Passive collection of ticks in New Hampshire reveals species-specific patterns of distribution and activity

Ticks and tick-borne diseases are increasing in the United States, including New Hampshire (NH). We report on the findings of an ongoing free crowdsourcing program spanning four years within NH. The date of tick’s submission was recorded along with species, sex, stage, location they were collected (translated into latitude and longitude), the activity the individual was doing when the tick was found, and host species. A total of 14,252 ticks belonging to subclass Acari, family Ixodidae and genera Ixodes, Dermacentor, Amblyomma, and Haemaphysalis was recorded from the period 2018–2021 throughout NH. A total of 2,787 Ixodes scapularis and 1,041 Dermacentor variabilis, were tested for the presence of Borrelia sp. (Spirochaetales: Spirochaetaceae), B. burgdorferi sensu lato, B. miyamotoi, B. mayonii, Babesia microti (Piroplasmida: Babesiidae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Francisella tularensis (Thiotrichales: Francisellaceae), and Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by PCR. For the I. scapularis ticks tested, the pathogen prevalence was 37% B. burgdorferi s.l. 1% B. miyamotoi, 6% A. phagocytophilum, and 5% Ba. microti. Only one D. variabilis resulted positive to F. tularensis. We created state-wide maps informing the differences of ticks as detailed by administrative divisions. Dermacentor variabilis peaked in June and I. scapularis peaked in May and October. The most reported activity by people with tick encounters was while walking/hiking, and the least was biking. Using the reported distribution of both species of ticks, we modeled their climate suitability in the target territory. In NH, I. scapularis and D. variabilis have distinct patterns of emergence, abundance, and distribution. Tick prevention is important especially during April–August when both tick species are abundant and active

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

MHC class II molecules influence antigen-specific CD4(+) T-lymphocyte responses primed by immunization and infection. CD4(+) T-cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale, and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2, and VirB10, candidates for inclusion in a multi-epitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2, and VirB10 T-cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes, defined by DRB3, DQA, and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T-cell proliferation assays with autologous antigen presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2, and seven representing seven or more epitopes in VirB10. Of eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition,three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2, and VirB10 peptide epitopes justify their testing as a multi-epitope vaccine against A. marginale

Immunogenicity and Linked Recognition of Anaplasma marginale Type IV Secretion System Proteins

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane (OM) that induces protection from infection and disease. However proteins that confer protective immunity and whether the protection requires linked T-cell and immunoglobulin G epitopes and/or interacting proteins are not known. Our goal was to target the conserved type IV secretion system (T4SS) to identify immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which B cells are optimally activated by helper T cells responding to the same or physically associated antigen. The T4SS is a membrane complex within many bacterial pathogens secreting virulence factors and promoting host cell invasion and intracellular survival. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11 and VirD4 were screened for their ability to induce IgG and stimulate CD4+ T cells from OM vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T cell responses in the majority of cattle, although animals with major histocompatibility complex class II (MHC class II) DRB3 RFLP types 3/16, 8/23, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals-specific IgG production may result from T cell help provided by responses to an interacting protein partner(s). Interacting partners were determined by far western blotting and confirmed by immunoprecipitation assays, and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. Since, MHC class II molecules influence antigen-specific CD4+ T-lymphocyte responses, specific bovine leukocyte antigens required for presentation of peptides from VirB9-1, VirB9-2, and VirB10 were also determined. Overlapping peptides spanning each protein were tested in T cell assays with autologous antigen presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified, of which, four DRA/DRB3 presented fifteen peptides, four DQA/DQB presented seven peptides, and three functional mixed isotype (DQA/DRB3) were identified. The immunogenicity, interactions, and broad MHC class II presentation of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked multi-epitope vaccine against A. marginale

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended

Anaplasma marginale Type IV Secretion System Proteins VirB2, VirB7, VirB11, and VirD4 Are Immunogenic Components of a Protective Bacterial Membrane Vaccine

Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serological responses of OM vaccinees to the OM proteome previously identified over 20 antigenic proteins, including three type IV secretion system (T4SS) proteins, VirB9-1, VirB9-2, and VirB10. Subsequent studies showed that these three proteins also stimulated CD4 + T-cell responses in OM vaccinees. The T4SS, composed of a complex of proteins spanning the inner and outer membranes of certain bacteria, is an important virulence factor but is relatively unexplored as a vaccine target. The goal of this study was to determine if additional T4SS proteins are immunogenic for animals immunized with the protective OM fraction of A. marginale. T4SS proteins expressed by in vitro transcription and translation were screened for stimulating proliferation of T cells from OM vaccinees, and immunogenic proteins were expressed as recombinant proteins in Escherichia coli and their immunogenicity was verified. VirB2, a putative VirB7, VirB11, and VirD4 were immunogenic for OM vaccinees expressing several common major histocompatibility complex (MHC) class II haplotypes. VirB2 is encoded by multiple genes that share a conserved central region, and epitope mapping revealed T-cell epitopes in this region. The discovery of novel immunogenic T4SS proteins recognized by outbred individuals with common MHC haplotypes further justifies evaluating the T4SS as a potential vaccine candidate for pathogenic bacteria

Association and Evidence for Linked Recognition of Type IV Secretion System Proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4 + T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale