Wayne L. Hynes

Identification of a Defensin from the Hemolymph of the American Dog Tick, Dermacentor variabilis

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.

Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: Immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae)

Abstract The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in >50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.

Molecular characterization and related aspects of the innate immune response in ticks.

Compared to insects, little is known about innate immunity in ticks. This chapter addresses the molecular processes that recognize non-self and the cellular and molecular processes mobilized to phagocytose, engulf, inhibit or kill invaders. We discuss the receptors that recognize pathogen associated molecular patterns (PAMPs) and the putative up-regulation of regulatory cascades that lead, ultimately, to cellular or molecular responses. We describe the molecular events that activate the cellular processes and the array of humoral factors that are mobilized against invading organisms, including antimicrobial peptides, proteases and protease inhibitors, lectins, coagulation factors and others. Special attention is directed to the antimicrobial activity of the midgut, the initial site of contact for microbes ingested with the blood. Blood feeding and digestion alone up-regulates an impressive array of proteins, e.g. oxidative stress reducing proteins, lectins, protease inhibitors, proteases, hydrolases, protein/lipid binding agents. Finally, we compare the innate immune responses of ticks with insects and other invertebrates and note deficiencies in our knowledge tick innate immunity.

The Plasma Pencil: A Source of Hypersonic Cold Plasma Bullets for Biomedical Applications

Cold plasma jets and their interaction with materials and media have recently been very active research topics. The low gas temperature enables applications in various fields, including medicine. In bioapplications, for example, it involves radiation and reactive species created by the plasma jet to modify or kill prokaryotic cells (bacteria) or eukaryotic cells (mammalian cells). In this paper, we present photographs of a pulsed cold plasma plume/jet that turned out to be a series of high-velocity plasma ldquobulletsrdquo or ldquopackets.rdquo These bullets travel at hypersonic speeds, and these transport with them chemically reactive species and deliver them to the surface of samples under treatment.

PCR amplification of streptococcal DNA using crude cell lysates

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.

Response of the Tick Dermacentor variabilis (Acari: Ixodidae) to Hemocoelic Inoculation of Borrelia burgdorferi (Spirochetales)

Abstract When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within <24 h. Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA. The hemocyte population increased ≈6 times within the first 6 h after inoculation compared with the uninoculated controls. In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes. Borreliacidal activity was demonstrated with cell-free hemolymph from D. variabilis. In vitro antimicrobial assays using hemolymph from borrelia-challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h. Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (≈15 kDa) (22% increase) and the induction of a ≈5.8 kDa peptide in the B. burgdorferi-challenged ticks. In contrast with the nonvector borne Bacillus subtilis, D. variabilis presented a rapid and robust response to challenge with cultured B. burgdorferi spirochetes and lead to their early elimination. The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed.

A defensin-like gene expressed in the black-legged tick, Ixodes scapularis

Abstract.  The black‐legged tick Ixodes scapularis Linnaeus (Acari: Ixodidae) is an important vector of microbial pathogens. Knowledge of the ticks innate immune response, particularly defensin and other antimicrobial peptides, is important for understanding how microbes survive in this tick. A defensin gene (slnA) from I. scapularis was obtained by reverse transcription–polymerase chain reaction (RT–PCR) using mRNA extracted from tissues of female ticks. RT–PCR indicated the gene was expressed in the midgut, haemocytes, and fat‐body, although no evidence of a peptide was found. Sequencing a cloned cDNA fragment revealed a 225 bp open reading frame encoding a 74 amino acid pre‐prodefensin, including the putative 38 amino acid mature peptide. Similarity between the defensin amino acid sequences of I. scapularis and Dermacentor variabilis (Say) (Acari: Ixodidae) was 62.2% for the pre‐prodefensin region; for the mature defensins from these two species the similarity was 78.9%, with the six cysteine residues being located in the same relative position. PCR amplification and sequencing of chromosomal DNA suggests that slnA, along with vsnA, the defensin gene from D. variabilis, does not contain any introns. This is in contrast to the defensins described for the soft tick, Ornithodoros moubata (sensu Walton) (Acari: Argasidae). The role of defensin in the innate immune response of I. scapularis following microbial invasions is discussed.

Rickettsia parkeri in gulf coast ticks, southeastern Virginia, USA.

We report evidence that Amblyomma maculatum tick populations are well established in southeastern Virginia. We found that 43.1% of the adult Gulf Coast ticks collected in the summer of 2010 carried Rickettsia parkeri, suggesting that persons living in or visiting southeastern Virginia are at risk for infection with this pathogen.

Virulence factors of the group A streptococci and genes that regulate their expression.

Group A streptococci produce a wide variety of extracellular (cell-associated and released) virulence factors. The function of these factors varies and includes roles in adhesion, spreading, tissue destruction, immune system evasion, and cell toxicity. How these factors are regulated with regard to one another is important for this organism s ability to bring about the variety of diseases this microbe is capable of causing. Therefore, along with the multitude of virulence factors, there are a number of regulatory systems that regulate, either directly or indirectly, the production of these factors and therefore influence the pathogenesis of group A streptococcal infections.

An arthropod defensin expressed by the hemocytes of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

Both soluble and cell-mediated components are involved in the innate immune response of arthropods. Injection of Borrelia burgdorferi, the Lyme disease agent, results in the secretion of defensin into the hemolymph of the ixodid tick, Dermacentor variabilis. The presence of the peptide is observed as early as 15 min post-challenge and remains present through 18 h post-challenge. As observed in insects and soft ticks, the transcript for defensin is detected as early as 1 h post-challenge in D. variabilis. RT-PCR resulted in an amplicon of 624 bp with a 225 bp region that translates to a 74 amino acid preprodefensin. The defensin encoding region was amplified, cloned and sequenced from the hemocytes. It appears as though defensin is stored in the granulocytes of the hemolymph and secreted into the hemolymph upon bacterial insult. The role of defensin as a contributing factor in determining vector competency is discussed.