Surveillance for Serological Evidence of Bourbon and Heartland Virus Infection in White-Tailed Deer and Feral Swine in Texas

Introduction

Over the past two decades in the United States, tick-borne diseases have increased from approximately 20,000 cases reported in 2004 to more than 50,000 cases in 2019 (Rosenberg et al., 2018). As an emerging public health threat with the potential burden of severe and fatal disease, the increase accounts for more than 75% of all vector-borne infections reported annually (Eisen and Eisen, 2018; Paddock et al., 2016; Rosenberg et al., 2018). The increasing incidence of tick-borne diseases has been attributed to an increase of tick-borne pathogens with 40% of 15 new pathogens discovered during the past two decades, and an increase in the population density and expansion of the geographic range of the tick vectors (Eisen and Eisen, 2018; Paddock et al., 2016). Improvements have been made in surveillance that has allowed for a more accurate assessment of the distribution and abundance of ticks and tick-borne pathogens and associated diseases (Eisen and Paddock, 2021; Mader et al., 2021). However, an accurate understanding of the current distribution of medically important ticks is hindered because tick surveillance is not standardized or routinely conducted across the United States. So, unless effective innovative countermeasures can be developed and utilized effectively, the rapidly increasing global trend of tick-borne diseases is likely to continue (Beard, Eisen and Eisen, 2021; Petersen, et al., 2019; Rosenberg et al., 2018).

Among the more recently discovered tick-borne pathogens, Bourbon virus (BRBV) and Heartland virus (HRTV) are the cause of febrile illnesses that may progress to severe and fatal diseases (Dembek et al., 2024). Since the discovery of BRBV (family Orthomyxoviridae; genus Thogotovirus) (Kawaoka and Palese, 2006) in 2014 in Kansas (Kosoy et al., 2015), this virus has been associated with four more cases, including two fatal cases (Brault et al., 2018; Cumbie et al., 2022; Roe et al., 2023). All of the cases have occurred in Kansas, Oklahoma, and Missouri. However, the detection of BRBV antibodies among humans in Missouri and North Carolina suggested that human cases may be underestimated (Bamunuarachchi et al., 2022; Zychowski et al., 2024). The lone star tick species (Amblyomma americanum) has been implicated as the vector of BRBV (Kosoy et al., 2015; Savage et al., 2017). Evidence based on a serosurvey in Missouri, North Carolina, and Virginia, involving several species of domestic and wild mammals revealed that the most likely vertebrate host involved in the maintenance and transmission cycle were white-tailed deer (WTD) (Odocoileus virginianus) and raccoon (Procyon lotor) (Jackson et al., Komar et al., 2020; Garba et al., 2024). HRTV (family Phenuiviridae; genus Bandavirus) (Adams et al., 2017) is transmitted by A. americanum and potentially other tick species (Savage et al., 2016). More than 60 cases of HRTV have occurred, including fatal cases in the Central and Eastern regions of the United States, with deaths from HRTV infections reported from Missouri, Tennessee, Oklahoma, and Virginia (Brault et al., 2018, Mantlo and Haley, 2023). Most of the evidence of infection in vertebrates has been reported from 13 states, including Arkansas, Georgia, Illinois, Indiana, Iowa, Kansas, Kentucky, Missouri, New York, North Carolina, Oklahoma, Tennessee, and Virginia (Riemersma and Komar, 2015). WTD and raccoons have been implicated most as vertebrate hosts of HRTV. The detection of HRTV antibody in raccoons is the only information available prior to this report on these tick-borne viruses in Texas (Riemersma and Komar, 2015).

As a preliminary effort to further understand the geographical distribution of BRBV and HRTV, this study was conducted to determine if there was serological evidence of infection by these viruses among feral swine (Sus scrofa) and WTD in Travis County, Texas.

