One Confirmed and 2 Suspected Cases of Heartland Virus Disease.

Heartland virus is a tickborne phlebovirus first identified in Missouri in 2009; 11 human cases have been reported in the literature. Reported hallmarks of infection have included fever, malaise, anorexia, gastrointestinal complaints, thrombocytopenia, neutropenia, and aminotransferase elevations. We report 1 confirmed and 2 suspected cases and discuss implications for case-finding.

Heartland Virus in Lone Star Ticks, Alabama, USA.

We detected Heartland virus (HRTV) in lone star nymphs collected in 2018 in northern Alabama, USA. Real-time reverse transcription PCR selective for the small segment of the HRTV genome and confirmatory sequencing of positive samples showed high identity with HRTV strains sequenced from Tennessee and Missouri.

A standardized method for the construction of a tick drag/flag sampling approach and evaluation of sampling efficacy.

Drag sampling and flagging are two of the most effective and widely applied techniques to monitor tick populations. Despite the importance of this sampling strategy, there is a lack of standardized protocols for the construction of an inexpensive tick drag/flag. To this end, we provide a step-by-step protocol that details the construction of a tick drag/flag. We provide evidence of efficacy by comparing results obtained over 3-months at 108 locations within the William B. Bankhead National Forest, Alabama, USA. Overall, our drag/flag sampling approach yielded 1127 larvae, 460 nymphs, and 53 adults for a total of 1640 ticks representing three species. We detected significant patterns in Amblyomma americanum abundance for nymphs and adults with greater counts in June (ß = 0.91 ± 0.36, 95% CI 0.55-1.27; ß = 2.44 ± 0.63, 95% CI 1.81-3.07, respectively) and July (ß = 0.73 ± 0.36, 95% CI 0.37-1.09; ß = 1.65 ± 0.66, 95% CI 0.99-2.31, respectively) as compared to August. We also detected a significant difference in tick captures by tick drag/flag fabric type with greater captures when muslin was used as compared to flannel (ß = 1.07 ± 0.06, 95% CI 1.01-1.13). Our goal is to provide instructions to assemble a highly effective tick drag/flag using minimal supplies. Evaluation and improvements of sampling techniques is essential to understand impacts of landscape management and larger stressors, such as climate change on tick populations but also for enhancing detection of invasive non-native species.

Novel Clinical and Pathologic Findings in a Heartland Virus-Associated Death.

We describe an investigation into a Heartland virus (HRTV)-associated death in Tennessee with novel clinical and pathologic findings. HRTV can cause rapidly fatal, widely disseminated infection with multisystem organ failure in patients without substantial comorbidities. We identified viral antigen in multiple organ tissues where it was not detected previously.

La Crosse Encephalitis Virus Infection in Field-Collected Aedes albopictus, Aedes japonicus, and Aedes triseriatus in Tennessee.

La Crosse virus (LACV) is a mosquito-borne virus and a major cause of pediatric encephalitis in the USA. La Crosse virus emerged in Tennessee and other states in the Appalachian region in 1997. We investigated LACV infection rates and seasonal abundances of the native mosquito vector, Aedes triseriatus, and 2 recently introduced mosquito species, Ae. albopictus and Ae. japonicus, in an emerging disease focus in Tennessee. Mosquitoes were collected using multiple trapping methods specific for Aedes mosquitoes at recent human case sites. Mosquito pools were tested via reverse transcriptase-polymerase chain reaction (RT-PCR) of the S segment to detect multiple Bunyamwera and California serogroup viruses, including LACV, as well as real-time RT-PCR of the M segment. A total of 54 mosquito pools were positive, including wild-caught adult females and laboratory-reared adults, demonstrating transovarial transmission in all 3 species. Maximum likelihood estimates (per 1,000 mosquitoes) were 2.72 for Ae. triseriatus, 3.01 for Ae. albopictus, and 0.63 for Ae. japonicus. We conclude that Ae. triseriatus and Ae. albopictus are important LACV vectors and that Ae. japonicus also may be involved in virus maintenance and transmission.

Seroepidemiologic study of human infections with spotted fever group Rickettsiae in North Carolina.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

An outbreak of bed bug infestation in an office building.

