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Theileriosis is a tick‐borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata‐infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%–20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch‐to‐batch variation, safety and ethical concerns. In this study, the suitability of serum‐free media for the cultivation of Theileria‐transformed cell lines was examined. Three commercial serum‐free media (HL‐1, ISF‐1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS‐containing RPMI‐1640 medium. ISF‐1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p =.0011). ISF‐1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p =.0004), whereas an Egyptian strain grew significantly faster in ISF‐1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p =.0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin‐free, serum‐free, protein‐free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF‐1 medium. The use of serum‐free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case‐by‐case basis. The relevance of Theileria cultivation in serum‐free media for applications such as vaccine development requires further examination. [ABSTRACT FROM AUTHOR]

To assess pastoralists' and agropastoralists' knowledge on Rift Valley fever (RVF), participatory epidemiological studies were conducted with 215 livestock keepers and 27 key informants in Napak, Butebo, Isingiro and Lyantonde districts, Uganda, between January and February 2022. Livestock keepers in all four districts had knowledge of RVF and even had local names or descriptions for it. Pastoralists and agropastoralists possessed valuable knowledge of RVF clinical descriptions and epidemiological risk factors such as the presence of infected mosquitoes, living in flood-prone areas, and excessive rainfall. RVF was ranked among the top ten most important cattle diseases. Pastoralists called RVF Lonyang , symbolizing a disease associated with jaundice, high fever, abortions in pregnant cows, and sudden death in calves. Key informants identified infected domestic animals, the presence of infected mosquitoes, livestock movement and trade, and infected wild animals as risk pathways for the introduction of RVF into an area. Drinking raw blood and milk was perceived as the most likely pathway for human exposure to RVF virus; while the highest consequence was high treatment costs. The results indicate that pastoralists provided key epidemiological information that could be essential for designing an effective national RVF surveillance and early warning system. [ABSTRACT FROM AUTHOR]

Background: Tick infestation is the major problem for animal health that causes substantial economic losses, particularly in tropical and subtropical countries. To better understand the spatial distribution of tick species and risk factors associated with tick prevalence in livestock in Pakistan, ticks were counted and collected from 471 animals, including 179 cattle, 194 buffaloes, 80 goats and 18 sheep, on 108 livestock farms in nine districts, covering both semi-arid and arid agro-ecological zones. Results: In total, 3,807 ticks representing four species were collected: Hyalomma anatolicum (n = 3,021), Rhipicephalus microplus (n = 715), Hyalomma dromedarii (n = 41) and Rhipicephalus turanicus (n = 30). The latter species is reported for the first time from the study area. Rhipicephalus microplus was the predominant species in the semi-arid zone, whereas H. anatolicum was the most abundant species in the arid zone. The overall proportion of tick-infested ruminants was 78.3% (369/471). It was highest in cattle (89.9%), followed by buffaloes (81.4%), goats (60.0%) and sheep (11.1%). The median tick burden significantly differed among animal species and was highest in cattle (median 58), followed by buffaloes (median 38), goats (median 19) and sheep (median 4.5). Female animals had significantly higher tick burdens than males and, in large ruminants, older animals carried more ticks than younger animals. The intensity of infestation was significantly lower in indigenous animals compared to exotic and crossbred cows. Analysis of questionnaire data revealed that the absence of rural poultry, not using any acaricides, traditional rural housing systems and grazing were potential risk factors associated with a higher tick prevalence in livestock farms. Conclusion: Absence of rural poultry, not performing acaricide treatments, traditional rural housing systems and grazing were important risk factors associated with higher tick prevalence in livestock farms. Age, gender, breed and animal species significantly affected the intensity of tick infestation. This report also describes the presence of R. turanicus in the Punjab Province of Pakistan for the first time. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Pakistan. [ABSTRACT FROM AUTHOR]

Nearly a century after the first reports of Rift Valley fever (RVF) were documented in Kenya, questions on the transmission dynamics of the disease remain. Specifically, data on viral maintenance in the quiescent years between epidemics is limited. We implemented a cross-sectional study in northern Kenya to determine the seroprevalence, risk factors, and ecological predictors of RVF in humans and livestock during an interepidemic period. Six hundred seventy-six human and 1,864 livestock samples were screened for anti-RVF Immunoglobulin G (IgG). Out of the 1,864 livestock samples tested for IgG, a subset of 1,103 samples was randomly selected for additional testing to detect the presence of anti-RVFV Immunoglobulin M (IgM). The anti-RVF virus (RVFV) IgG seropositivity in livestock and humans was 21.7% and 28.4%, respectively. RVFV IgM was detected in 0.4% of the livestock samples. Participation in the slaughter of livestock and age were positively associated with RVFV exposure in humans, while age was a significant factor in livestock. We detected significant interaction between rainfall and elevation's influence on livestock seropositivity, while in humans, elevation was negatively associated with RVF virus exposure. The linear increase of human and livestock exposure with age suggests an endemic transmission cycle, further corroborated by the detection of IgM antibodies in livestock. [ABSTRACT FROM AUTHOR]

