Out of Asia? Expansion of Eurasian Lyme borreliosis causing genospecies display unique evolutionary trajectories.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens.

Range Expansion of Ixodes scapularis and Borrelia burgdorferi in Ontario, Canada, from 2017 to 2019.

Range expansion of the vector tick species, Ixodes scapularis, has been detected in Ontario over the last two decades. This has led to elevated risk of exposure to Borrelia burgdorferi, the bacterium that causes Lyme disease. Previous research using passive surveillance data suggests that I. scapularis populations establish before the establishment of B. burgdorferi transmission cycles, with a delay of ∼5 years. The objectives of this research were to examine spatial and temporal patterns of I. scapularis and its pathogens from 2017 to 2019 in southwestern, eastern, and central Ontario, and to explore patterns of B. burgdorferi invasion. Over the 3-year study period, drag sampling was conducted at 48 sites across Ontario. I. scapularis ticks were tested for B. burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia species, including Babesia microti and Babesia odocoilei, and Powassan virus. I. scapularis was detected at 30 sites overall, 22 of which had no history of previous tick detection. B. burgdorferi was detected at nine sites, eight of which tested positive for the first time during this study and five of which had B. burgdorferi detected concurrently with initial tick detection. Tick and pathogen hotspots were identified in eastern Ontario in 2017 and 2018, respectively. These findings provide additional evidence on the range expansion and population establishment of I. scapularis in Ontario and help generate hypotheses on the invasion of B. burgdorferi in Ontario. Ongoing public health surveillance is critical to monitor changes in I. scapularis and its pathogens in Ontario.

Clinical Tick-Borne Encephalitis in a Roe Deer (Capreolus capreolus L.).

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

Evaluation of two artificial infection methods of live ticks as tools for studying interactions between tick-borne viruses and their tick vectors.

Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR-/- or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.

Molecular detection of dugbe orthonairovirus in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Nigeria.

Dugbe orthonairovirus (DUGV), a tick-borne zoonotic arbovirus, was first isolated in 1964 in Nigeria. For over four decades, no active surveillance was conducted to monitor the spread and genetic variation of DUGV. This study detected and genetically characterized DUGV circulating in cattle and their infesting ticks (Amblyomma and Rhipicephalus (Boophilus)) in Kwara State, North-Central Nigeria. Blood and or ticks were collected from 1051 cattle at 31 sampling sites (abattoirs and farms) across 10 local government areas of the State. DUGV detection was carried out by RT-qPCR, and positive samples sequenced and phylogenetically analysed. A total of 11824 ticks, mostly A. variegatum (36.0%) and R. (B.) microplus (63.9%), were obtained with mean tick burden of 12 ticks/cattle. Thirty-four (32 A. variegatum and two R. (B.) microplus) of 4644 examined ticks were DUGV-positive, whereas all of the cattle sera tested negative for DUGV genome. Whole genome sequence (S, M and L segments) and phylogenetic analyses indicate that the positive samples shared up to 99.88% nucleotide identity with and clustered around the Nigerian DUGV prototype strain IbAr 1792. Hence, DUGV with high similarity to the previously characterised strain has been detected in Nigeria. To our knowledge, this is the first report of DUGV in North-Central Nigeria and the most recent information after its last surveillance in 1974.

Effect of Vegetation on the Abundance of Tick Vectors in the Northeastern United States: A Review of the Literature.

Tick-borne illnesses have been on the rise in the United States, with reported cases up sharply in the past two decades. In this literature review, we synthesize the available research on the relationship between vegetation and tick abundance for four tick species in the northeastern United States that are of potential medical importance to humans. The blacklegged tick (Ixodes scapularis) (Say; Acari: Ixodidae) is found to be positively associated with closed canopy forests and dense vegetation thickets, and negatively associated with open canopy environments, such as grasslands or old agricultural fields. The American dog tick (Dermacentor variabilis) (Say; Acari: Ixodidae) has little habitat overlap with I. scapularis, with abundance highest in grasses and open-canopy fields. The lone star tick (Amblyomma americanum) (Linnaeus; Acari: Ixodidae) is a habitat generalist without consistent associations with particular types of vegetation. The habitat associations of the recently introduced Asian longhorned tick (Haemaphysalis longicornis) (Neumann; Acari: Ixodidae) in the northeastern United States, and in other regions where it has invaded, are still unknown, although based on studies in its native range, it is likely to be found in grasslands and open-canopy habitats.