Since 2000, resurgence in bed bugs has occurred in the U.S. Reports of infestations of homes, hospitals, hotels, and offices have been described. On September 1, 2011, complaints of itching and bites among workers in an office were reported to the Tennessee Department of Health. A retrospective cohort study and environmental assessments were performed in response to the complaints. Canines certified to detect live bed bugs were used to inspect the office and arthropod samples were collected. Of 76 office workers, 61 (80%) were interviewed; 39 (64%) met the case definition. Pruritic maculopapular lesions were consistent with arthropod bites. One collected arthropod sample was identified as a bed bug by three entomologists. Exposures associated with symptoms included working in a cubicle in which a canine identified bed bugs (risk ratio [RR]: 1.8; 95% confidence interval [CI]: 1.3-3.6), and self-reported seasonal allergies (RR: 1.6, 95% CI: 1.0-2.4). Bed bugs represent a reemerging and challenging environmental problem with clinical, psychological, and financial impacts.

Utilization of a zoo for mosquito (Diptera: Culicidae) diversity analysis, arboviral surveillance, and blood feeding patterns.

Zoos provide a unique opportunity to study mosquito feeding ecology as they represent areas where exotic animals, free-roaming native animals, humans, and mosquito habitats overlap. Therefore, these locations are a concern for arbovirus transmission to both valuable zoo animals and human visitors. We sampled mosquitoes in and around The Nashville Zoo at Grassmere in Tennessee, USA, over 4 months in 2020 using 4 mosquito trap methods and 12 sampling locations. Mosquitoes were identified to species, Culex mosquitoes were analyzed for arboviruses, and all engorged mosquitoes were preserved for host usage analysis. We captured over 9,000 mosquitoes representing 27 different species, including a new species record for Davidson County, TN (Culex nigripalpus Theobald). Minimum infection rates for West Nile virus (WNV) (Flaviviridae: Flavivirus), St. Louis encephalitis virus (Flaviviridae: Flavivirus), and Flanders virus (Hapavirus: Rhabdoviridae) were 0.79, 0, and 4.17, respectively. The collection of 100 engorged mosquitoes was dominated by Culex pipiens pipiens Linnaeus (38%), Culex erraticus Dyar and Knab (23%), and Culex pipiens pipiens-Culex pipiens quinquefasciatus hybrids (10%). Host DNA from 84 engorged mosquitoes was successfully matched to a variety of host species (n = 23), with just 8 species belonging to the zoo. Wild birds were the most frequently fed upon host, in particular northern cardinals (Cardinalis cardinalis L. Passeriformes: Cardinalidae), which are competent WNV reservoirs. Taken together, our results demonstrate the utility of zoos as sentinels for emerging pathogens, for studying wildlife and human risk of zoonotic diseases, and for assessing vector diversity.

Eastern equine encephalitis in Tennessee: 2002-2008.

Human and equine outbreaks caused by eastern equine encephalomyelitis virus (EEEV) typically occur in North America adjacent to coastal wetlands associated with the presence of Culiseta melanura (Coquillet) mosquitoes. Eastern equine encephalomyelitis (EEE) is an emerging disease in Tennessee, as the first records of equine disease began in 2002. In 2006 we trapped and tested mosquitoes for EEEV at hardwood swamps in western Tennessee that were at the epicenter of a multi-equine outbreak in fall of 2005. Additionally, the Tennessee Valley Authority tested mosquito pools collected in Tennessee swamps from 2000 to 2007 for the presence of arboviruses. Two pools of EEEV positive Culex erraticus (Dyer and Knab) mosquitoes were found (one each in 2003 and 2004) in a county adjacent to where the 2005 outbreak occurred. In 2008, another EEE outbreak involving multiple horses occurred in West Tennessee. A brain specimen was collected from a horse during this outbreak and the first isolate of EEEV from Tennessee was obtained. In total, 74,531 mosquitoes collected from 2000 to 2008 were tested via polymerase chain reaction and VecTest for EEEV. The traditional enzootic vector, Cs. melanura, was found in low numbers at all collection sites. Cx. erraticus, however, was consistently found in high numbers and was the only mosquito species in which EEEV was detected. We suggest that EEE transmission may be maintained by Cx. erraticus in a nontraditional cycle. We discuss the importance of a nontraditional cycle from the perspective of EEEV adaptation and emergence.

Rickettsia parkeri in Amblyomma americanum ticks, Tennessee and Georgia, USA.

To determine the geographic distribution of the newly recognized human pathogen Rickettsia parkeri, we looked for this organism in ticks from Tennessee and Georgia, USA. Using PCR and sequence analysis, we identified R. parkeri in 2 Amblyomma americanum ticks. This rickettsiosis may be underdiagnosed in the eastern United States.

Use of hamster-baited no. 17 Trinidad mosquito traps at an eastern equine encephalomyelitis focus in Tennessee.

