EVIDENCE OF PROTOZOAN AND BACTERIAL INFECTION AND CO-INFECTION AND PARTIAL BLOOD FEEDING IN THE INVASIVE TICK HAEMAPHYSALIS LONGICORNIS IN PENNSYLVANIA

Abstract

The Asian longhorned tick, Haemaphysalis longicornis, an invasive tick species in the United States, has been found actively host-seeking while infected with several human pathogens. Recent work has recovered large numbers of partially engorged, host-seeking H. longicornis, which together with infection findings raises the question of whether such ticks can reattach to a host and transmit pathogens while taking additional bloodmeals. Here we conducted molecular blood meal analysis in tandem with pathogen screening of partially engorged, host-seeking H. longicornis to identify feeding sources and more inclusively characterize acarological risk. Active, statewide surveillance in Pennsylvania from 2020 to 2021 resulted in the recovery of 22/1,425 (1.5%) partially engorged, host-seeking nymphal and 5/163 (3.1%) female H. longicornis. Pathogen testing of engorged nymphs detected 2 specimens positive for Borrelia burgdorferi sensu lato, 2 for Babesia microti, and 1 co-infected with Bo. burgdorferi s.l. and Ba. microti. No female specimens tested positive for pathogens. Conventional PCR blood meal analysis of H. longicornis nymphs detected avian and mammalian hosts in 3 and 18 specimens, respectively. Mammalian blood was detected in all H. longicornis female specimens. Only 2 H. longicornis nymphs produced viable sequencing results and were determined to have fed on black-crowned night heron, Nycticorax nycticorax. These data are the first to molecularly confirm H. longicornis partial blood meals from vertebrate hosts and Ba. microti infection and co-infection with Bo. burgdorferi s.l. in host-seeking specimens in the United States, and the data help characterize important determinants indirectly affecting vectorial capacity. Repeated blood meals within a life stage by pathogen-infected ticks suggest that an understanding of the vector potential of invasive H. longicornis populations may be incomplete without data on their natural host-seeking behaviors and blood-feeding patterns in nature.

Populations of the Asian longhorned tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), native to eastern Asia, are increasing rapidly and continuing to spread since first being detected in the northeast United States in 2017 (Rainey et al., 2018). For instance, in Pennsylvania, H. longicornis was first detected in 2019, and immediately thereafter, active surveillance of public areas in the southeastern part of the state revealed the further distribution of this tick species, accounting for 56%, 10%, 34%, and 32% of field-collected specimens (n = 796) from Bucks, Chester, Montgomery, and Philadelphia counties, respectively (Pennsylvania Department of Environmental Protection [DEP] statewide surveillance, unpubl. data). Subsequent surveys of the same counties indicate that populations of H. longicornis have increased substantially and now account for 82%, 56%, 46%, and 54%, respectively, of all collected ticks (n = 3,543) from these regions in 2022. More broadly, confirmed local H. longicornis tick populations have increased from 78 counties across 12 states to 178 counties across 17 states between August 2019 and August 2022 (USDA, 2022). Diverse host choice in conjunction with parthenogenetic reproduction will likely facilitate the further spread of H. longicornis into other regions of the United States (Raghavan et al., 2019).

In addition to increasing and expanding populations, H. longicornis is associated with numerous pathogens of veterinary and human health concern in its native and introduced ranges (Zhao et al., 2020). Establishment in the United States has necessitated the implementation of robust surveillance programs for H. longicornis to include the study of its distribution, population dynamics, and infection status (Beard et al., 2018; Pritt, 2020). Research on pathogens of veterinary importance has been primarily focused on Theileria orientalis, the causative agent of bovine theileriosis, given demonstrated vector competence of H. longicornis for foreign isolates (Watts et al., 2016). These efforts have mostly sought to evaluate the prevalence of the T. orientalis Ikeda-VA strain in host-seeking specimens and the vector competence of U.S. populations of H. longicornis for this pathogen to determine potential threats to local livestock health and production (Thompson et al., 2020; Dinkel et al., 2021). Similarly, pathogens of public health importance have been screened in actively questing H. longicornis to determine capacity for acquisition from local hosts. To date, Bourbon virus RNA has been detected in all life stages of H. longicornis in Virginia, and Borrelia burgdorferi sensu stricto (s.s.) DNA and the human pathogenic variant of Anaplasma phagocytophilum (Ap-ha) have been identified in adult and nymphal specimens in Pennsylvania (Price et al., 2021a, 2022a; Cumbie et al., 2022a). Collectively, these findings along with increasing abundance and continued range expansion highlight the need for further epidemiological investigations to determine the specific role of H. longicornis in the tick-borne disease landscape.

Despite reports identifying pathogens in field-collected specimens, experimental vector competence studies have demonstrated that although H. longicornis larvae can acquire pathogens from infected hosts in laboratory settings, they lose the infection during the molt to the nymphal stage, suggesting that transstadial transmission of Bo. burgdorferi s.s. and A. phagocytophilum (Ap-ha) is inefficient (Breuner et al., 2020; Levin et al., 2021). Although these studies suggest that H. longicornis is unlikely to play a major role in the transmission of the aforementioned pathogens, a recent report from active tick surveillance in Pennsylvania (Price et al., 2022b) and observations in Connecticut have documented that a significant proportion of the ticks recovered are partially engorged, host-seeking H. longicornis compared to native tick species. These findings, presenting primary evidence of repeated, successful questing events, indicate that H. longicornis could potentially acquire pathogens during an initial, partial blood meal from an infected host and successfully transmit to a second host during a succeeding meal within the same life stage, an important caveat to the results of experimental transmission studies (Eisen, 2018).

Here we used molecular assays to investigate the status of infection for H. longicornis with a particular focus on the foremost tick-borne pathogens found in the native tick Ixodes scapularis Say (Bo. burgdorferi, A. phagocytophilum, Babesia microti), the prevalence of partial blood feeding in questing specimens, and host range for partially engorged, host-seeking ticks collected during active surveillance in several Pennsylvania counties.

MATERIALS AND METHODS

Field surveillance and laboratory procedures followed Price et al. (2022b). Briefly, active tick surveillance was conducted in all 67 Pennsylvania counties intermittently from 1 October 2020 to 8 April 2021 and weekly from 1 May to 31 August 2021, periods coinciding with peak adult and nymphal I. scapularis and all life stages of H. longicornis activity (Tufts et al., 2019; Piedmonte et al., 2021). Consistent with Centers for Disease Control and Prevention (CDC) recommendations, sampling sites included public areas in deciduous forests, habitats conducive to I. scapularis and H. longicornis tick activity (CDC, 2018; Thompson et al., 2021; Cumbie et al., 2022b). Questing ticks were collected by dragging a 1 m² white felt cloth across the forest floor over vegetation/leaf litter for ≥100 m per collection occasion. Ticks captured on cloths were removed at 10 m intervals and transferred into labeled, ethanol-filled vials for transport to the laboratory. Ticks were then identified to species using a Nikon SMZ-800N stereomicroscope (Nikon Instruments Inc., Melville, New York) and morphological keys (Keirans and Litwak, 1989; Egizi et al., 2019), and classified by life stage, sex, and degree of engorgement (unengorged, partial, full; Fig. 1). Initial engorgement characterization by darkened abdomen and swollen opisthosoma was further refined with body and scutum length measurements using NIS-Elements software (Nikon Instruments) to estimate Scutal index (Yeh et al., 1995), an anatomical relationship developed for I. scapularis, but functional for other species to estimate feeding duration (e.g., Kopsco et al., 2020).

Figure 1.

Light microscopy images of dorsal (A) and ventral (B) aspect and scanning electron microscopy images of dorsal (C) and ventral (D) aspect of adult Haemaphysalis longicornis collected in Pennsylvania. Color version available online.