An integrated overview of the bacterial flora composition of Hyalomma anatolicum, the main vector of CCHF.

The microbial flora associated with Hyalomma anatolicum ticks was investigated using culture-dependent (CD) and independent (next generation sequencing, NGS) methods. The bacterial profiles of different organs, development stages, sexes, and of host cattle skins were analyzed using the CD method. The egg and female gut microbiota were investigated using NGS. Fourteen distinct bacterial strains were identified using the CD method, of which Bacillus subtilis predominated in eggs, larval guts and in adult female and male guts, suggesting probable transovarial transmission. Bacillus velezensis and B. subtilis were identified in cattle skin and tick samples, suggesting that skin is the origin of tick bacteria. H.anatolicum males harbour lower bacterial diversity and composition than females. The NGS analysis revealed five different bacterial phyla across all samples, Proteobacteria contributing to >95% of the bacteria. In all, 56611sequences were generated representing 6,023 OTUs per female gut and 421 OTUs per egg. Francisellaceae family and Francisella make up the vast majority of the OTUs. Our findings are consistent with interference between Francisella and Rickettsia. The CD method identified bacteria, such B. subtilis that are candidates for vector control intervention approaches such paratransgenesis whereas NGS revealed high Francisella spp. prevalence, indicating that integrated methods are more accurate to characterize microbial community and diversity.

A Comparison of Questing Substrates and Environmental Factors That Influence Nymphal Ixodes pacificus (Acari: Ixodidae) Abundance and Seasonality in the Sierra Nevada Foothills of California.

In California, the western blacklegged tick, Ixodes pacificus Cooley and Kohls, is the principal vector of the Borrelia burgdorferi sensu lato (sl) complex (Spirochaetales: Spirochaetaceae, Johnson et al.), which includes the causative agent of Lyme disease (B. burgdorferi sensu stricto). Ixodes pacificus nymphs were sampled from 2015 to 2017 at one Sierra Nevada foothill site to evaluate our efficiency in collecting this life stage, characterize nymphal seasonality, and identify environmental factors affecting their abundance and infection with B. burgdorferi sl. To assess sampling success, we compared the density and prevalence of I. pacificus nymphs flagged from four questing substrates (logs, rocks, tree trunks, leaf litter). Habitat characteristics (e.g., canopy cover, tree species) were recorded for each sample, and temperature and relative humidity were measured hourly at one location. Generalized linear mixed models were used to assess environmental factors associated with I. pacificus abundance and B. burgdorferi sl infection. In total, 2,033 substrates were sampled, resulting in the collection of 742 I. pacificus nymphs. Seasonal abundance of nymphs was bimodal with peak activity occurring from late March through April and a secondary peak in June. Substrate type, collection year, month, and canopy cover were all significant predictors of nymphal density and prevalence. Logs, rocks, and tree trunks had significantly greater nymphal densities and prevalences than leaf litter. Cumulative annual vapor pressure deficit was the only significant climatic predictor of overall nymphal I. pacificus density and prevalence. No associations were observed between the presence of B. burgdorferi sl in nymphs and environmental variables.

Human-tick encounters as a measure of tickborne disease risk in lyme disease endemic areas.