Background: In Europe, feline vector-borne infections are gaining importance because of the changing climate, expanding habitats of potential vectors and expanding pathogen reservoirs. The main objective of this study was to assess the prevalence of vector-borne pathogens (VBPs) in stray cats in Zaragoza, Spain, and to investigate potential risk factors for infection, including feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). Methods: Blood samples from stray cats presented to the veterinary faculty in Zaragoza between February 2020 and 2022 were tested by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Ehrlichia canis, Rickettsia spp., haemotropic Mycoplasma spp., Hepatozoon spp., Leishmania infantum, piroplasms and microfilariae at the LABOKLIN laboratory. The cats were also tested for FeLV and FIV by PCR. Results: Nearly half of the cats (158/332, 47.6%) were positive for at least one VBP. Hepatozoon spp. were detected in 25.6%, haemotropic Mycoplasma spp. in 22.9%, B. henselae in 9.3% and L. infantum in 2.1% of the cats. Male sex had a statistically significant association with test results for haemotropic Mycoplasma spp. (odds ratio 1.38 [1.21;1.57]); regionality with Hepatozoon spp., B. henseale and FIV; and seasonality with Hepatozoon spp., haemotropic Mycoplasma spp., L. infantum and FeLV (P ≤ 0.05 each). A strong positive correlation was reported for the amount of rainfall and the number of cats that tested positive for Hepatozoon spp. (ρ = 753, P = 0.05). None of the cats tested positive for A. phagocytophilum, A. platys, E. canis, Rickettsia spp., piroplasms, or microfilariae. Co-infections with multiple VBPs were detected in 56 out of 332 cats (16.9%). Thirty-one of the 332 cats included in the study (9.3%) tested positive for FeLV (6.9%) and for FIV (3.6%). In 20/31 cats (64.5%) that tested positive for FeLV/FIV, coinfections with VBP were detected (P = 0.048, OR 2.15 [0.99; 4.64]). Conclusions: VBPs were frequently detected in stray cats in Zaragoza. In particular, regionality and seasonality had a statistically significant association with PCR results for most VBPs included in the study. Highlights: Nearly half of the cats tested positive for at least one vector-borne pathogen by PCR Hepatozoon felis, haemotropic Mycoplasma, Bartonella and Leishmania were detected Stray cats should be monitored to prevent and manage potential zoonotic diseases [ABSTRACT FROM AUTHOR]

Nearly a century after the first reports of Rift Valley fever (RVF) were documented in Kenya, questions on the transmission dynamics of the disease remain. Specifically, data on viral maintenance in the quiescent years between epidemics is limited. We implemented a cross-sectional study in northern Kenya to determine the seroprevalence, risk factors, and ecological predictors of RVF in humans and livestock during an interepidemic period. Six hundred seventy-six human and 1,864 livestock samples were screened for anti-RVF Immunoglobulin G (IgG). Out of the 1,864 livestock samples tested for IgG, a subset of 1,103 samples was randomly selected for additional testing to detect the presence of anti-RVFV Immunoglobulin M (IgM). The anti-RVF virus (RVFV) IgG seropositivity in livestock and humans was 21.7% and 28.4%, respectively. RVFV IgM was detected in 0.4% of the livestock samples. Participation in the slaughter of livestock and age were positively associated with RVFV exposure in humans, while age was a significant factor in livestock. We detected significant interaction between rainfall and elevation's influence on livestock seropositivity, while in humans, elevation was negatively associated with RVF virus exposure. The linear increase of human and livestock exposure with age suggests an endemic transmission cycle, further corroborated by the detection of IgM antibodies in livestock. [ABSTRACT FROM AUTHOR]

Background Culicoides spp. play an important role in the transmission of several vector-borne pathogens such as Bluetongue and Schmallenberg virus in Europe. To better understand the biology of local Culicoides species, a study divided into three parts was performed in northeast Germany to elucidate the feeding activity patterns (study A), preferential landing and feeding sites (study B) and host feeding preferences (study C) of Culicoides spp. using cattle and sheep as baits. Methods In study A, the activity of Culicoides spp. was monitored over a 72 h period by collecting insects at regular intervals from the interior of drop traps with cattle or sheep standing inside. In study B, Culicoides spp. were directly aspirated from the coat and fleece of cattle and sheep during the peak activity period of Culicoides. In study C, Culicoides spp. were collected using drop traps with either cattle or sheep standing inside and located 10 m apart. Results In study A, 3,545 Culicoides midges belonging to 13 species were collected, peak activity was observed at sunset. In study B, 2,024 Culicoides midges were collected. A significantly higher number of midges was collected from the belly and flank of cattle in comparison to their head region. In study C, 3,710 Culicoides midges were collected; 3,077 (83%) originated from cattle and 633 (17%) from sheep. Nearly half (46.7%) of the midges collected from cattle were engorged, significantly more than the number of engorged midges collected from sheep (7.5%). Culicoides from the Obsoletus complex (C. obsoletus and C. scoticus) were the most common Culicoides species encountered, followed by C. punctatus. Other species identified were C. dewulfi, C. chiopterus, C. pulicaris, C. lupicaris, C. pallidicornis, C. subfascipennis, C. achrayi, C. stigma, C. griseidorsum and C. subfagineus, the last two species are reported for the first time in Germany. Engorged C. chiopterus were collected in relatively high numbers from sheep, suggesting that this species may have a preference for sheep. Conclusions An insight into the feeding behaviour of local Culicoides species under field conditions in northeast Germany was obtained, with implications for the implementation of control measures and midge-borne disease risk analysis. [ABSTRACT FROM AUTHOR]

The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in-field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase. [ABSTRACT FROM AUTHOR]