We set out no. 17 Trinidad traps baited with hamsters at a swamp in Tennessee, where recent eastern equine encephalomyelitis (EEE) equine outbreaks had occurred, to determine which mosquito species at this site were attracted to these traps. We also set out CO2-baited CDC light traps for comparison. Of 11 species present in CO2-baited CDC light traps, only Culex (Melanoconion) erraticus (Dyar and Knab) blood fed on hamsters in the Trinidad traps. Significantly less Cx. erraticus mosquitoes entered Trinidad traps per trap night (mean = 17.6, median = 6.0) compared with CDC traps (mean = 38.7, median = 35.0). Advantages and disadvantages in using hamster-baited no. 17 Trinidad traps compared with CO2-baited CDC light traps to capture Cx. erraticus mosquitoes are discussed. Further understanding of the feeding behavior and ecology of this potential vector is warranted.

Vector competence of eastern and western forms of Psorophora columbiae (Diptera: Culicidae) mosquitoes for enzootic and epizootic Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) continues to circulate enzootically in Mexico with the potential to re-emerge and cause disease in equines and humans in North America. We infected two geographically distinct mosquito populations of eastern Psorophora columbiae form columbiae (Chiapas, Mexico and Texas, United States) and one mosquito population of western Psorophora columbiae form toltecum (California, United States) with epizootic and enzootic IE VEEV and epizootic IAB VEEV. We detected no differences between epizootic and enzootic IE viruses in their ability to infect any of the mosquito populations analyzed, which suggested that neither species selects for epizootic IE viruses. Psorophora columbiae f. columbiae (Texas) were significantly less susceptible to infection by epizootic IE than Ps. columbiae f. columbiae (Mexico). Psorophora columbiae f. toltecum populations were more susceptible than Ps. columbiae f. columbiae populations to epizootic IE and IAB viruses.

Evaluation of a Spotted Fever Group Rickettsia Public Health Surveillance System in Tennessee.

Spotted fever group (SFG) rickettsioses are endemic in Tennessee, with ∼2,500 cases reported during 2000-2012. Because of this substantial burden of disease, we performed a three-part evaluation of Tennessee's routine surveillance for SFG rickettsioses cases and deaths to assess the system's effectiveness. Tennessee Department of Health (TDH) SFG rickettsioses surveillance records were matched to three patient series: 1) patients with positive serologic specimens from a commercial reference laboratory during 2010-2011, 2) tertiary medical center patients with positive serologic tests during 2007-2013, and 3) patients identified from death certificates issued during 1995-2014 with SFG rickettsiosis-related causes of death. Chart reviews were performed and patients were classified according to the Council of State and Territorial Epidemiologists' case definition. Of 254 SFG Rickettsia-positive serologic specimens from the reference laboratory, 129 (51%) met the case definition for confirmed or probable cases of rickettsial disease after chart review. The sensitivity of the TDH surveillance system to detect cases was 45%. Of the 98 confirmed or probable cases identified from the medical center, the sensitivity of the TDH surveillance system to detect cases was 34%. Of 27 patients identified by death certificates, 12 (44%) were classified as confirmed or probable cases; four (33%) were reported to TDH, but none were correctly identified as deceased. Cases of SFG rickettsioses were underreported and fatalities not correctly identified. Efforts are needed to improve SFG rickettsiosis surveillance in Tennessee.

Challenges to Arboviral Surveillance in Tennessee: Health-Care Providers' Attitudes and Behaviors.

Surveillance of arboviruses depends on health-care providers' ability to diagnose and report human cases of disease. The purposes of this study were to assess Tennessee providers' 1) self-efficacy toward diagnosis and management, 2) clinical practices, and 3) variation in these measures by provider characteristics. A survey was e-mailed to 13,851 providers, of which 916 (7%) responded. Respondents diagnosed more arboviruses in the previous year than were recorded in surveillance records, an indication of underreporting. Respondents had low to moderate self-efficacy toward diagnosis and management of arboviruses. Although more than 70% (N = 589) used paired serology, only 46% (N = 396) asked patients to return for a convalescent specimen draw within the correct time frame. One of the most commonly reported barriers to testing was uncertainty about which tests to order. Providers working in family medicine and urgent care, nurse practitioners, and those at outpatient facilities had lower rates of high self-efficacy than their counterparts working in other settings and from other specialties. Clinical practices were influenced by specialty, designation, setting, and geography but not by years of experience. Education to improve arboviral surveillance in Tennessee is warranted. Topics could include proper diagnosis and management, appropriate testing and overcoming barriers to testing, and public health reporting.

Human Infections by Multiple Spotted Fever Group Rickettsiae in Tennessee.