All engorged specimens were shipped to the Connecticut Agricultural Experiment Station (CAES, New Haven, Connecticut) to corroborate morphological identifications, screen for pathogens, and identify blood meal sources.

On receipt at CAES, ticks were removed from ethanol and rinsed twice with deionized water. DNA was extracted from whole specimens using DNAzol BD (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer's recommendations with minor modifications. Briefly, each tick was placed in a 2-ml microcentrifuge tube, to which 400 μl of DNAzol BD and 20 μl of proteinase K were added. Each specimen was either macerated in an MM300 Mixer Mill (Retsch, Newtown, Pennsylvania) or homogenized manually using the straightened end of an autoclaved paper clip. Homogenates were then incubated at 70 C for 10 min and centrifuged at 20,817 g for 10 min. After the addition of 3 μl of polyacryl carrier (Molecular Research Center), DNA was precipitated using 200 μl of 100% EtOH. DNA pellets were then washed twice with 75% EtOH before elution in 30 μl of deionized water or 1× TE buffer. All samples were stored at −20 C until use.

To corroborate the morphological identification of specimens, DNA of 3 ticks was used to amplify the 18S rRNA region (Mangold et al., 1997) using the following primer pair: 5′-CTGGTTGATCCTGCCAGTAG-3′ (forward) and 5′-CTTCCGCAGGTTCACCTACG-3′ (reverse) (von Dohlen and Moran, 1995; McDiarmid et al., 2000). Each PCR reaction mix consisted of 3 μl of DNA, 4 μl 10 × Qiagen PCR Buffer (15 mmol/L MgCl₂), 0.8 μl dNTP mix (10 mmol/L), 2 μl each of forward and reverse primers (0.1–0.5 μmol/L), 0.2 μl Taq DNA polymerase (1.25 U/reaction), and 28 μl deionized water in a total volume of 40 μl. All PCR assays were performed in a Veriti Thermal Cycler (Applied Biosystems, Foster City, California). PCR thermal cycler conditions included initial denaturation at 95 C for 1 min, 32 cycles at 94 C for 1 min, 62 C for 1 min, 72 C for 1 min, followed by a final extension at 72 C for 10 min.

Following genetic identification, partially engorged ticks were screened for evidence of infection with Bo. burgdorferi sensu lato (s.l.) using the following primers for the flagellin gene: 5′-ACATATTCAGATGCAGACAGAGGT-3′ (forward) and 5′-GCAATCATAGCCATTGCAGATTGT-3′ (reverse) (Barbour et al., 1996), and the following primers for the 16S rRNA gene: 5′-CTGGCAGTGCGTCTTAAGCA-3′ (forward) and 5′-GTATTCACCGTATCATTCTGATATAC-3′ (reverse) (Gazumyan et al., 1994). Thermal cycling conditions for flagellin primers included initial denaturation at 95 C for 5 min, 32 cycles at 95 C for 30 sec, 62 C for 30 sec, 68 C for 30 sec, followed by a final extension at 68 C for 5 min (Little et al., 2019; Pokutnaya et al., 2020). Cycling conditions for 16S rRNA primers included initial denaturation at 95 C for 10 min, 35 cycles at 95 C for 1 min, 56 C for 1 min, 72 C for 1 min and 20 sec, followed by a final extension at 72 C for 7 min (Little et al., 2019; Pokutnaya et al., 2020). Screening for evidence of infection with A. phagocytophilum was conducted using the following specific primer set for the 16S rRNA gene: 5′-CACATGCAAGTCGAACGGATTATTC-3′ (forward) and 5′-TTCCGTTAAGAAGGATCTAATCTCC-3′ (reverse) (Steiner et al., 2006; Lee et al., 2014). The cycling conditions were as follows: initial denaturation at 95 C for 2 min, 40 cycles at 94 C for 30 sec, 60 C for 30 sec, 72 C for 1 min, and a final extension at 72 C for 5 min (Pokutnaya et al., 2020). To screen for Ba. microti, the following primers targeting the 16S rDNA gene were used: 5′-CTTAGTATAAGCTTTTATACAGC-3′ (forward) and 5′-ATAGGTCAGAAACTTGAATGATACA-3′ (reverse) (Persing et al., 1992). Thermal cycling conditions were 95 C for 10 min, followed by 35 cycles of 94 C for 1 min, 57 C for 1 min, 72 C for 1 min, and a final extension of 72 C for 10 min (Pokutnaya et al., 2020).