Abstract: Tick proteins functioning in vital physiological processes such as blood meal uptake, digestion and reproduction are potential targets for anti-tick vaccines, since vaccination could inhibit these essential functions and ultimately affect tick survival. In this study we identified metzincin metalloproteases from Rhipicephalus microplus as potential vaccine candidates since they are implicated as essential to blood-cavity formation, bloodmeal digestion and reproduction in ixodid ticks. Eight transcripts encoding proteins that contain the characteristic metzincin zinc-binding motif HEXXHXXG/NXXH/D and a unique methionine containing “methionine-turn” were identified from native and in-house assembled R. microplus expressed sequence tag (EST) databases. These were representative of five reprolysin-like and three astacin-like metzincin metalloproteases. Reverse transcription-PCR analysis indicated that the reprolysins were most abundantly expressed in the salivary glands, whereas the astacins were most abundant in the midgut and ovaries. In vivo gene silencing was performed to assess a possible phenotype of these metalloproteases during adult female R. microplus blood feeding and reproduction. RNA interference (RNAi) against two of the reprolysins and one of the astacins significantly affected the average egg weight and oviposition rate. Evidently, this reverse genetic approach enabled the evaluation of the overall vital impact of tick proteins. Finally, integrated real time-PCR studies also revealed an extensive cross organ network between the R. microplus metzincin transcripts, supporting the use of a combinatorial metzincin-based anti- R. microplus vaccine. [Copyright &y& Elsevier]

Abstract: Tick control on livestock relies principally on the use of acaricides but the development of acaricide resistance and concerns for environmental pollution underscore the need for alternative control methods, for instance through the use of anti-tick vaccines. Two commercial vaccines based on the recombinant Bm86 protein from Rhipicephalus (Boophilus) microplus ticks were developed. Partial protection of the Bm86 vaccine against other Rhipicephalus (Boophilus) and Hyalomma tick species suggests that the efficacy of a Bm86-based vaccine may be enhanced when based on the orthologous recombinant Bm86 antigen. We therefore identified and analysed the Bm86 homologues from species representing the main argasid and ixodid tick genera, including two from the prostriate Ixodes ricinus tick species. A novel protein from metastriate ticks with multiple epidermal growth factor (EGF)-like domains which is structurally related to Bm86 was identified by using a 3′ rapid amplification of cDNA ends (3′-RACE) method with a degenerate primer based on a highly conserved region of Bm86 and its orthologues. This second protein was named ATAQ after a part of its signature peptide. Quantitative reverse transcriptase-PCR showed that ATAQ proteins are expressed in both midguts and Malpighian tubules, in contrast to Bm86 orthologues which are expressed exclusively in tick midguts. Furthermore, expression of this protein over the life stages of R. microplus and Rhipicephalus appendiculatus was more continuous compared with Bm86. Although a highly effective vaccine antigen, gene silencing of Bm86 by RNA interference (RNAi) produced only a weak phenotype. Similarly the RNAi phenotype of Rhipicephalus evertsi evertsi females in which the expression of Ree86, ReeATAQ or a combination of both genes was silenced by RNAi did not differ from a mock-injected control group. The vaccine potential of ATAQ proteins against tick infestations is yet to be evaluated. [ABSTRACT FROM AUTHOR]

Abstract: The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis. [Copyright &y& Elsevier]

Background: Bovine anaplasmosis has been reported in several European countries, but the vector competency of tick species for Anaplasma marginale from these localities has not been determined. Because of the wide distributional range of Dermacentor reticulatus within Europe and the major role of Dermacentor spp. as a vector of A. marginale in the United States, we tested the vector competency of D. reticulatus for A. marginale. Results: Male D. reticulatus were allowed to feed for 7 days on a calf persistently infected with a Zaria isolate of A. marginale, after which they were removed and held off-host for 7 days. The ticks were then allowed to feed a second time for 7 days on a susceptible tick-naïve calf. Infection of calf No. 4291 was detected 20 days post exposure (p.i.) and confirmed by msp4 PCR. Thirty percent of the dissected acquisition fed ticks was infected. In addition, A. marginale colonies were detected by light microscopy in the salivary glands of the acquisition fed ticks. Transmission of A. marginale to calf No. 9191 was confirmed by examination of Giemsa-stained blood smears and msp4 PCR. Ticks were dissected after transmission feeding and presence of A. marginale was confirmed in 18.5% of the dissected ticks. Conclusion: This study demonstrates that D. reticulatus males are competent vectors of A. marginale. Further studies are needed to confirm the vector competency of D. reticulatus for other A. marginale strains from geographic areas in Europe. [ABSTRACT FROM AUTHOR]

Abstract: Outbreaks of autochthonous babesiosis, caused by Babesia canis, occurred in The Netherlands in the spring and autumn of 2004 affecting 23 dogs. Nineteen animals recovered after treatment, whereas four dogs died. Adult Dermacentor reticulatus ticks collected from these dogs indicate that canine babesiosis could become endemic in The Netherlands. [Copyright &y& Elsevier]

