinical variables at admission were selected. For each variable, the best cut-offs for the risk of poor outcome were identified using receiver operating characteristic (ROC) curves. Finally, a stepwise logistic regression model was performed. A total of 106 patients with a mean age of 71.1 (9.8) years were included. The mean maximum expiratory volume in the first second (FEV1)(%) was 45.2%, and the mean COPD assessment test (CAT) score at admission was 24.8 (7.1). At 3 months, 39 (36.8%) patients demonstrated poor outcomes: death (2.8%), readmission (20.8%) or new exacerbation (13.2%). Variables included in the logistic model were: previous hospital admission, FEV1 < 45%, Charlson ≥ 3, hemoglobin (Hb)<13 g/L, PCO2 ≥ 46 mmHg, fibrinogen ≥ 554 g/L, C-reactive protein (CRP)≥45 mg/L, leukocyte count < 9810 × 109/L, purulent sputum, long-term oxygen therapy (LTOT) and CAT ≥ 31 at admission. The final model showed that Hb < 13 g/L (OR = 2.46, 95%CI 1.09-6.36), CRP ≥ 45 mg/L (OR = 2.91, 95%CI: 1.11-7.49) and LTOT (3.07, 95%CI: 1.07-8.82) increased the probability of poor outcome up to 82.4%. Adding a CAT ≥ 31 at admission increased the probability to 91.6% (AUC = 0.75; p = 0.001). Up to 36.8% of COPD patients had a poor outcome within 3 months after hospital discharge, with low hemoglobin and high CRP levels being the risk factors for poor outcome. A high CAT at admission increased the predictive value of the model.

Bronchodilator reliever use and its association with the economic and humanistic burden of COPD: a propensity-matched study.

BACKGROUND AND AIMS: Short-acting bronchodilators are normally used as supplemental relief medication for breakthrough symptoms in COPD patients. The objective of this cross-sectional study was to assess if more frequent vs infrequent use of relief medication in maintenance-treated COPD patients, split by the severity dyspnea, was associated with an increase in the overall disease burden. METHODS: A population-based cross-sectional survey (Adelphi DSP) was conducted among patients with COPD in five European countries. Information was collected on demographic and clinical characteristics, reliever inhaler use, dyspnea (mMRC), health status (CAT, EQ-5D), sleep quality (JSEQ) and healthcare resource use including moderate-severe COPD exacerbations, physician visits, COPD medications and other COPD related resources. The humanistic and economic burden was compared between patients with infrequent reliever use (<1 occasion/week) and more frequent use (≥ 1 occasion/week). The association between increased reliever use and economic burden was also examined after matching patients based on propensity-scores balancing demographic and disease burden characteristics. RESULTS: Among the 1373 COPD patients prescribed a reliever inhaler, 29% reported using reliever medication ≥1 occasion/week. In the unmatched cohort, more frequent reliever use (n = 377) compared to infrequent use (n = 996) was linked to poorer health status (CAT: 25.7 vs 20.0; p < .0001; EQ-5D-3L: 0.63 vs 0.82; p < .0001) and poorer sleep quality (JSEQ: 8.6 vs 4.6 units; p < .0001). More frequent reliever use was also associated with higher annual rates of moderate/severe exacerbations (1.6 vs 1.0 events/year; p < .0001) and respiratory specialist visits (2.8 vs 2.2 events/year; p = .0001). In the propensity-score matched population, more frequent reliever use was also associated with significantly higher annual costs for COPD management (€5,034 vs €3,705, p = .0327) compared to patients with infrequent reliever use. CONCLUSION: In moderate-to-severe COPD, more frequent reliever use is associated with increased exacerbation risk and increased management costs.

Tick holocyclotoxins trigger host paralysis by presynaptic inhibition.

Ticks are important vectors of pathogens and secreted neurotoxins with approximately 69 out of 692 tick species having the ability to induce severe toxicoses in their hosts. The Australian paralysis tick (Ixodes holocyclus) is known to be one of the most virulent tick species producing a flaccid paralysis and fatalities caused by a family of neurotoxins known as holocyclotoxins (HTs). The paralysis mechanism of these toxins is temperature dependent and is thought to involve inhibition of acetylcholine levels at the neuromuscular junction. However, the target and mechanism of this inhibition remain uncharacterised. Here, we report that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracellular calcium. Previous study was conducted using extracts from tick salivary glands, while the present study is the first to use pure toxins from I. holocyclus. Our findings provide greater insight into the mechanisms by which these toxins act to induce paralysis.