To identify vertebrate sources of blood meals in partially engorged ticks, PCR assays were conducted to amplify portions of the cytochrome b gene of mitochondrial DNA using avian- and mammalian-specific primer pairs, as previously described (Molaei and Andreadis, 2006; Molaei et al., 2006, 2007). The avian primers included 5′-GACTGTGACAAAATCCCNTTCCA-3′ (forward) and 5′-GGTCTTCATCTYHGGY TTACAAGA-3′ (reverse) and yielded fragments of roughly 508 base pairs (bp). Mammalian primers were 5′-CGAAGCTTGATATGAAAAACCATCGTTG-3′ (forward) and 5′-TGTAGTTRTCWGGGTCHCCTA-3′ (reverse) with an amplicon size of 772 bp. PCR thermal cycler conditions for avian primers included initial denaturation at 95 C for 5 min, 36 cycles at 94 C for 30 sec, 60 C for 30 sec, 72 C for 30 sec, followed by a final extension at 72 C for 7 min. PCR thermal cycler conditions for mammalian primers included initial denaturation at 95 C for 10 min, 36 cycles at 94 C for 30 sec, 55 C for 45 sec, 72 C for 1 min and 30 sec, followed by a final extension at 72 C for 7 min. A Taq PCR Core Kit (Qiagen, Valencia, California) was used for all reactions consistent with the manufacturer's recommendations.

PCR amplicons were purified using the Qiagen PCR Purification Kit (Qiagen), and purified products were sequenced using a 3730xl DNA Analyzer (Applied Biosystems) at the Keck Sequencing Facility, Yale University (New Haven, Connecticut). Sequences were subsequently annotated using ChromasPro version 2.1.8 (Technelysium Pty Ltd., South Brisbane, Queensland, Australia). Identification of blood meal sources and confirmation of tick species were determined by comparing annotated sequences to publicly available records in the GenBank database (NCBI) via BLAST search.

RESULTS

During active statewide surveillance in Pennsylvania from 2020 to 2021, 3,244 nymphal and 3,678 female I. scapularis, and 1,425 nymphal and 163 female H. longicornis specimens were recovered from 2,267 separate collection events. Of these, 22 (1.5%) nymphal and 5 (3.1%) female H. longicornis were identified as partially engorged. We also collected 7 (0.2%) partially engorged I. scapularis nymphs; no engorged female specimens of this species were detected.

The 3 specimens randomly used for genetic identification were confirmed to be H. longicornis by sequencing the PCR-amplified products of the 18S rRNA in forward and reverse directions. BLAST searches of the annotated sequences revealed greater than 96% identity with other H. longicornis sequences of this gene in GenBank (GenBank accession nos. JQ346680.1, JQ346681.1, MW767831.1). Two of the annotated sequences from our study exhibited greater than 97% similarity to the aforementioned records in GenBank and were subsequently submitted to the database (accession nos. OM331798 and OM333163).