Background: Hepatozoon canis is a protozoal agent that is known to be transmitted by oral uptake of H. canis-infected Rhipicephalus sanguineus sensu lato ticks in dogs. Vertical transmission of H. canis has only been described once in a study evaluating dogs from Japan. The aim of this study was to investigate the parasitological status of puppies from a bitch that had tested positive for Hepatozoon spp. prior to giving birth. Findings: A 4-year-old, female, pregnant dog imported from Italy (Sardinia) to Germany showed clinical signs of lethargy and tachypnoea and tested positive for H. canis by PCR. The dog gave birth to eight puppies, one of which was stillborn and another that had to be reanimated. Haematology, buffy coat analysis and a biochemistry profile were performed for each dog. EDTA-blood of the surviving seven puppies and bone marrow, liver, spleen, amniotic fluid, and umbilical cord of the stillborn puppy was tested for the presence of Hepatozoon spp. by PCR. The mother and the seven surviving puppies tested positive for H. canis by PCR at day 62 post-partum. Gamonts were detected in all dogs by buffy coat evaluation. Haematological and biochemistry results revealed mild abnormalities. In the stillborn puppy, spleen, umbilical cord, and amniotic fluid were positive for H. canis. Conclusion: The results confirm that vertical transmission is a possible route of H. canis infection in dogs, demonstrated by molecular detection of the pathogen in the stillborn puppy. In the seven surviving puppies, vertical transmission was the most likely transmission route. A potential impact of the level of parasitaemia on the health of puppies, as well as its pathogenesis, should be investigated further. [ABSTRACT FROM AUTHOR]

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance. [ABSTRACT FROM AUTHOR]

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions. [ABSTRACT FROM AUTHOR]

Background: Hyalomma marginatum and H. rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa to central Asia but may also be found in Central and Northern Europe through introduction by migratory birds. Methods: Ticks were collected while feeding or crawling on animals and humans, or from the environment, in different regions in Germany, between 2019 and 2021 in a citizen science study and from 2022 to 2023 in the wake of this study. Results: From 2019 to 2023, a total of 212 Hyalomma adult ticks were detected in Germany. This included 132 H. marginatum and 43 H. rufipes ticks sent to research institutions and 37 photographic records that were only identified to genus level. The number of detected ticks varied over the years, with the highest number of 119 specimens recorded in 2019, followed by 57 in 2020. Most of the specimens were collected from horses, while some were collected from other animals, humans or found crawling on human clothes or other objects inside or outside houses. The screening of 175 specimens for Crimean-Congo hemorrhagic fever virus and of 132 specimens for Babesia/Theileria spp. by PCR gave negative results, while human-pathogenic Rickettsia were detected in 44% (77/175) of the total samples. Subsequent amplicon sequencing and phylogenetic analysis of representative samples determined the species of 41 Rickettsia aeschlimannii and one R. slovaca sequences. Conclusions: Analysis of climatic factors indicated a significantly higher probability of Hyalomma occurrence at locations with higher average spring temperature during the years 2019 and 2020 compared to randomly generated pseudo-absence locations. Dry and hot conditions probably facilitated Hyalomma nymphs' survival and molting into adults during these years. [ABSTRACT FROM AUTHOR]

Ticks are blood-sucking ectoparasites and can transmit various pathogens of medical and veterinary relevance. The life cycle of ticks can be completed under laboratory conditions on experimental animals, but the artificial feeding of ticks has attracted increased interest as an alternative method. This study represents the first report on the successful in vitro feeding of all life stages of two-host tick species, Hyalomma scupense and Hyalomma excavatum, and the three-host tick Hyalomma dromedarii. The attachment and engorgement rates of adults were 84% (21/25) and 76% (19/25) for H. scupense females. For adult H. excavatum and H. dromedarii, 70% (21/30) and 34.4% (11/32) of the females attached and all attached females successfully fed to repletion. The oviposition rates of the artificially fed females were 36.4%, 57.1% and 63.1% for H. dromedarii, H. excavatum and H. scupense, respectively, with a reproductive efficiency index varying between 44.3 and 60.7%. For the larvae, the attachment and engorgement rates were 44.2% (313/708) and 42.8% (303/708) for H. dromedarii, 70.5% (129/183) and 56.8% (104/183) for H. excavatum and 92.6% (113/122) and 55.7% (68/122) for H. scupense. The attachment and engorgement rates for the nymphs were 90.2% (129/143) and 47.6% (68/143) for H. dromedarii, 66.7% (34/51) and 41.2% (21/51) for H. excavatum, and 44.1% (30/68) and 36.8% (25/68) for H. scupense. Molting rates of the immature stages varied between 71.3% (216/303) and 100% (68/68) for the larvae and between 61.9% (13/21) and 96% (24/25) for the nymphs. The successful in vitro feeding of all stages of the three Hyalomma species makes this method a valuable tool for tick research, with potential applications in studies on the pathogens transmitted by these tick species such as Theileria annulata. [ABSTRACT FROM AUTHOR]

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick's enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum. Author summary: The South African bont tick Amblyomma hebraeum is an important vector of heartwater, a debilitating disease of livestock with devastating economic and social impact in sub-Saharan Africa. The causative agent of heartwater is the intracellular proteobacterium Ehrlichia ruminantium. To date a single decades-old live vaccine constitutes the main strategy of controlling heartwater infection but is ineffective against heterologous strains of the pathogen in the field. In our study we delineate the molecular mechanism of A. hebraeum tick midgut genes in response to E. ruminantium infection. We performed comparative analyses of infected midgut transcriptomes of engorged nymphs, unfed and partly-fed adult ticks with their corresponding age-matched uninfected controls. We present differentially expressed genes that may act as barriers or gateways for E. ruminantium in tick midgut-cell entry and warrant further investigations in order to develop novel intervention strategies such as anti-tick vaccine candidate molecules. Previous transcriptomic studies have only described E. ruminantium genes induced during infection in vitro and at the tick bite-site or salivary glands. [ABSTRACT FROM AUTHOR]