Materials and Methods

Results

Discussion

Only two species of ticks, I. scapularis (n = 83) and A. americanum (n = 58), were collected in 2020–2021 from feral swine and two species, including 196 I. scapularis and 11 D. albipictus were collected from WTD during 2019–2021 in Travis County, Texas. Why A. americanum was not collected from WTD as one of this tick’s preferred hosts is unknown (Paddock and Yabsley, 2007). However, most of the A. americanum were collected from feral swine during the warmer month of the fall and early spring season that may explain why this species was not collected from WTD that were only sampled during the cooler winter months. While information on the seasonal distribution A. americanum in Travis County, Texas, was not available, our findings were consistent with a study in southeast Kansas that showed a distinct seasonal distribution pattern with peak activity of A. americanum nymphs in May, adult in June, larvae in August and then the abundance declined through October and the winter season (Hroobi et al., 2021). In contrast, the population density of I. scapularis peaked during the early to midwinter months.

While our study did not involve testing these ticks for BRBV and HRTV, serum samples obtained from feral swine were negative for antibody to BRBV, and 12% (9/75) had antibody to HRTV. Of the samples obtained from WTD, all were negative for neutralizing antibody to BRBV, and 6.6% (5/75) were positive for HRTV antibody. The antibody titers for HRTV in feral swine and WTD ranged from 1:20 to 1:160 using a 70% reduction in the virus plaque-forming units. These titers were higher than those reported for five HRTV experimentally infected fawn deer with titers of 1:8 for one deer, 1:16 for three deer, and 1:128 for one deer at day 14 post infection (Clarke et al., 2018a). Our titers ranged from being lower to comparable for 103 of 1428 antibody-positive wild animals that had titers >1:40 using 70% reduction in plaque-forming units (Riemersma and Komar, 2015). The animals included 55 WTD, 33 raccoons, 11 coyotes, and 4 moose that were sampled in 13 states. Also, our observed titers ranged from being lower to comparable for HRTV antibody detected in 5% (38/761) of wild animals with titers ranging from 1:40 to ≥1:320 using 80% reduction in plaque-forming units (Garba et al., 2024). While the antibody titers were comparable with the results reported by others, further evidence that the antibody was elicited by HRTV was because the serum samples were heat-treated to avoid nonspecific reactivity, and the reactivity of the serum samples was detected using virus-specific neutralization tests in at least two separate evaluations. Also, control negative samples used in the assays had no appreciable neutralizing antibodies in the assay. Although there are Phleboviruses related to HRTV in the United States including Sunday Canyon virus, Rio Grande virus, and Lone Star virus, the results of comparative neutralization tests indicated that HRTV was serologically distinguishable from these Phleboviruses but does not exclude the possibility that there are other undiscovered related viruses Bosco-Lauth et al., 2015 Riemersma and Komar, 2015; Lambert et al., 2015). Overall, the isolation of HTRV from the tick’s species would have been more conclusive evidence that this virus was enzootic in Travis County. However, this was a preliminary survey, and the overall low infection rate (IR) of the BRBV and HRTV in field-collected ticks precluded efforts to include virus isolation attempts for these viruses in the lone star ticks collected in our survey. For example, multiple surveys indicated that the IR for HRTV in A. americanum in Georgia was 0.46/1000 ticks and none for BRBV (Romer et al., 2022). The rate for HRTV was 1.4/1000 during 2012 in Missouri (Savage et al., 2013) and 1.8/1000 in 2013 in Missouri (Savage et al., 2016). All 14,193 ticks collected during 2016 in eastern Kansas were negative for BRBV and HRTV (Jackson et al., 2019), and another survey during the same year in eastern Kansas, the IR for HRTV in A. americanum nymphs was 0.42 or 1 in 2381 nymphs (Savage et al., 2016). The rate for HRTV among A. americanum was 9.46/1000 in Illinois (Tuten et al., 2020). In New York, the BRBV IR ranged from 0% to 0.77/1000 in A. americanum (Dupuis et al., 2023) and in a subsequent survey, the HRTV rate was 1.1/1000 in A. americanum ticks (Dupuis et al., 2021). The IR for BRBV in A. americanum was 0.31/1000 for one site during 2013 in Missouri, and at another site during the same year was 7.35/1000 (Savage et al., 2016). In another study in Missouri, the BRBV IR was 0.32 for nymphs/1000 and 0.07/1000 for adults (Savage et al., 2017). In Kansas, the BRBV IR in A. americanum was 0.25/1000 (Savage et al., 2018). Therefore, with these low rates, it is not likely that either BRBV or HRTV would have been isolated from the 58 A. americanum collected during this survey.