Of the 22 partially engorged nymphal H. longicornis screened for evidence of infection with I. scapularis–borne pathogens prevalent in the region, 2 tested positive for Bo. burgdorferi s.l., 2 for Ba. microti, and none for A. phagocytophilum (Table I). One of the specimens of H. longicornis was co-infected with Bo. burgdorferi s.l. and Ba. microti. No partially engorged female specimens of this species tested positive for pathogens (n = 5). We also tested 7 partially engorged I. scapularis nymphs from this survey, and of these, 3 were positive for Bo. burgdorferi, 2 for Ba. microti, and 1 for A. phagocytophilum. One of the I. scapularis specimens was co-infected with Bo. burgdorferi s.l. and Ba. microti, and another one was co-infected with A. phagocytophilum and Ba. microti.

Table I.

Engorged Haemaphysalis longicornis collected from active surveillance in Pennsylvania and tested for blood meal and pathogens.

Specimen	Life stage	Date collected	Municipality	County	Borrelia burgdorferi	Anaplasma phagocytophilum	Babesia microti
1	Nymph	21 May 2021	Lower Southampton Township	Bucks
2	Nymph	5 Apr 2021	Northampton Township	Bucks			†
3*	Nymph	5 Apr 2021	Northampton Township	Bucks	†		†
4	Nymph	5 Apr 2021	Northampton Township	Bucks
5*	Nymph	5 Apr 2021	Northampton Township	Bucks
6	Nymph	5 Apr 2021	Northampton Township	Bucks
7	Nymph	19 May 2021	Northampton Township	Bucks
8	Nymph	19 May 2021	Northampton Township	Bucks
9	Nymph	19 May 2021	Northampton Township	Bucks
10	Nymph	21 May 2021	Lower Southampton Township	Bucks
11	Nymph	3 May 2021	Northampton Township	Bucks
12	Nymph	11 May 2021	Upper Providence Township	Montgomery
13	Nymph	16 Jun 2021	Lower Southampton Township	Bucks
14	Nymph	25 Jun 2021	West Whiteland Township	Chester
15	Nymph	3 May 2021	Northampton Township	Bucks
16	Nymph	6 May 2021	Falls Township	Bucks
17	Nymph	16 Jun 2021	Northampton Township	Bucks
18	Nymph	13 May 2021	West Whiteland Township	Chester	†
19	Nymph	8 Jul 2021	Philadelphia City	Philadelphia
20	Nymph	16 Jun 2021	Lower Southampton Township	Bucks
21	Nymph	13 Apr 2021	Bristol Township	Bucks
22	Nymph	10 Aug 2021	West Whiteland Township	Chester
23	Female	10 Aug 2021	West Whiteland Township	Chester
24	Female	18 Aug 2021	Philadelphia City	Philadelphia
25	Female	24 Aug 2021	Philadelphia City	Philadelphia
26	Female	10 Aug 2021	West Whiteland Township	Chester
27	Female	24 Aug 2021	Philadelphia City	Philadelphia

^*Specimens for which host species were successfully identified.

^†Specimens tested positive for pathogens.

DNA from all 34 partially engorged tick specimens produced positive results when amplified by conventional PCR using universal vertebrate primers for blood meal analysis. Of 7 I. scapularis nymphs used to conduct blood meal analysis, 1 positive PCR result was obtained using avian primers and 1 was obtained using mammalian primers. The PCR assays of 22 H. longicornis nymphs revealed 3 positive results using avian primers and 18 positive results using mammalian primers, and those of the 5 females produced no amplification using avian primers and 5 positive results using mammalian primers. Despite our success in PCR amplification and our best efforts to sequence these amplicons in both directions, only 2 H. longicornis nymphs produced viable sequencing results and were determined to have fed on black-crowned night heron, Nycticorax nycticorax Linnaeus. All other sequences produced inconclusive blood meal results due to poor sequence quality, lower percent identity in comparison to available sequences in GenBank, or no representation of host sequences in GenBank.