Entomological measures have long served as proxies for human risk of Lyme disease (LD) and other tickborne diseases (TBDs) in endemic areas of the United States, despite conflicting results regarding the correlation between these measures and human disease outcomes. Using data from a previous TBD intervention study in Connecticut, Maryland and New York, we evaluated whether human-tick encounters can serve as an accurate proxy for risk of TBDs in areas where LD and other Ixodes scapularis-transmitted infections are common. Among 2,590 households consisting of 4,210 individuals, experiencing a tick encounter was associated with an increased risk of both self-reported (RR = 3.17, 95% CI: 2.05, 4.91) and verified TBD (RR = 2.60, 95% CI: 1.39, 4.84) at the household level. Household characteristics associated with experiencing any tick encounter were residence in Connecticut (aOR = 1.86, 95% CI: 1.38, 2.51) or New York (aOR = 1.66, 95% CI: 1.25, 2.22), head of household having a graduate level education (aOR = 1.46, 95% CI: 1.04, 2.08), owning a pet (aOR = 1.80, 95% CI: 1.46, 2.23) and a property size of 2 acres or larger (aOR = 2.30, 95% CI: 1.42, 3.70). Results for individual characteristics were similar to those for households. Future prevention studies in LD endemic areas should consider using human-tick encounters as a robust proxy for TBD risk.

Detection of Hyalomma rufipes in a recently arrived asylum seeker to the EU.

The importation of novel tick species to Europe and the emergence of tick-borne diseases have been of rising concern over the last decades. In May 2019, a total of 349 asylum seekers arrived in Malta by boat. Public health syndromic surveillance was conducted on all migrant boat arrivals. The incidental finding of a tick with anomalous morphology in a newly arrived migrant in Malta prompted an epidemiological investigation. Morphological identification of the tick followed by species identification using keys specific to North Africa was conducted and molecular testing for Crimean Congo haemorrhagic fever virus (CCHFV) was performed. Detailed interview and clinical examination of the case were conducted on arrival and follow-up interviews were undertaken 1- and 4-weeks post-arrival. A Hyalomma rufipes tick was identified on the chest of a 28-year-old male from Sudan. The patient reported malaise and headache on arrival. No further symptoms were reported during follow-up. There was no evidence of previous CCHFV infection or the presence of other ticks or pathogens on the patient. The investigation revealed that the H. rufipes tick had likely been acquired in Libya. This is the first report of the presence of a H. rufipes tick, the main vector for CCHFV, on a recently arrived migrant in Europe. This event highlights the importance of increasing awareness on the risk of tick-borne infections among recently arrived migrants in the Mediterranean countries and the need to consider tick screening as part of the health screening offered in the EU.

Behavioral characteristics and endosymbionts of two potential tularemia and Rocky Mountain spotted fever tick vectors.

Due to climate change-induced alterations of temperature and humidity, the distribution of pathogen-carrying organisms such as ticks may shift. Tick survival is often limited by environmental factors such as dryness, but a predicted hotter and wetter world may allow the expansion of tick ranges. Dermacentor andersoni and D. variabilis ticks are morphologically similar, co-occur throughout the Inland Northwest of Washington State, U.S.A., and both can be injected with pathogenic Rickettsia and Francisella bacteria. Differences in behavior and the potential role of endosymbiotic Rickettsia and Francisella in these ticks are poorly studied. We wanted to measure behavioral and ecological differences between the two species and determine which, if any, Rickettsia and Francisella bacteria - pathogenic or endosymbiotic - they carried. Additionally, we wanted to determine if either tick species may be selected for if the climate in eastern Washington becomes wetter or dryer. We found that D. andersoni is more resistant to desiccation, but both species share similar questing behaviors such as climbing and attraction to bright light. Both also avoid the odor of eucalyptus and DEET but not permethrin. Although both tick species are capable of transmitting pathogenic species of Francisella and Rickettsia, which cause tularemia and Rocky Mountain Spotted Fever, respectively, we found primarily non-pathogenic endosymbiotic strains of Francisella and Rickettsia, and only one tick infected with F. tularensis subspecies holarctica.

Records of ticks (Acari: Ixodidae) on humans and distribution of spotted-fever cases and its tick vectors in Paraná State, southern Brazil.

