Experimental colonization and persistence of Francisella tularensis in Dermacentor variabilis and Amblyomma americanum

Francisella tularensis causes tick-transmitted tularemia epizootics in rodent and rabbit hosts and incidental infections in humans. The objective of this study was to develop a F. tularensis tick colonization model for elucidating the salient features of its biology in tick vectors. The first two studies reported herein describe the systematic study of F. tularensis ssp. holarctica strain LVS colonization in the tick vectors Dermacentor variabilis and Amblyomma americanum as well as describing the capillary feeding (CF) method of colonizing the different stages of these ticks. Post capillary feeding (PCF), level of colonization was determined by CFU determinations of tick minceate. Transmission of F. tularensis from larvae to nymph was seen in both tick species, but only A. americanum nymphs maintained F. tularensis for longer periods of time (168 days PCF). Transstadial transmission from nymph to adult was also demonstrated in both the tick species, but only D. variabilis ticks maintained F. tularensis colonization after molting from nymphs to adults. For CF adults, F. tularensis initial colonization of the gut disseminated to hemolymph and salivary glands in three weeks and 24 h PCF for D. variabilis and A. americanum respectively. Colonization of adult D. variabilis ticks persisted up to 6 months PCF (longest time point in study). Transovarial transmission was not observed in either tick species. However, colonized D. variabilis adult females transferred F. tularensis to surface layer of eggs but not to hatched larvae. The extrinsic incubation period (time to secretion in saliva), determined by intra-hemocel injection of F. tularensis in D. variabilis and A. americanum was 4 and 2 days, respectively. The ID50 for mice for intraperitoneal injection of F. tularensis from adult D. variabilis salivary glands versus laboratory culture F. tularensis were 2 and 43 CFU, respectively. Both tick species appear competent as experimental vectors for F. tularensis with D. variabilis adults and A. americanum nymphs better adapted for long term persistence of F. tularensis. The role of these ticks as a possible inter-epizootic reservoir of F. tularensis is also discussed. The final chapter of this dissertation addresses the possible role of chitin as a nutrient source for F. tularensis in the tick life cycle

Application of Bi-cell Surface Plasmon Resonance for the Detection of Aptamer Mediated Thrombin Capture in Serum

Using the bi-cell surface plasmon resonance instrument, we were able to detect thrombin in the concentration range of 500 femtomoles to 2 picomoles with thrombin aptamer as the capture probe. The lowest limit of detection was 250 femtomoles. The affinity of the aptamer for prothrombin was also investigated, which was detected at 2 picomoles. Using serum samples, 1 picomole of thrombin was detected. We proved that an aptamer could be used as a capture ligand in bi-cell SPR instrument. This model could be used in a clinical setting for the real time detection of diagnostically relevant biomolecules.Veterinary Pathobiolog

Subolesin expression in response to pathogen infection in ticks

<p>Abstract</p> <p>Background</p> <p>Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, <it>Anaplasma marginale</it>. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with <it>Anaplasma</it>, <it>Ehrlichia</it>, <it>Rickettsia</it>, <it>Babesia </it>or <it>Theileria </it>spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in <it>Dermacentor variabilis </it>males exposed to various pathogens by capillary feeding (CF).</p> <p>Results</p> <p>Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with <it>A. marginale</it>. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in <it>D. variabilis </it>infected with <it>A. marginale </it>and other tick-borne pathogens resulted in lower infection levels, while infection with <it>Francisella tularensis </it>increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in <it>D. variabilis </it>exposed to <it>Escherichia coli</it>, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism.</p> <p>Conclusions</p> <p>Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.</p

Biology of Francisella tularensis Subspecies holarctica Live Vaccine Strain in the Tick Vector Dermacentor variabilis

Background: The c-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis. Methodology/Principal Findings: Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.860.8610 1 and 1.160.03610 3 CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42 % of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50 % of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then t

Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis.

The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 10(4) CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 10(3) to 10(8) CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 10(1) to 10(5) CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger mammals which are not involved in maintenance of sylvatic tularemia

Apparent prevalence and selected risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci in bulk tank milk of dairy herds in Indiana, Ohio, and Michigan

The purpose of this study was to determine the apparent prevalence and risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci (NASM) in bulk tank milk (BTM) obtained from 300 dairy farms that belong to a cooperative collecting milk from Indiana, Michigan, and Ohio. Dairy field personnel recorded information about selected farm level risk factors and collected and froze BTM samples (n = 300) that were sent to Michigan State University researchers. Milk samples were thawed at room temperature and pre-enriched by adding 1 to 4 mL of Mueller-Hinton broth supplemented with 6.5% NaCl and incubated at 37°C for 24 h. Subsequently, 10 µL was plated on mannitol salt agar and Mueller-Hinton agar supplemented with 2.5% NaCl containing 2 mg/L oxacillin and 20 mg/L aztreonam. Colonies that grew on the selective media were subcultured on blood agar and identified using MALDI-TOF mass spectrometry. Phenotypic methicillin resistance was tested using cefoxitin disk diffusion. Conventional PCR was used to detect mecA and mecC in phenotypically resistant isolates. Of 550 isolates that were obtained from mannitol salt agar plates and 10 isolates from Mueller-Hinton agar plates, 16 species of NASM accounted for 84% of staphylococci, while S. aureus accounted for the remaining 16%. Among S. aureus, 4 isolates from 4 farms (1.3%) demonstrated phenotypic resistance to methicillin resistance but none carried mecA or mecC genes. Among NASM, 45 isolates from 40 farms (13.3%) demonstrated phenotypic resistance to methicillin. However, only 13 NASM isolates (7 Mammaliicoccus sciuri, 2 Staphylococcus haemolyticus, 1 Mammaliicoccus fleuretti, 1 Staphylococcus epidermidis, 1 Staphylococcus saprophyticus, and 1 Staphylococcus hyicus) from 13 farms were positive for mecA, whereas all were negative for mecC. Thus, the prevalence of mecA-positive NASM in BTM was 4.3%. Based on molecular results, this study demonstrated a low prevalence of methicillin resistance NASM from BTM samples collected from farms in the Upper Midwest. Dairy farms that contained ≤200 lactating cows and had swine located on the farm had a higher prevalence of methicillin-resistant NASM than smaller farms that did not contain swine

LVS colonization of adult <i>A</i>. <i>americanum</i>.

<p>The filled circles are capillary tube fed adults. The calculated mean CFU/tick for colonized unfed adults for each time point is represented by the horizontal line. For each time point, n was 11; for day 168, n was 29.</p

Tissue dissemination of LVS in adult <i>A</i>. <i>americanum</i> post inoculation.

<p>Solid black bar—gut, white bar with dots–hemolymph, white bar with diagonal lines—salivary glands, and white bars with cross marks–saliva (CFU/μl of saliva). For each time point, n was 5. Error bars indicate standard deviation. For day 2 post-capillary tube feeding salivary glands were not collected.</p

Quantum and duration of colonization and transstadial transmission of LVS experimental design.

<p>Larvae, nymphs and adults <i>A</i>. <i>americanum</i> in batches of various sizes were capillary fed tick meal containing LVS. Ingestion of capillary fed tick meal was determined for nymph and adult ticks by post-feeding weight gains of ≥0.3mg. Nymphs and adults showing <0.3mg weight gains were excluded from further analysis. The overall rate of ticks positive for LVS for each tick life cycle stage capillary tube fed LVS is given as total number of ticks which were positive for LVS (+LVS)/total number of ticks tested.</p