Studies on the microbiota of ticks have promoted hypotheses about the combined effects of the bacterial community, its functional contributions to the tick's physiology or probable competition effects with some tick-borne pathogens. However, knowledge on the origin of the microbiota of newly hatched larvae is missing. This study aimed to elucidate the source(s) of the microbiota in unfed tick larvae, addressing the composition of the "core microbiota" and the best ways to decontaminate eggs for microbiota studies. We applied laboratory degree bleach washes and/or ultraviolet light treatments on engorged Rhipicephalus australis females and/or their eggs. No significant effects of these treatments on the reproductive parameters of females and the hatching rates of eggs were observed. However, the different treatments did show striking effects on the composition of the microbiota. The results indicated that bleach washes disrupted the internal tick microbiota in females, implying that bleach may have entered the tick and subsequently affected the microbiota. Furthermore, the analyses of results demonstrated that the ovary is a main source of tick microbiota, while the contribution of Gené's organ (a part of the female reproductive system that secretes a protective wax coat onto tick eggs) or the male's spermatophore requires further investigation. Further studies are needed to identify best practice protocols for the decontamination of ticks for microbiota studies. [ABSTRACT FROM AUTHOR]

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo–derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo–derived variant p104 genes. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi sensu lato , the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus , which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine. [ABSTRACT FROM AUTHOR]

Rickettsiella species are bacterial symbionts that are present in a great variety of arthropod species, including ixodid ticks. However, little is known about their genetic diversity and distribution in Ixodes ricinus, as well as their relationship with other tick-associated bacteria. In this study, we investigated the occurrence and the genetic diversity of Rickettsiella spp. in I. ricinus throughout Europe and evaluated any preferential and antagonistic associations with Candidatus Midichloria mitochondrii and the pathogens Borrelia burgdorferi sensu lato and Borrelia miyamotoi. Rickettsiella spp. were detected in most I. ricinus populations investigated, encompassing a wide array of climate types and environments. The infection prevalence significantly differed between geographic locations and was significantly higher in adults than in immature life stages. Phylogenetic investigations and protein characterization disclosed four Rickettsiella clades (I–IV). Close phylogenetic relations were observed between Rickettsiella strains of I. ricinus and other arthropod species. Isolation patterns were detected for Clades II and IV, which were restricted to specific geographic areas. Lastly, although coinfections occurred, we did not detect significant associations between Rickettsiella spp. and the other tick-associated bacteria investigated. Our results suggest that Rickettsiella spp. are a genetically and biologically diverse facultative symbiont of I. ricinus and that their distribution among tick populations could be influenced by environmental components. [ABSTRACT FROM AUTHOR]

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed. [ABSTRACT FROM AUTHOR]

Background: Soft ticks (Ixodida: Argasidae) are medically important ectoparasites that mainly feed on birds and mammals, which play a key role in their geographic distribution and dispersion. Despite their importance, studies on soft ticks are scarce for many regions and countries of the world, including Pakistan. Methods: In this study, 2330 soft ticks—179 larvae (7.7%), 850 nymphs (36.4%), 711 males (30.5%) and 590 females (25.3%)—were collected from animal shelters in 18 locations within five districts of Khyber Pakhtunkhwa, Pakistan. A subset of the collected ticks was processed for DNA extraction and polymerase chain reaction (PCR) for the amplification of tick 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1), and rickettsial 16S rDNA gene fragments. The obtained sequences were used for the construction of a phylogenetic tree. Results: All the specimens were morphologically identified as Ornithodoros, and were morphologically similar to Ornithodoros tholozani. The genus was confirmed by sequencing partial 12S rDNA, 16S rDNA and cox1 gene fragments. Additionally, a Rickettsia sp. was detected in some of the collected ticks by PCR targeting 16S rDNA. The morphological relatedness of the tick specimens with O. tholozani was confirmed by phylogenetic analysis, in which the Ornithodoros sp. clustered with Ornithodoros tholozani and Ornithodoros verrucosus, both of which belong to the subgenus Pavlovskyella and have been previously reported from Israel, Ukraine and Iran. The phylogenetic tree also indicated that the Ornithodoros sp. from Pakistan corresponds to an undetermined species. Furthermore, the associated Rickettsia sp. grouped with the limoniae group of Rickettsia species previously reported from Argas japonicus ticks from China. Conclusions: This is the first molecular study of an Ornithodoros species from Pakistan. Further studies are essential to confirm its identity and possible pathogenicity with regard to its associated microorganisms in the studied region. [ABSTRACT FROM AUTHOR]

Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic. The aim of this study was to identify diverse tick microbiomes, which may contain as-yet unidentified pathogens, using a metagenomic approach. DNA prepared from bacteria/archaea-enriched fractions obtained from seven tick species, namely Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus and Ixodes ricinus, was subjected to pyrosequencing after whole-genome amplification. The resulting sequence reads were phylotyped using a Batch Learning Self-Organizing Map (BLSOM) program, which allowed phylogenetic estimation based on similarity of oligonucleotide frequencies, and functional annotation by BLASTX similarity searches. In addition to bacteria previously associated with human/animal diseases, such as Anaplasma, Bartonella, Borrelia, Ehrlichia, Francisella and Rickettsia, BLSOM analysis detected microorganisms belonging to the phylum Chlamydiae in some tick species. This was confirmed by pan-Chlamydia PCR and sequencing analysis. Gene sequences associated with bacterial pathogenesis were also identified, some of which were suspected to originate from horizontal gene transfer. These efforts to construct a database of tick microbes may lead to the ability to predict emerging tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding tick biology, including vector competency and interactions with pathogens and symbionts. [ABSTRACT FROM AUTHOR]

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs. [ABSTRACT FROM AUTHOR]

A 12-year old Elo dog was presented with recurring symptoms of conjunctivitis in November 2019. A single whitish nematode was found upon inspection of the eye and identified as a Thelazia callipaeda male. The morphological identification of the eye worm was supported by analysis of a partial cytochrome c oxidase I (cox1) gene sequence. The dog lived in Lower Saxony, northwestern Germany, and had not visited regions known to be endemic for T. callipaeda. This suggests that a local transmission cycle of this zoonotic nematode may exist in Germany. [ABSTRACT FROM AUTHOR]

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this. [ABSTRACT FROM AUTHOR]

Recent studies reported the detection of DNA from tick‐borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi‐locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district. [ABSTRACT FROM AUTHOR]

Background: The bacterium Coxiella burnetii is the etiological agent of Q fever and is mainly transmitted via inhalation of infectious aerosols. DNA of C. burnetii is frequently detected in ticks, but the role of ticks as vectors in the epidemiology of this agent is still controversial. In this study, Ixodes ricinus and Dermacentor marginatus adults as well as I. ricinus nymphs were fed on blood spiked with C. burnetii in order to study the fate of the bacterium within putative tick vectors. Methods: Blood-feeding experiments were performed in vitro in silicone-membrane based feeding units. The uptake, fecal excretion and transstadial transmission of C. burnetii was examined by quantitative real-time PCR as well as cultivation of feces and crushed tick filtrates in L-929 mouse fibroblast cells and cell-free culture medium. Results: Ticks successfully fed in the feeding system with engorgement rates ranging from 29% (D. marginatus) to 64% (I. ricinus adults). Coxiella burnetii DNA was detected in the feces of both tick species during and after feeding on blood containing 105 or 106 genomic equivalents per ml blood (GE/ml), but not when fed on blood containing only 104 GE/ml. Isolation and cultivation demonstrated the infectivity of C. burnetii in shed feces. In 25% of the I. ricinus nymphs feeding on inoculated blood, a transstadial transmission to the adult stage was detected. Females that molted from nymphs fed on inoculated blood excreted C. burnetii of up to 106 genomic equivalents per mg of feces. Conclusions: These findings show that transstadial transmission of C. burnetii occurs in I. ricinus and confirm that I. ricinus is a potential vector for Q fever. Transmission from both tick species might occur by inhalation of feces containing high amounts of viable C. burnetii rather than via tick bites. [ABSTRACT FROM AUTHOR]

A Hypoderma larva was removed from a painful swelling in the lumbar region of a 17-month-old male alpaca kept on a farm in the Brandenburg district, eastern Germany. Morphological analysis and sequencing of the 18S rRNA gene demonstrated it was a second instar larvae of Hypoderma diana. The main host of H. diana is the roe deer (Capreolus capreolus). This is the first description of hypodermosis caused by H. diana in a camelid species. [ABSTRACT FROM AUTHOR]

Tick‐borne diseases (TBDs) have a large impact on animal health and the livelihood of livestock owners, particularly in developing countries. Although climatic and ecological conditions in Pakistan may favour the transmission of tick‐borne pathogens (TBPs), only a few systematic studies have been carried out on TBPs and the diseases that they cause in this country. To improve our understanding of the distribution of TBPs, 3,807 ticks were collected from ruminants (n = 369) on 108 livestock farms (semi‐arid zone = 36, arid zone = 72) in Punjab Province. After morphological identification ticks were pooled into 405 pools (Hyalomma anatolicum = 300, Rhipicephalus microplus = 89, Hyalomma dromedarii = 9, Rhipicephalus turanicus = 7) based on their species, locality of collection, and the host. DNA from each pool was screened by a Reverse Line Blot (RLB) hybridization assay for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia, and Theileria species. DNA from at least one TBP was found in 142 (35.1%) pools. Among the positive pools, 91 (64.1%) had a mixed infection with two or more TBPs, whereas 51 (35.9%) pools were infected with a single TBP. The detected pathogens not only included species that were known to be endemic in Pakistan, such as Theileria annulata (6.7%), Theileria orientalis (3.5%), Anaplasma marginale (5.7%), Anaplasma centrale (2.7%), Anaplasma ovis (1.5%), Babesia bigemina (0.7%), and Babesia bovis (0.2%), but also several TBPs that had not been reported to occur in Pakistan before. This included Ehrlichia minasensis (3.2%), an Anaplasma platys‐like organism (1.2%), Babesia occultans (0.2%), and Rickettsia massiliae (0.2%), as well as two previously uncharacterized species: Ehrlichia sp. Multan (18.0%) and Anaplasma sp. (BL099‐6) (2.22%). The pathogenicity of these novel species remains to be examined. This study shows that a much broader spectrum of TBPs is present in Pakistan than previously thought, including several zoonotic pathogens. [ABSTRACT FROM AUTHOR]

