The invasive tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was first documented to infest sheep in New Jersey in 2017 (Rainey et al. 2018) and is now known to be widely established in the eastern United States, where it parasitizes a wide range of species of mammals and birds (Tufts et al. 2019, USDA 2024). In the United States, this tick species was demonstrated to be an experimental vector for, and naturally infected with, Theileria orientalis Ikeda, a causative agent of theileriosis in cattle (Thompson et al. 2020, Dinkel et al. 2021, Dye-Braumuller et al. 2023). However, this species failed to experimentally demonstrate the ability to vector Babesia bovis (Poh et al. 2024). With regard to human pathogens, H. longicornis was shown to be an experimental vector for Heartland virus, Powassan virus, and the Rocky Mountain spotted fever agent, Rickettsia rickettsii (Stanley et al., 2020, Raney et al. 2022a, 2022b), but evidence for natural infection with these disease agents is still lacking. Conversely, H. longicornis has occasionally been found naturally infected with causative agents of anaplasmosis (Anaplasma phagocytophilum) and Lyme disease (Borrelia burgdorferi sensu stricto; Spirochaetales: Spirochaetaceae) (Price et al. 2022a, 2022b, 2023, Eleftheriou et al. 2024), but experimental studies indicate this tick is either incapable or ineffective as a vector of these disease agents (Breuner et al. 2020, Levin et al. 2021). There also was a recent report of Bourbon virus RNA being detected in H. longicornis ticks collected from Virginia (Cumbie et al. 2022), but the experimental transmission of this virus has not been demonstrated.

Breuner et al. (2020) showed that H. longicornis larvae could acquire Borrelia burgdorferi s.s. while feeding to completion on infected Mus musculus mice (56% of 32 examined fed larvae were infected), but that the infection was lost during the molt to the nymphal stage (0% of 520 examined molted nymphs were infected). However, there have been subsequent reports of occasional natural infection of H. longicornis nymphs and adults with B. burgdorferi s.s. or B. burgdorferi sensu lato (Price et al. 2021, 2023, Dye-Braumuller et al. 2023). Partially engorged questing nymphs and adults have also been collected by drag sampling (Price et al. 2022b, 2023). These findings are noteworthy as previous experimental work showed that Ixodes scapularis Say (Acari: Ixodidae) larvae and nymphs having taken a partial blood meal on a B. burgdorferi s.s.-infected rodent host were able to reattach to a new host and feed to completion, and the partially fed infected nymphs were also able to transmit B. burgdorferi s.s. to the naïve hosts on which they completed their blood meal (Piesman 1991, Shih and Spielman 1993).

In this study, we sought to similarly evaluate if H. longicornis nymphs can acquire B. burgdorferi s.s. during interrupted feedings when allowed to remain attached for only 48 or 72 h on confirmed infected M. musculus mice as opposed to the 96–120 h required to feed to repletion based on previous work (Breuner et al. 2020). Assessments of the partially fed nymphs included determination of weight and engorgement index, compared to unfed nymphs, to confirm feeding, and testing by a multiplex PCR amplicon sequencing assay for the presence of B. burgdorferi s.s.

First, 10 CD-1 M. musculus mice (Charles River Laboratories, Wilmington, MA) were infected with the B. burgdorferi s.s. B31 strain (originally isolated from I. scapularis ticks collected on Shelter Island, NY). This was accomplished by exposing the mice to feeding by infected I. scapularis nymphs (B. burgdorferi s.s.-infected I. scapularis colony; Centers for Disease Control and Prevention, Fort Collins, CO): up to 20 nymphs with an estimated B. burgdorferi s.s. infection rate of 90% was introduced to each mouse. Eight mice were then confirmed infected with B. burgdorferi s.s. by culture of ear tissue biopsies that were taken 3 wk after nymphal tick infestation; cultures were grown in BSK II media (in-house) at 34°C and aliquots were examined by dark-field microscopy (400× magnification).

Next, H. longicornis nymphs were allowed to take partial blood meals from the 8 infected mice. Feeding capsules were applied to the mice, as previously described by Breuner et al. (2020), to facilitate attachment and recovery of the ticks. The specific pathogen-free H. longicornis nymphs used were obtained from a colony started with ticks from Westchester County, NY, and maintained at the Medical Entomology Laboratory of the Centers for Disease Control and Prevention, Atlanta, GA. Four mice each were assigned to the 48 h or 72 h feeding duration groups, with each mouse having 20 nymphs initially introduced into its feeding capsule. If capsules became dislodged during the feeding period and ticks were able to escape into the mouse holding cage, the mice were removed from the study. Mice were euthanized at either 48 or 72 h after the ticks were introduced, the capsules were removed, and all ticks that were not attached to the carcass and found loose in the cage were removed and discarded. Only ticks that were found attached to the mouse carcass at the time of euthanasia were included in the further steps of the study. Mouse carcasses were held for 24 h after euthanasia in a tick drop-off cage to allow ticks to detach naturally from the deceased host (Piesman 1991). Tick attachment to the deceased host was assessed at 4 and 24 h after euthanasia, and all detached ticks were collected from the water at the bottom of the drop-off cage. If ticks had not detached naturally from the host at 24 h after euthanasia, they were removed from the carcass with forceps (each mouse had 1–2 ticks manually removed after 24 h).

