全沟硬蜱围食膜初步观察研究

本工作用光镜和电镜技术对全沟硬蜱的围食膜进行了初步观察研究。结果表明，饥饿幼虫、若虫、成虫以及吸血期雄性成虫不具围食膜。吸血期幼虫、若虫及雌性成虫最晚分别于吸血开始后20、20及18小时出现了单层结构的围食膜；缓慢吸血期围食膜的厚度持续增加，而在快速吸血期其厚度却会显著减小。饱血后发育期围食膜不断增厚，愈来愈拱曲，与肠壁的距离不断扩大，并形成多层结构；至少分别于饱血后12、25及11天，仍能在幼虫、若虫及雌性成虫肠腔观察到完好状态的围食膜。全沟硬蜱是我国北方地区多种血液传播病原体的重要媒介，其围食膜的发现及围食膜形态变化的初步描述，对进一步认识这些病原体在全沟硬蜱体内迁移扩散规律、发育动态以及向宿主的传播途径，可提供有益的启示。     

蜱传性疾病--莱姆病螺旋体研究进展

莱姆病是一种蜱传性人兽共患病,在我国北方各省广泛流行,其病原体为伯氏疏螺旋体。莱姆病可使皮肤、心脏、关节和神经系统等多种组织器官受损。本文综述了莱姆病螺旋体的形态、种下分类、生化特征、传播媒介和贮存宿主等方面的研究进展"     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Proteomic profiling of plasma-derived small extracellular vesicles: a novel tool for understanding the systemic effects of tick burden in cattle

Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.     

Transmission of Theileriosis in the Traditional Farming Sector in the Southern Province of Zambia during 1995–1996

The incidence of first contact with the protozoan Theileria parva was determined in three traditional cattle herds in the Southern Province of Zambia in 1995 and 1996. The majority of first contacts occurred during the dry season in June, July and August, at a time of nymphal activity and in the absence of Rhipicephalus appendiculatus adults, indicating that larva to nymph transmission plays a more prominent role than nymph to adult transmission under the prevailing conditions.     

Detection of Invasive Borrelia burgdorferi Strains in North‐Eastern Piedmont,Italy

Following reports of human cases of Lyme borreliosis from the Ossola Valley, a mountainous area of Piemonte, north‐western Italy, the abundance and altitudinal distribution of ticks, and infection of these vectors with Borrelia burgdorferi sensu lato were evaluated. A total of 1662 host‐seeking Ixodes ricinus were collected by dragging from April to September 2011 at locations between 400 and 1450 m above sea level. Additional 104 I. ricinus were collected from 35 hunted wild animals (4 chamois, 8 roe deer, 23 red deer). Tick density, expressed as the number of ticks per 100 m², resulted highly variable among different areas, ranging from 0 to 105 larvae and from 0 to 22 nymphs. A sample of 352 ticks (327 from dragging and 25 from wild animals) was screened by a PCR assay targeting a fragment of the 16S rRNA gene of B. burgdorferi s.l. Positive samples were confirmed with a PCR assay specific for the 5S‐23S rRNA intergenic spacer region and sequenced. Four genospecies were found: B. afzelii (prevalence 4.0%), B. lusitaniae (4.0%), B. garinii (1.5%) and B. valaisiana (0.3%). Phylogenetic analysis based on the ospC gene showed that most of the Borrelia strains from pathogenic genospecies had the potential for human infection and for invasion of secondary body sites.     

New method using quantitative PCR to follow the tick blood meal and to assess the anti-feeding effect of topical acaricide against Rhipicephalus sanguineus on dogs

A 28-day study was conducted to assess the dynamic of blood feeding by Rhipicephalus sanguineus ticks on dogs treated or not with a novel topical combination of fipronil, amitraz and (S)-methoprene. Dogs were infested weekly through exposure to ticks in crates for 4 h. Ticks were then counted in the crates at 2 h and 4 h post dog exposure. Ticks were also counted and removed from the dogs at 2 h, 4 h, 6 h, 12 h and 24 h post tick exposure. The inhibition of blood feeding was assessed by both tick quantification and designing and performing a quantitative PCR (qPCR) to detect the canine hydroxymethylbilane synthase (HMBS) gene in ticks. The percentage of repellency sensu lato based on the ticks collected in crates at 2 h varied from 4.7% at day 28 to 48.3% at day 7. The immediate mortality rate of the ticks expelled at 2 h varied from 1.5% at day 21 to 31.7% at day 7. The efficacy calculation showed that the acaricidal combination started to kill ticks in as little as 2 h. The average efficacy reached 90.0% at 12 h post crate challenges and 100% at 24 h post exposure in crates. The inclusion of an internal amplification control was used to ensure that no significant template-derived PCR inhibition (≤6.2%) affected the overall results. The reduction of blood feeding was significant at 4 h (>80.0%) and >99.0% at 24 h post tick exposure in the crate. The high repellency rate and the lethal efficacy of CERTIFECT^® resulted in significantly fewer live attached ticks, consequently reducing blood intake and fluid exchanges.     

Effect of new ethyl and methyl carbamates on Rhipicephalus microplus larvae and adult ticks resistant to conventional ixodicides

The effects of six new synthetic carbamates on fully engorged females of four Rhipicephalus microplus strains (one reference strain susceptible to conventional ixodicides, two strains multiresistant to ixodicides and one tick field isolate) were compared. In addition, the effect of two other new synthetic carbamates was tested on larvae from the same strains. The first six tested carbamates reduced egg laying and inhibited egg hatching in the four studied strains (P < 0.05). Compared with untreated females, the eggs produced by the treated engorged female ticks of all strains had a dark, dry, opaque appearance and were less adherent. The remaining two tested carbamates induced larval mortality in all of the evaluated strains. The three studied R. microplus strains displayed 50% resistance ratios (RR₅₀) of less than 2 when compared to the susceptible reference strain. These results demonstrate that both carbamates with a larvicidal effect and carbamates that inhibit egg laying and embryo development are efficacious against tick strains that are resistant to commercial ixodicides, no cross resistance was observed.     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.