The long feeding duration of ixodid ticks and need for regular blood changes turns the artificial feeding of ticks into a tedious process. To reduce the number of blood changes, a semi-automated system (SAS) for the artificial feeding of hard ticks was developed and evaluated. It consisted of a glass feeding reservoir that can accommodate six tick feeding chambers. A peristaltic pump was used to pump blood through the feeding reservoir, which was changed once daily. Groups of Dermacentor reticulatus and Ixodes ricinus adults were fed simultaneously in both the SAS and a conventional in vitro feeding system. In the conventional system, feeding chambers were hung inside a glass beaker filled with blood that was replaced twice daily. Dermacentor reticulatus adults fed in the SAS obtained significantly higher engorgement weights. Although engorgement rates between both systems were comparable, significantly more SAS-fed females laid fertile egg batches. The egg batch weight of SAS-fed females was also significantly higher. In contrast, the engorgement rate and fecundity of SAS-fed I. ricinus were significantly reduced in comparison to ticks fed in the conventional system. This reduction was likely to be caused by fungal infestation, which could spread between feeding chambers in the SAS. Although the SAS reduced the workload compared to the conventional feeding system and showed promising results for the in vitro feeding of D. reticulatus adults, measures to prevent fungal infestations in the SAS should be considered in future studies. [ABSTRACT FROM AUTHOR]

Tick-borne diseases (TBDs) cause significant losses among livestock and impact the livelihoods of resource-poor farming communities worldwide. In Ethiopia, detailed studies on the epidemiology of tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to determine the prevalence and species composition of bovine TBPs of veterinary significance in local cattle populations. A comprehensive cross-sectional epidemiological study was conducted in cattle populations of Illubabor zone in Southwestern Ethiopia from June to August 2013. For this purpose, blood samples were collected from 392 cattle. A combination of polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay was employed for the detection of TBPs in these samples. The PCR/RLB results of the 392 blood samples indicated a high overall prevalence of 96.9% for TBPs, including Theileria mutans (66.1%), Theileria orientalis (51.8%), Anaplasma sp. Omatjenne (25.5%), Anaplasma marginale (14.5%), Babesia bigemina (14.0%) and Theileria velifera (13.0%) and minor occurrences of Ehrlichia ruminantium (0.5%) and Ehrlichia minasensis (0.26%). Moreover, three novel Anaplasma genotypes were detected in bovine blood samples. A phylogenetic analysis revealed that they most likely represent three, but at least two, new species. The prevalence of the three novel Anaplasma species, preliminary designated as Anaplasma sp. Hadesa, Anaplasma sp. Saso and Anaplasma sp. Dedessa, was 12.5%, 14.3% and 5.6%, respectively. Overall, a total of 227 cattle (57.9%) were found to be co-infected with two or more TBPs simultaneously and 86 different species combinations were observed. The findings show a very high burden of infection of cattle with TBPs in Ethiopia. The high frequency of co-infections suggests that clinical manifestations might be complex. Further research is required to determine the pathogenicity, host cell types and vector of the three novel Anaplasma species identified in this study. [ABSTRACT FROM AUTHOR]

Background: The development of standardized in vitro feeding methods for ixodid ticks has been hampered by their complex feeding behaviour and the long duration of their blood meal. In this study, we aimed to optimize several parameters for the in vitro feeding of adult Dermacentor reticulatus. Methods: Ticks were fed on heparinized bovine blood collected at a slaughterhouse, using a modified silicone membrane feeding assay. Effects on tick feeding and fecundity of different blood meal treatments (freezing, irradiation, addition of antibiotics), ambient conditions (increased CO2 concentration) and phagostimulant use (addition of 2 g/l and 4 g/l glucose to the blood meal) were systematically evaluated. Results: Although fungal growth occurred more frequent in feeding units of ticks fed on defrosted blood, the attachment rate, engorgement mass and fecundity of females fed on defrosted blood did not significantly differ from that of ticks fed on fresh blood. A reduction in the fecundity of female D. reticulatus ticks was observed when ticks were fed with gamma-irradiated blood or untreated blood compared to blood treated with gentamycin. Both the engorgement mass and fecundity increased when ticks were fed at a 5% CO2 level. A non-significant increase in the engorgement mass and engorgement rate of D. reticulatus was observed when blood was supplemented with 4 g glucose per litre compared to 2 g/l. Conclusion: An artificial feeding method was adapted for the feeding of adult D. reticulatus ticks. Of all parameters tested, only the artificial feeding at 5% CO2 levels resulted in a significant increase (P < 0.05) in the engorgement mass and fecundity of female D. reticulatus ticks. The supplementation of blood with antibiotics resulted in a significantly higher tick fecundity in comparison to ticks fed with untreated or irradiated blood. [ABSTRACT FROM AUTHOR]