The partially fed H. longicornis nymphs we recovered were assessed for evidence of engorgement and infection with B. burgdorferi s.s. All the ticks that dropped from each mouse were weighed as a group as they did not weigh enough to register individually using our microbalance (Ohaus Explorer E01140, Parsippany, NJ). The total weight was divided by the number of ticks to obtain an average weight (mg) per tick. A group of unfed H. longicornis nymphs were also weighed as a comparison group. An engorgement index (the ratio between total body length and scutum length) was determined for each individual tick recovered from the mice (Fig. 1), and for unfed ticks serving as a comparison, following Price et al. (2022b) and based on the engorgement index 1 of Yeh et al. (1995). The scutal length and body length were measured using Olympus cellSens Imaging Software (Olympus, Waltham, MA). All collected partially fed ticks were assessed for B. burgdorferi s.s. infection, based on detection of the Borrelia flaB target (Hojgaard et al. 2020) and using a multiplex polymerase chain reaction (PCR) amplicon sequencing assay described by Osikowicz et al. (2024).

Animal use and experimental procedures were conducted in accordance with an approved protocol on file with the Centers for Disease Control and Prevention, Division of Vector-Borne Diseases, Institutional Animal Care and Use Committee.

Of the 80 H. longicornis nymphs placed on 4 mice (N01, N04, N06, and N07 were removed from the study due to their capsules becoming dislodged) 57 (71%) nymphs were found to be attached at the time of mouse euthanasia at either 48 or 72 h after the nymphs were introduced onto the mice (Table 1). The nymphal attachment rate was higher for the 48 h group at 80% for both mice, compared to 60% and 65% for the 2 mice in the 72 h group (Table 1). The median engorgement index of each group ranged from 2.98 (2.88–3.17) for unfed nymphs to 3.04 (2.94–3.20) and 3.06 (2.88–3.23) for nymphs attached for 48 h and 3.34 (3.00–3.57) and 3.79 (2.94–4.30) for nymphs fed on the 2 mice in the 72 h attachment group (Table 1 and Figure 1). The scutal indices reported by Price et al. (2022b) for unfed (2.98) and engorged (3.40) H. longicornis nymphs were similar to the engorgement indices for unfed ticks (2.98), and ticks that fed on mice for 72 h (3.34 and 3.79 for the 2 individual mice) described in this study. The mean weight was 0.22 mg for unfed nymphs, increasing to 0.33 and 0.35 mg for nymphs attached to each of 2 mice for 48 h and 0.56 and 0.60 mg for nymphs attached to each of 2 mice for 72 h group (Table 1). The mean weights of partially engorged nymphs having been attached for either 48 or 72 h in this study (0.33–0.60 mg) were much lower than reported for fully engorged and naturally detached nymphs (3.03 mg) in a previous study (Zheng et al. 2011). This is not surprising as ixodid ticks tend to ingest the majority of the blood meal in the last 12–36 h of their natural feeding period (Balashov 1972), and our study aimed to interrupt the blood meal before the nymphs had ingested enough blood to be able to molt to adults without taking in additional blood. In our study, the partially fed nymphs that had detached themselves from the deceased hosts were found on the underside of the lid, and sides of the cage. Although we cannot know if given the opportunity the partially fed ticks would have attached to a subsequent host, these ticks appeared active and presumably were seeking a second host.

Unexpectedly, B. burgdorferi s.s. was detected in a higher number of engorged nymphs (2/32) at the 48 h attachment timepoint compared to the 72 h timepoint (0/25), despite nymphs removed at 72 h being attached for 24 h longer (Table 1). This result contrasts with the finding of Piesman (1991) for I. scapularis (I. dammini) larvae, where each increase in larval feeding time (from 8 to 16, 24, 48, and ≥72 h) on an infected mouse corresponded to an increased infection rate for B. burgdorferi s.s., and the infection rate of partially fed larvae had reached 68% (16/24 ticks) at the 48 h timepoint. Moreover, the percentages of infected partially fed H. longicornis nymphs in our study of 0–12% (0/12, 0/13, 0/16, and 2/16 ticks; Table 1) across individual mice was much lower than the 56% (18/32 ticks) reported for fully fed H. longicornis larvae (having typically completed their blood meals in 96–120 h) in a previous study with the same strain of B. burgdorferi s.s. (Breuner et al. 2020). It also is noteworthy that the nymphs acquired B. burgdorferi s.s. from only 1 of the 2 confirmed infected mice exposed to nymphal feeding for 48 h and from none of the 2 infected mice with nymphs feeding for 72 h (Table 1). We have no clear explanation for this surprising finding. Based on previous studies, H. longicornis appears to be less effective in acquiring B. burgdorferi s.s. from infected hosts compared to I. scapularis, and we cannot rule out the possibility that the mouse from which the nymphs acquired spirochetes had a higher bacteremia than the other mice at the time of nymphal feeding. The small sample size for examined partially fed nymphs (12–16 per mouse) also could have yielded stochastic results, and further investigation would be required to evaluate the relationship between feeding duration and the infection rate.

We conclude that the potential for H. longicornis nymphs to acquire B. burgdorferi s.s. via interrupted feeding in the first 72 h of a blood meal on an infected host is low. However, it remains a plausible scenario of how field-collected questing H. longicornis nymphs that have naturally detached from an infected host before completing their blood meal can be infected with B. burgdorferi s.s. DNA, without the need for transstadial transmission of spirochetes from the larval stage to have occurred. It remains unknown if H. longicornis ticks containing B. burgdorferi s.s. DNA from a partial blood meal can maintain viable spirochetes, or if they could infect another competent host while completing their blood meal.