DISCUSSION

Our work reported herein aimed to identify the potential of tick-borne pathogens and host associations of partially engorged, actively questing H. longicornis to elucidate its feeding behavior and more inclusively characterize acarological risk. We report the identification of H. longicornis showing evidence of a single infection with Ba. microti and co-infection with Bo. burgdorferi s.l. Although our molecular assays detected Bo. burgdorferi s.l., a complex that contains several species with varied pathogenic capacities (Eisen, 2020), the vast majority (>95%) of Bo. burgdorferi–positive I. scapularis nymphs collected from active surveillance in Pennsylvania are Bo. burgdorferi s.s., as determined by a specific outer surface protein A (OspA) qPCR assay (Pennsylvania DEP, unpubl. data; Dibernardo et al., 2014). In addition, we molecularly confirm partial blood feeding of this invasive tick species on vertebrate hosts. While Bo. burgdorferi DNA has previously been detected in questing H. longicornis in the United States (Price et al., 2021a), to our knowledge, Ba. microti has been found only in host-seeking specimens of this species in its native range of eastern Asia (Zhang et al., 2017; Hong et al., 2019). These findings, therefore, represent the first records of H. longicornis infection with Ba. microti and co-infection with Bo. burgdorferi s.l. and Ba. microti in the United States.

Although reports of partially blood-fed native ticks from active surveillance are rare, the occurrence of this phenomenon in H. longicornis is significantly greater (see Price et al., 2022b), which offers a unique opportunity to examine feeding and pathogen dynamics in tandem. Of the pathogens simultaneously identified in field-collected ticks from the northeastern United States, co-infections with Bo. burgdorferi and Ba. microti are the most common (Johnson et al., 2017; Edwards et al., 2019). Statewide active nymphal I. scapularis surveillance in Pennsylvania during 2022, for instance, yielded only 3.1% (45/1,434) of specimens infected with Ba. microti, and of these 56% (25/45) were co-infected with Bo. burgdorferi s.l. (Pennsylvania DEP, unpubl. data). Borrelia burgdorferi and Ba. microti are both maintained in small mammalian reservoirs, the most notable of which is the white-footed mouse (Peromyscus leucopus Rafinesque) (Goethert et al., 2021). This rodent species is infected with Ba. microti in high rates in central Pennsylvania (26%) (Rocco et al., 2020), and there are increasing reports of H. longicornis collected from the white-footed mice (Thompson et al., 2021; Poje et al., 2022). Laboratory experiments have previously demonstrated the ability of larval and nymphal H. longicornis to acquire Ba. microti by blood feeding on infected white-footed mice (Kusakisako et al., 2015). These findings support that pathogen acquisition by H. longicornis in our study has likely occurred via feeding on an infected host. Furthermore, Wu et al. (2017) demonstrated transstadial maintenance of Ba. microti and transmission to naïve rodents by infected H. longicornis nymphs, indicating their potential as a vector of this parasite. This finding is particularly important for Pennsylvania where babesiosis incidence has significantly increased, averaging nearly 4 new cases annually over the past 15 yr (Pennsylvania Department of Health [DOH], unpubl. data), a pattern consistent with localized observations in the state (Acosta et al., 2013; Liu et al., 2019; Ingram and Crook, 2020) and across the northeastern United States (e.g., Stafford et al., 2014; Goethert et al., 2018). Though considerable, the increase in Pennsylvania is likely an underestimate because babesiosis is not a reportable infection (although efforts are underway to amend Pennsylvania Code Title 28, Chapter 27, and include it in the list of reportable diseases; L. Lind, Pennsylvania DOH, pers. com.). These findings are especially disconcerting considering a recent systematic review of clinical features of human Ba. microti infection that documents severe manifestations and complications including splenic infarction and a 6% mortality rate among infected patients (Dumic et al., 2020). Simultaneous human infection of Ba. microti with Bo. burgdorferi especially has been found to heighten disease severity and duration (Diuk-Wasser et al., 2016). Severe babesiosis presentations have been documented even in immunocompetent patients in southeastern Pennsylvania (Genda et al., 2016). More robust pathogen screening of H. longicornis populations is essential to determine if and how the increasing abundance of this invasive and vector-competent tick affects pathogen maintenance in vertebrate host populations and/or the continuing emergence of babesiosis in the state (Ingram and Crook, 2020). Further investigations are also needed to assess the frequency of H. longicornis feeding on small mammals that serve as pathogen reservoirs to determine the tick's role as a potential enzootic vector.