Our results revealed that antibody to HRTV, but not to BRBV, was detected in feral swine parasitized by A. americanum and moreso by I. scapularis, and that antibody to HRTV, but not to BRBV, were detected in WTD parasitized predominately by I. scapulari. Although this and other species of ticks, such as Amblyomma maculatum, Dermacentor variabilis, Haemaphysalis leporispalustris, and Ixodes dentatus, have been tested, BRBV and HRTV were only detected in A. americanum ticks until the recent isolation of BRBV from Haemaphysalis longicornis (Cumbie et al., 2022; Dembek et al., 2024; Dupuis et al., 2021; Kosoy et al., 2015; Roe et al., 2023; Savage et al., 2017, 2018). Multiple isolations of BRBV from H. longicornis in several counties of Virginia suggested that other tick species may be vectors of this virus (Cumbie et al., 2022). Of relevance, H. longicornis is a vector of Dabie virus, a bandavirus (Li et al., 2022) that is closely related to HRTV and can transmit HRTV transovarially (Raney et al., 2022). Also, in northern New England, serological evidence of HRTV infection was detected in WTD sampled in Maine, Vermont, and New Hampshire (Riemersma and Komar, 2015) where established populations of lone star ticks are unknown (Springer et al., 2014). Our observation that 12% of serum samples obtained from feral swine were positive for antibody to HRTV is the first observation of possible infection of this vertebrate species by HRTV. Further studies are needed to evaluate the validity of this observation and the possible role of feral swine in the maintenance and transmission cycle of HRTV. In addition, 6.6% (5/75) of the WTD had antibody to HRTV that with the exception of Florida (6.1%) was lower than the rates reported among WTD during surveys conducted in 12 states in the central, eastern, and southern regions of the United States (Clarke et al., 2018b; Dembek et al., 2024 ; Dupuis et al., 2021; Garba et al., 2024; Riemersma and Komar, 2015). These surveys revealed that the HRTV seroprevalence rate by state for WTD was 14% (15/104) in Georgia, 6.1% (4/65) in Florida, 11% (7/63) in Maine, 18% (4/22) in New Hampshire, 9.7% (7/72) in Vermont, 41% (13/42) in North Carolina, 8.5% (21/247) in Virginia, 9.8% (67/686) in New York, 40% (2/5) in Oklahoma, 22% (7/32), in Arkansas, 20% (1/5), in South Carolina, and 24% (6/25) in Louisiana. As reported in this survey involving seroprevalence rate for HRTV among wildlife in 13 states (Riemersma and Komar, 2015), antibody was detected in 4.7% (4/85) raccoons as the only information available for Texas prior to this report on the potential vertebrate hosts of HRTV in Texas (Riemersma and Komar, 2015). These positive raccoons involved a survey that was conducted in 22 counties during 2010 in Southwestern Texas. Our observation of serological evidence of HRTV extends the known distribution of this virus to Central Texas and includes feral swine and WTD as additional species implicated in the state as vertebrate host of the virus. Overall, among the many wildlife species with serological evidence of HRTV infection that was reported during the above surveys, these findings in Texas are consistent with the overall observations that WTD and raccoons were implicated most frequently as potential amplifying hosts of HRTV (Clarke et al., 2018b; Riemersma and Komar, 2015).