Brazilian spotted fever (BSF) is the deadliest rickettsiosis in the world. Although the epidemiology of the disease has been established in Brazil, there are still limited data available on distribution of tick vectors and tick species parasitizing humans in the country, particularly in Paraná State. The State of Paraná is located in the southern region of the country and is covered by two biomes: Atlantic rainforest and Cerrado. Thus, the aims of this study were i) to map the distribution of SF tick vectors, ii) to describe and map the distribution of human parasitism by ticks, and iii) to map the distribution of fatal and non-fatal spotted fever (SF) cases in Paraná State, southern Brazil. Data were reviewed and compiled from previous published reports, and also from two scientific collections of Paraná State. SF cases were retrieved from the Brazilian Notifiable Diseases Information System. A total of 50 cases of human parasitism by ticks were recorded, with a total of 64 (22 males, 12 females, 30 nymphs) ticks collected. The following 12 tick species were identified: Amblyomma aureolatum, Amblyomma brasiliense, Amblyomma calcaratum, Amblyomma dubitatum, Amblyomma incisum, Amblyomma longirostre, Amblyomma ovale, Amblyomma parkeri, Amblyomma scalpturatum, Amblyomma sculptum, Haemaphysalis juxtakochi and Rhipicephalus sanguineus sensu lato (s.l.). The most prevalent tick species associated to cases of human parasitism were A. sculptum (13/50; 26 %), A. aureolatum (10/50; 20 %), A. brasiliense (5/50; 10 %), A. ovale (5/50; 10 %) and A. parkeri (4/50; 8%). A total of 51 non-fatal and five fatal SF cases were recorded. Data from this study highlights the need for monitoring ticks parasitizing humans aiming early detection of tick-borne diseases cases, particularly BSF in Paraná State, southern Brazil.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Climate change implications for the distribution of the babesiosis and anaplasmosis tick vector, Rhipicephalus (Boophilus) microplus.

Climate change ranks among the most important issues globally, affecting geographic distributions of vectors and pathogens, and inducing losses in livestock production among many other damaging effects. We characterized the potential geographic distribution of the ticks Rhipicephalus (Boophilus) microplus, an important vector of babesiosis and anaplasmosis globally. We evaluated potential geographic shifts in suitability patterns for this species in two periods (2050 and 2070) and under two emissions scenarios (RCPs 4.5 and 8.5). Our results anticipate increases in suitability worldwide, particularly in the highest production areas for cattle. The Indo-Malayan region resulted in the highest cattle exposure under both climate change projections (2050), with increases in suitability of > 30%. This study illustrates how ecological niche modeling can be used to explore probable effects of climate change on disease vectors, and the possible consequences on economic dimensions.

Could climate trends disrupt the contact rates between Ixodes ricinus (Acari, Ixodidae) and the reservoirs of Borrelia burgdorferi s.l.?

This study addresses the modifications that future climate conditions could impose on the transmission cycles of Borrelia burgdorferi s.l. by the tick Ixodes ricinus in Europe. Tracking the distribution of foci of a zoonotic agent transmitted by vectors as climate change shapes its spatial niche is necessary to issue self-protection measures for the human population. We modeled the current distribution of the tick and its predicted contact rates with 18 species of vertebrates known to act as reservoirs of the pathogen. We approached an innovative way for estimating the possibility of permanent foci of Borrelia afzelii or Borrelia garinii tracking separately the expected spatial overlap among ticks and reservoirs for these pathogens in Europe. Environmental traits were obtained from MODIS satellite images for the years 2002-2017 (baseline) and projected on scenarios for the years 2030 and 2050. The ratio between MODIS baseline/current interpolated climatologies (WorldClim), and the ratio between MODIS-projected year 2050 with five climate change scenarios for that year (WorldClim) revealed no significant differences, meaning that projections from MODIS are reliable. Models predict that contact rates between the tick and reservoirs of either B. garinii or B. afzelii are spatially different because those have different habitats overlap. This is expected to promote different distribution patterns because of the different responses of both groups of reservoirs to environmental variables. Models for 2030 predict an increase in latitude, mainly in the circulation of B. garinii, with large areas of expected permanent contact between vector and reservoirs in Nordic countries and central Europe. However, climate projections for the year 2050 predict an unexpected scenario of contact disruption. Though large areas in Europe would be suitable for circulation of the pathogens, the predicted lack of niche overlap among ticks and reservoirs could promote a decrease in permanent foci. This development represents a proof-of-concept for the power of jointly modeling both the vector and reservoirs in a common framework. A deeper understanding of the unanticipated result regarding the year 2050 is needed.

