Daniel E. Sonenshine

Electron microscopic investigation of the effects of diabetes mellitus on the Achilles tendon

Fine structural changes in the Achilles tendons of patients with long-term diabetes mellitus were investigated. All patients had clinical and electrophysiological evidence of diabetic neuropathy and had ulceration and/or Charcot neuroarthropathy. Several differences between tendons of diabetic (n = 12) and nondiabetic (n = 5) individuals were observed by electron microscopy. In diabetics, these differences included increased packing density of collagen fibrils, decreases in fibrillar diameter, and abnormal fibril morphology. In one diabetic patient, individual collagen fibrils were tightly apposed so that many areas of tendon appeared as a single mass of closely adhering fibrillae. In addition, foci in which collagen fibrils appeared twisted, curved, overlapping and otherwise highly disorganized were common in specimens from most patients (11 of 12). These morphologic abnormalities in the Achilles tendons of diabetics appear to reflect a poorly known process of structural reorganization that may be the result of nonenzymatic glycation expressed over many years. Such structural changes could contribute to the tightening of the Achilles tendor a phenomenon consistent with clinical observations of extreme shortening of the Achilles tendon-gastrocnemius-soleus complex common in advanced diabetic neuropaths. In patients with diabetic neuropathy, tendon shortening causes severe equinus that may precipitate serious ulceration, stress fractures, and Charcot collapse of the foot. However, in nondiabetics, the fine structure of the Achilles tendon appears normal, consistent with the finding that the ultrastructural changes result from diabetes rather than neuropathy.

Rickettsial Infection in Dermacentor variabilis (Acari: Ixodidae) Inhibits Transovarial Transmission of a Second Rickettsia

Abstract This study examined the ability of ticks to maintain multiple species of spotted fever group rickettsiae via transovarial transmission. Using a capillary feeding method, previously established Rickettsia montana- and Rickettsia rhipicephali-infected cohorts of Dermacentor variabilis (Say) were exposed to R. rhipicephali and R. montana, respectively, in two reciprocal challenge experiments. Eggs collected from individual females, for two successive generations, of each cohort were assessed for rickettsial infection by polymerase chain reaction for each challenge experiment. Assessment of the eggs from challenged ticks identified that both R. montana- and R. rhipicephali-infected ticks were refractory to their respective challenge rickettsiae. The prechallenged infection rate for both F1 and F2 generations (100%) of the R. montana-infected cohort was resistant to transovarial transmission of the second rickettsia species, and only R. montana was detected in the eggs of F1 = (50%) and F2 = (74%) challenged females. The R. rhipicephali-infected cohort maintained a lower level of infection (20%) in the population and did not transovarially transmit the challenge species, however, detectable levels of infection were lost after the first generation. Second-generation ticks, no longer infected with R. rhipicephali, became susceptible to infection with R. montana and female ticks (≈4%) were able to transmit R. montana to their progeny. The resistance of the ovaries to co-infection and apparent host-specific nature of infection suggests that rickettsial infection of tick ovaries may alter the molecular expression of the oocytes so as to preclude secondary infection with other rickettsiae.

Exploring the mialome of ticks: an annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae)

BackgroundTicks are obligate blood feeders. The midgut is the first major region of the body where blood and microbes ingested with the blood meal come in contact with the ticks internal tissues. Little is known about protein expression in the digestive tract of ticks. In this study, for analysis of global gene expression during tick attachment and feeding, we generated and sequenced 1,679 random transcripts (ESTs) from cDNA libraries from the midguts of female ticks at varying stages of feeding.ResultsSequence analysis of the 1,679 ESTs resulted in the identification of 835 distinct transcripts, from these, a total of 82 transcripts were identified as proteins putatively directly involved in blood meal digestion, including enzymes involved in oxidative stress reduction/antimicrobial activity/detoxification, peptidase inhibitors, protein digestion (cysteine-, aspartic-, serine-, and metallo-peptidases), cell, protein and lipid binding including mucins and iron/heme metabolism and transport. A lectin-like protein with a high match to lectins in other tick species, allergen-like proteins and surface antigens important in pathogen recognition and/or antimicrobial activity were also found. Furthermore, midguts collected from the 6-day-fed ticks expressed twice as many transcripts involved in bloodmeal processing as midguts from unfed/2-day-fed ticks.ConclusionThis tissue-specific transcriptome analysis provides an opportunity to examine the global expression of transcripts in the tick midgut and to compare the gut response to host attachment versus blood feeding and digestion. In contrast to those in salivary glands of other Ixodid ticks, most proteins in the D. variabilis midgut cDNA library were intracellular. Of the total ESTs associated with a function, an unusually large number of transcripts were associated with peptidases, cell, lipid and protein binding, and oxidative stress or detoxification. Presumably, this is consistent with their role in intracellular processing of the blood meal and response to microbial infections. The presence of many proteins with similar functions is consistent with the hypothesis that gene duplication contributed to the successful adaptation of ticks to hematophagy. Furthermore, these transcripts may be useful to scientists investigating the role of the tick midgut in blood-meal digestion, antimicrobial activity or the transmission of tick-borne pathogens.

Passage of host serum components, including antibody, across the digestive tract of Dermacentor variabilis (Say).

(14 wk postinfection), the death rates were 36 and 40% in group A and B respectively, compared to 8% in the noninfected control (Table I). Metacercarial production was high and continued for at least 50 days after the prepatent period (Table I). The procedure described appears to be valuable and useful for the cultivation of k postinfection), the d ath rates w re 36 40% in group A and B resp ctively, comed to 8% in the no infect d control (Table aquatic species of Lymnaea and for mass producti n of metacercariae of Fasciola gigantica. The m thod is simple and time saving and makes possible mass production of snails. The growth rate of juvenile snails was fast and the mortality of both noninfected and infected snails was low. ic species of Lymnaea nd for mass proio of metacercariae of Fasciola gi antie ethod is simple and time saving and s possible mas production of snails. The t rate of juvenile snails was fast and the

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.

Infection and Transovarial Transmission of Rickettsiae in Dermacentor variabilis Ticks Acquired by Artificial Feeding

In this study we examined the efficiency of an in vitro feeding technique using glass microcapillaries as a method of establishing rickettsiae-infected lines of ticks. To quantify the volume ingested by ticks during microcapillary feeding, the incorporation of radiolabeled amino acids in tick gut and hemolymph was calculated. Fifteen of 18 ticks consumed between 0.06 and 6.77 microl. However, ingestion of fluid was not correlated to weight gain during capillary feeding. Uninfected and partially fed laboratory-reared female Dermacentor variabilis ticks were exposed to either Rickettsia montana- or Rickettsia rhipicephali-infected Vero cells via microcapillary tubes, returned to rabbit hosts, and allowed to feed to repletion. All tissues collected from ticks allowed to feed overnight on rickettsiae-infected fluids were found to be infected when examined by IFA. When rickettsiae-infected and uninfected capillary-fed ticks were allowed to feed to repletion and lay eggs, no significant differences in mean engorgement weight or fecundity was observed. When we assessed the efficiency of transovarial transmission of rickettsiae by ticks that imbibed rickettsiae-infected cells by polymerase chain reaction (PCR) and IFA, infection was detected by PCR in the eggs from 85% of the ticks exposed to R. montana and 69% of the ticks exposed to R. rhipicephali. Rickettsial genes were not amplified in samples of the uninfected controls. Examination by IFA of egg samples from females exposed to rickettsiae-infected cells identified rickettsiae in 100% of the samples tested, while the uninfected controls were negative.

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.