While detection of pathogen nucleic acids from partially engorged ticks does not demonstrate vector competence, it does establish pathogen acquisition through blood feeding from local hosts and continued maintenance of pathogen DNA while questing, though the viability of infections was not assayed. Furthermore, as only marginal blood components remain after molting, which are detectable with extremely sensitive PCR assays (see Goethert et al., 2021), our analysis is reflective of blood meals acquired within stadia. Therefore, these data provide molecular evidence for H. longicornis interrupted feeding events and indicate multiple successful host acquisitions, as simulated through drag recovery (Nyrhilä et al., 2020). Additionally, the prevalence of H. longicornis repeated feedings may be even greater as the analysis herein could not distinguish blood acquired from different individuals of the same species (Tahir et al., 2020).

Our finding of partially engorged H. longicornis infected with both Ba. microti and Bo. burgdorferi s.l. is of epidemiological significance as repeated blood meals within stadia by pathogen-infected ticks may decouple transstadial transmission efficiency, or lack thereof (e.g., Breuner et al., 2020), from vector competence (Eisen, 2020). That is, the successful reattachment to a host(s), by these partially engorged H. longicornis infected from an earlier, interrupted blood meal, presents a potential pathogen transmission route, exclusive of the tick's capacity to retain infection through the molt, characterizing one of a suite of behavioral, environmental, and genetic determinants indirectly affecting vectorial capacity (De la Fuente et al., 2017). Our results suggest that an understanding of the vector potential of invasive H. longicornis populations may be incomplete without data on their natural host-seeking behaviors and blood-feeding patterns in nature, information attainable only through active surveillance studies.

Molecular analysis of nymphal and adult H. longicornis specimens detected avian (11%) and mammal (85%) host blood. Sequencing of positive amplicons was challenging, and only 2 H. longicornis nymphs yielded successful identification of black-crowned night heron. This finding, representing a new host record, provides more insight into local host use and contributes to the growing lists of wildlife used by H. longicornis (USDA, 2022). This host is of particular interest as night herons are considered to have a cosmopolitan distribution and are migratory in Pennsylvania (Gross, 1923). The population dynamics of this confirmed host thus have the potential to facilitate H. longicornis dispersal and further their invasive range and establishment (Tsao et al., 2021). Nonetheless, mammals, while reported to be preferred hosts (Tufts et al., 2019), were a much larger contributor to incompletely fed H. longicornis. This could be attributable to differential host immune responses and/or grooming capacity/behaviors and warrants further study. Additional sequencing efforts will be necessary to describe specific reservoir host species. Despite limited sequencing information, these data are among the first to identify a vertebrate source in blood meals from host-seeking H. longicornis and provide molecular confirmation of morphological engorgement characterization and thereby more explicit evidence of partial blood-feeding phenomena.

Overall, this study provides important evidence for interrupted feeding of H. longicornis via molecular confirmation of partial blood meals of vertebrate hosts and represents the first U.S. detection of Ba. microti infection and simultaneous detection of Ba. microti and Bo. burgdorferi s.l. in this invasive tick species. The rapidly progressing invasion of H. longicornis in the United States presents an ecological disturbance and disease threat. The incorporation of blood-feeding behavior and pathogen screening in active surveillance efforts can improve understanding of potential environmental impacts and public health risks associated with H. longicornis expansion (Beard et al., 2018). Our findings highlight the growing importance of recognizing the H. longicornis invasion within an ecological and epidemiological context, which should encourage active surveillance to include density measures (e.g., Price et al., 2021b), pathogen (co)infection rates, and human-tick encounters to develop a baseline of risk and inform integrated management strategies.