Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA

<p>Abstract</p> <p>Background</p> <p>The epidemiology of <it>E. ruminantium </it>infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia.</p> <p>Methods</p> <p>We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting <it>E. ruminantium </it>infection was also assessed.</p> <p>Results</p> <p>The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable <it>A. variegatum </it>infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher <it>E. ruminantium </it>prevalence in the animals with increasing age and both the Spearman's rank test (<it>r</it>_{<it>s </it>}= -0.1512; P = 0.003) and <it>kappa </it>statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays.</p> <p>Conclusion</p> <p>The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.</p

Defining the concept of ‘tick repellency’ in veterinary medicine

Although widely used, the term repellency needs to be employed with care when applied to ticks and other periodic or permanent ectoparasites. Repellency has classically been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from its source. However, for crawling arthropods such as ticks, the term commonly subsumes a range of effects that include arthropod irritation and consequent avoiding or leaving the host, failing to attach, to bite, or to feed. The objective of the present article is to highlight the need for clarity, to propose consensus descriptions and methods for the evaluation of various effects on ticks caused by chemical substances

Vector-borne and other pathogens of potential relevance disseminated by relocated cats

Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat’s lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27 + memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement. </p

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27 + memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement. </p

Abundance estimation of Ixodes ticks (Acari: Ixodidae) on roe deer (Capreolus capreolus)

Despite the importance of roe deer as a host for Ixodes ticks in central Europe, estimates of total tick burden on roe deer are not available to date. We aimed at providing (1) estimates of life stage and sex specific (larvae, nymphs, males and females, hereafter referred to as tick life stages) total Ixodes burden and (2) equations which can be used to predict the total life stage burden by counting the life stage on a selected body area. Within a period of 1½ years, we conducted whole body counts of ticks from 80 hunter-killed roe deer originating from a beech dominated forest area in central Germany. Averaged over the entire study period (winter 2007–summer 2009), the mean tick burden per roe deer was 64.5 (SE ± 10.6). Nymphs were the most numerous tick life stage per roe deer (23.9 ± 3.2), followed by females (21.4 ± 3.5), larvae (10.8 ± 4.2) and males (8.4 ± 1.5). The individual tick burden was highly aggregated (k = 0.46); levels of aggregation were highest in larvae (k = 0.08), followed by males (k = 0.40), females (k = 0.49) and nymphs (k = 0.71). To predict total life stage specific burdens based on counts on selected body parts, we provide linear equations. For estimating larvae abundance on the entire roe deer, counts can be restricted to the front legs. Tick counts restricted to the head are sufficient to estimate total nymph burden and counts on the neck are appropriate for estimating adult ticks (females and males). In order to estimate the combined tick burden, tick counts on the head can be used for extrapolation. The presented linear models are highly significant and explain 84.1, 77.3, 90.5, 91.3, and 65.3% (adjusted R2) of the observed variance, respectively. Thus, these models offer a robust basis for rapid tick abundance assessment. This can be useful for studies aiming at estimating effects of abiotic and biotic factors on tick abundance, modelling tick population dynamics, modelling tick-borne pathogen transmission dynamics or assessing the efficacy of acaricides