Although neutralizing antibody to BRBV was not detected in WTD and feral swine in this survey, neutralizing antibody to this virus was detected in several wildlife species, including 86% (12/14) of WTD and 50% (31/62) among raccoon in Missouri (Jackson et al., 2019), as high as 66.5% in New York (Dupuis et al., 2023) and 56% (18/32) of WTD in North Carolina (Komar et al., 2020). In a more recent survey, the seroprevalence rate for BRBV in Virginia among WTD was 13.3% (33/249) and 11.6% (31/112) for raccoons (Garba et al., 2024). The seroprevalence rate for BRBV during the fall of 2021 and 2022 in St. Louis, Missouri, was 90% (9/10) for raccoons, 54% (7/13) for WTD, 100% for bobcats (Lynx rufus), 100% (1/1) for red foxes (Vulpes vulpes), and 14% (1/7) for opossums (Didelphis virginiana) (Bamunuarachchi et al., 2024). The rate during the spring of 2022 in St. Louis was 100% (3/3) for coyotes, 93% (14/15) for raccoons, 79% (11/14) for WTD, 100% (2/2) for bobcats, and 0% (0/11) for opossums. Although the seroprevalence rates in WTD and raccoons for BRBV were lower than for HRTV in these animals, both species were implicated more frequently among all of the wildlife species as potential amplifying vertebrate hosts for these viruses (Hao et al., 2022; Jackson et al., 2019; Komar et al., 2020; Roe et al., 2023). The common occurrence of serological evidence of infection of these viruses in many different wildlife species, especially WTD, helped to validate our findings of HRTV infection among WTD in Central Texas. The failure to detect antibody to BRBV in Central Texas during this survey and during the previous survey in 22 counties in southwest Texas (Riemersma and Komar, 2015), suggested that this virus has not yet spread to these areas of Texas, or that the virus was not detected because of the limited sampling. As described above, serological evidence has implicated several wildlife species as potential amplifying host of HRTV and BRBV with WTD and raccoons being the most frequently implicated vertebrate species. While our findings of serological evidence of HRTV infection in WTD were consistent with previous observations, experimental infection of WTD, raccoons, goats, chickens, rabbits, and hamsters with HRTV failed to produce viremia, but elicited a limited neutralizing antibody response in all animals as evidence of infection (Bosco-Lauth et al., 2016; Clarke et al., 2018b). As a result, these findings and the observations that HTRV virus has never been isolated from a mammalian wildlife species (Bosco-Lauth et al., 2016; Feng et al., 2024; McMullan et al., 2012; Muehlenbachs et al., 2014) led to the conclusion that these and other vertebrate species were not competent amplifying host (Clarke et al., 2018b; Mantlo and Haley, 2023). Although serological evidence of BRBV infection has been detected in several wildlife species and a recent report indicated that BRBV RNA was detected in a coyote (Bamunuarachchi et al., 2024), the amplifying host of BRBV remains unknown (Dupuis et al., 2023). These results suggested that HRTV and most likely BRBV could be maintained in infected A. americanum via vertical transmission (Godsey et al., 2016), and that transmission could occur without the need for virus replication in the vertebrate host from infected tick-to-uninfected ticks while cofeeding on a vertebrate host (Godsey et al., 2021). Cofeeding that avoided the need for a viremic host has been described as a mechanism for the transmission of Thogoto virus (Jones et al., 1987), and Crimean–Congo hemorrhagic fever virus (Gargili et al., 2017), thus suggesting that A. americanum may be the sole reservoir for both HRTV and BRBV or that other unidentified vertebrate species may serve as reservoirs (Mantlo et al., 2023).

Conclusions

As indicated above, the only previous report for Texas regarding the prevalence of BRBV and HRTV was the detection of HRTV antibody in raccoons in Southwestern Texas. In Travis County, Texas, A. americanum and I. scapularis were the tick species more commonly found parasitizing WTD and feral swine, thus, these vertebrate species were serving as competent hosts for ticks that are a public health concern for humans and animals. Whether or not these animals were serving as reservoir hosts for BRBV and HRTV is unknown. Furthermore, because these viruses have not been isolated from any wild or domestic animals except for the detection of BRBV viral RNA in a coyote, the question of vertebrate reservoir(s) remains inconclusive. The very high prevalence of HRTV antibody among WTD and raccoons in northwestern Missouri may provide an opportunity to target these species for sampling for virus isolation as part of the wildlife-serosurveillance program to understand more conclusively the vertebrate species involved as possible reservoir hosts for HRTV (Bosco-Lauth et al., 2015). Finally, the preliminary findings of this survey implied that HRTV was enzootic in Travis County, Texas. This observation and the reported detection of HRTV in raccoons in Southwestern Texas should be considered as evidence for further ecological and epidemiological studies because of the potential of this virus to cause severe and fatal human infections.