Patience is not always a virtue: effects of terrain complexity on the host-seeking behaviour of adult blacklegged ticks, Ixodes scapularis, in the presence of a stationary host.

Blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), are the primary vectors of Lyme disease in the U.S.A. In this study, adult ticks were observed on public trails exhibiting increasing levels of terrain complexity with a potential host nearby. The goal of this study was to (a) examine the extent to which adult ticks may actively search (vs. sit-and-wait) for a nearby host, (b) determine whether or not ticks could locate the position of the host in natural conditions and (c) determine the role of terrain complexity on the distances ticks travelled in a short period of time (30 min). Results indicate that, when a potential stationary host is within 50 cm, ticks will utilize an active-search strategy. The majority of ticks moved in the direction of the host in natural conditions. Finally, ticks in a less complex terrain were more active and travelled greater horizontal distances than ticks in a more complex terrain. In conclusion, the use of an active-search approach would likely increase the foraging success of ticks, especially in terrains with minimal complexity, near host animals that have stopped to rest or feed, reinforcing that humans should be vigilant about checking for ticks after being outdoors.

Dermacentor reticulatus is a vector of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV; family Flaviviridae) is the most medically important tick-borne virus in Europe and Asia. Ixodes ricinus and I. persulcatus ticks are considered to be the main vector ticks of TBEV in nature due to their specific ecological associations with the vertebrate hosts. Nevertheless, recent TBEV prevalence studies in ticks suggest that Dermacentor reticulatus ticks might play a relevant role in the maintenance of TBEV in nature. The goal of this study was to evaluate the vector competency of D. reticulatus for TBEV through experimental tick infections and comparative in vivo transmission studies involving D. reticulatus and I. ricinus ticks. We observed that after a transcoxal micro-capillary inoculation, adult female D. reticulatus ticks efficiently replicated TBEV during the observed period of 21 days. The mean virus load reached up to 2.5 × 105 gene copies and 6.4 × 104 plaque forming units per tick. The infected D. reticulatus ticks were able to transmit the virus to mice. The course of infection in mice was comparable to the infection after a tick bite by I. ricinus while the virus spread and clearance was slightly faster. Moreover, D. reticulatus ticks were capable of tick-to-tick non-viraemic transmission of TBEV to the Haemaphysalis inermis nymphs during co-feeding on the same animal. The co-feeding transmission efficiency was overall slightly lower (up to 54 %) in comparison with I. ricinus (up to 94 %) and peaked 1 day later, at day 3. In conclusion, our study demonstrated that D. reticulatus is a biologically effective vector of TBEV. In line with the recent reports of its high TBEV prevalence in nature, our data indicate that in some endemic foci, D. reticulatus might be an underrecognized TBEV vector which contributes to the expansion of the TBEV endemic areas.

Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis.

BACKGROUND: The tick Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated. METHODS: Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi). RESULTS: The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased. CONCLUSION: The three enzymes all play important roles in embryonic development of H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.

Documentation of the Expansion of the Gulf Coast Tick (Amblyomma maculatum) and Rickettsia parkeri: First Report in Illinois.

The Gulf Coast tick, Amblyomma maculatum, is of public and veterinary health concern, as it is the primary vector of Rickettsia parkeri and Hepatozoon americanum, causative agents of Rickettsiosis and American canine hepatozoonosis. The Gulf Coast tick's range has expanded over the last 50 yr into the mid-Atlantic states, and its expansion is expected to continue northward. We are reporting the presence of A. maculatum for the first time in Illinois, including a total of 18 specimens collected at 6 different sites during surveys in 2013 and 2019. Fourteen of these specimens were screened for Rickettsia parkeri, which resulted in the detection of this bacteria in 8 samples from 4 counties. By depositing these specimens in scientific collections, we provide materialistic evidence of their establishment in 2 counties. We urge health officials to rely on and use scientific collections to document the expansion of these and other vectors across the country. Additionally, we recommend that health practitioners become aware of the clinical similarities between Rocky Mountain Spotted Fever (caused by Rickettsia rickettsii) and "tidewater" fever (caused by R. parkeri).