Introduction: We previously identified tick salivary lectin pathway inhibitor (TSLPI) in Ixodes scapularis, a vector for Borrelia burgdorferi sensu stricto (s.s.) in North America. TSLPI is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement pathway through interference with mannose binding lectin (MBL) activity. Since Ixodes ricinus is the predominant vector for Lyme borreliosis in Europe and transmits several complement sensitive B. burgdorferi sensu lato (s.l.) strains, we aimed to identify, describe, and characterize the I. ricinus ortholog of TSLPI. Methods: We performed (q)PCRs on I. ricinus salivary gland cDNA to identify a TSLPI ortholog. Next, we generated recombinant (r)TSLPI in a Drosophila expression system and examined inhibition of the MBL complement pathway and complement-mediated killing of B. burgdorferi s.l. in vitro. Results: We identified a TSLPI ortholog in I. ricinus salivary glands with 93% homology at the RNA and 89% at the protein level compared to I. scapularis TSLPI, which was upregulated during tick feeding. In silico analysis revealed that TSLPI appears to be part of a larger family of Ixodes salivary proteins among which I. persulcatus basic tail salivary proteins and I. scapularis TSLPI and Salp14. I. ricinus rTSLPI inhibited the MBL complement pathway and protected B. burgdorferi s.s. and Borrelia garinii from complement-mediated killing. Conclusion: We have identified a TSLPI ortholog, which protects B. burgdorferi s.l. from complement-mediated killing in I. ricinus, the major vector for tick-borne diseases in Europe. [ABSTRACT FROM AUTHOR]

Human tick-borne diseases that are transmitted by Ixodes ricinus, such as Lyme borreliosis and tick borne encephalitis, are on the rise in Europe. Diminishing I. ricinus populations in nature can reduce tick exposure to humans, and one way to do so is by developing an anti-vector vaccine against tick antigens. Currently, there is only one anti-vector vaccine available against ticks, which is a veterinary vaccine based on the tick antigen Bm86 in the gut of Rhipicephalus microplus. Bm86 vaccine formulations cause a reduction in the number of Rhipicephalus microplus ticks that successfully feed, i.e. lower engorgement weights and a decrease in the number of oviposited eggs. Furthermore, Bm86 vaccines reduce transmission of bovine Babesia spp. Previously two conserved Bm86 homologues in I. ricinus ticks, designated as Ir86-1 and Ir86-2, were described. Here we investigated the effect of a vaccine against recombinant Ir86-1, Ir86-2 or a combination of both on Ixodes ricinus feeding. Recombinant Ixodes ricinus Bm86 homologues were expressed in a Drosophila expression system and rabbits were immunized with rIr86-1, rIr86-2, a combination of both or ovalbumin as a control. Each animal was infested with 50 female adults and 50 male adults Ixodes ricinus and tick mortality, engorgement weights and egg mass were analyzed. Although serum IgG titers against rIr86 proteins were elicited, no effect was found on tick feeding between the rIr86 vaccinated animals and ovalbumin vaccinated animals. We conclude that vaccination against Bm86 homologues in Ixodes ricinus is not an effective approach to control Ixodes ricinus populations, despite the clear effects of Bm86 vaccination against Rhipicephalus microplus. [ABSTRACT FROM AUTHOR]

Ixodes ricinus transmits bacterial, protozoal and viral pathogens, causing disease and forming an increasing health concern in Europe. ANTIDotE is an European Commission funded consortium of seven institutes, which aims to identify and characterize tick proteins involved in feeding and pathogen transmission. The knowledge gained will be used to develop and evaluate anti-tick vaccines that may prevent multiple human tick-borne diseases. Strategies encompassing anti-tick vaccines to prevent transmission of pathogens to humans, animals or wildlife will be developed with relevant stakeholders with the ultimate aim of reducing the incidence of tick-borne diseases in humans. [ABSTRACT FROM AUTHOR]

Background: Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes. Results: The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont. Conclusions: We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans. [ABSTRACT FROM AUTHOR]

Background: 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, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). Results: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis 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 D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitzlike protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens. [ABSTRACT FROM AUTHOR]

Background: Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations. Results: In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35-42%) and purity (80-85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins. Conclusion: These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa. [ABSTRACT FROM AUTHOR]

Background: Subolesin is an evolutionary conserved protein that was discovered recently in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development. In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin plays a role in gene expression, and therefore affects multiple cellular processes. The objective of this study was to provide evidence for the role of subolesin in gene expression. Results: Two subolesin-interacting proteins were identified and characterized by yeast two-hybrid screen, co-affinity purification and RNA interference (RNAi). The effect of subolesin knockdown on the tick gene expression pattern was characterized by microarray analysis and demonstrated that subolesin RNAi affects the expression of genes involved in multiple cellular pathways. The analysis of subolesin and interacting protein sequences identified regulatory motifs and predicted the presence of conserved protein kinase C (PKC) phosphorylation sites. Conclusion: Collectively, these results provide evidence that subolesin plays a role in gene expression in ticks. [ABSTRACT FROM AUTHOR]

Distinct pathogenic and epidemiological features underlie different Theileria parva strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of T. parva strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the cytochrome b gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except T. parva-Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other Theileria species: T. annulata, T. taurotragi, and T. lestoquardi, with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven T. parva haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking T. parva mitochondrial haplotypes from this study will provide insight into the parasite's epidemiological dynamics and underpin current control efforts. [ABSTRACT FROM AUTHOR]

Background: Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. Methods: Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1α gene. Results: Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3–5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P = 0.002281). Microsatellite analysis identified 15 different genotypes. Conclusions: The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests. [ABSTRACT FROM AUTHOR]

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens. [ABSTRACT FROM AUTHOR]