Rocky Mountain spotted fever is the most common tick-borne disease in Tennessee. However, Rickettsia rickettsii has rarely been isolated from endemic ticks, suggesting rickettsioses may be caused by other species. A total of 56 human serum samples that were serologically positive for exposure to Rickettsia were obtained from commercial laboratories in 2010 and 2011. In addition, 20 paired sera from patients with encephalitis and positive Rickettsia serology were obtained from the Tennessee Unexplained Encephalitis Surveillance (TUES) study. Using an immunofluorescence assay, reactivity of the sera to R. rickettsii, Rickettsia montanensis, Rickettsia parkeri, and Rickettsia amblyommii was tested, and a comparison of endpoint titers was used to determine the probable antigen that stimulated the antibody response. Cross-absorption was conducted for 94.8% (N = 91) of the samples due to serologic cross-reactivity. Of the commercial laboratory samples, 55.4% (N = 31) had specific reactivity to R. amblyommii and 44.6% (N = 25) were indeterminate. Of the paired TUES samples, 20% (N = 4) had specific reactivity to R. amblyommii, 5% (N = 1) to R. montanensis, and 5% (N = 1) to R. parkeri Patients with specific reactivity to R. amblyommii experienced fever (75%), headache (68%) and myalgia (58%). Rash (36%) and thrombocytopenia (40%) were less common. To our knowledge, this is the first time R. amblyommii has been reported as a possible causative agent of rickettsioses in Tennessee.

Potential role of sylvatic and domestic African mosquito species in dengue emergence.

Dengue virus 2 (DENV-2) strains that circulate in sylvatic habitats of Senegal and other parts of west Africa are believed to represent ancestral forms that evolved into endemic/epidemic strains that now circulate widely in urban areas of the tropics. Previous studies suggested that the evolution of the endemic/epidemic strains was mediated by adaptation to the peridomestic mosquito vectors Aedes aegypti and Ae. albopictus. We conducted experimental infections using sylvatic and peridomestic Senegalese mosquitoes, and both sylvatic and urban DENV-2 strains to determine if endemic DENV-2 adaptation was vector species specific, and to assess ancestral vector susceptibility. Aedes furcifer and Ae. luteocephalus, probable sylvatic vectors, were highly susceptible to both sylvatic and urban DENV-2 strains. In contrast, sylvatic Ae. vittatus and both sylvatic and peridomestic populations of Ae. aegypti were relative refractory to all DENV-2 strains tested. These results indicate that adaptation of DENV-2 to urban vectors did not result in a loss of infectivity for some African sylvatic vectors. Implications for dengue emergence in west Africa are discussed.

Meconial peritrophic matrix structure, formation, and meconial degeneration in mosquito pupae/pharate adults: histological and ultrastructural aspects.

The noncellular peritrophic matrix (PM) that forms around the food bolus in the midgut of many arthropod species may influence the fate of ingested microbes. In mosquitoes, PMs have been identified in the pupal as well as larval and adult stages. In pupae, the PMs surround the meconium, the sloughed larval midgut epithelium. Meconial PM1 (MPM1) forms early in the pupal stadium, and a second meconial PM (MPM2) sometimes forms around the time of adult emergence. A recent study suggests that MPMs contribute to the sterilization of the adult midgut by sequestering microorganisms ingested during the larval stage, which, along with remaining meconial material, are egested after adult emergence. We have compared MPM1 formation and patterns of meconial degeneration in representative species in five mosquito genera and identified a temporal association between MPM1 formation, meconial degeneration, and apolysis. Ultrastructural study of MPM1 and MPM2 in Aedes aegypti (L.) revealed that MPM1 seems to be structurally different from either the larval or adult PMs, whereas MPM2 more closely resembles PM formed around a bloodmeal in adult females. Our results are consistent with the microbial sequestration role.

Dengue among American missionaries returning from Jamaica, 2012.

Dengue is an acute febrile illness caused by any of four mosquito-transmitted dengue virus (DENV) types. Dengue is endemic in Jamaica, where an epidemic occurred in 2012. An investigation was conducted by multiple agencies for 66 missionaries traveling from nine US states to Jamaica after 1 missionary from the group was confirmed to have dengue. Travelers were offered diagnostic testing, and a survey was administered to assess knowledge, behaviors, and illness. Of 42 survey respondents, 9 (21%) respondents reported an acute febrile illness during or after travel to Jamaica. Of 15 travelers that provided serum specimens, 4 (27%) travelers had detectable anti-DENV immunoglobulin M antibody, and 1 traveler also had DENV-1 detected by reverse transcriptase polymerase chain reaction. Recent or past infection with a DENV was evident in 93% (13 of 14) missionaries with available sera. No behavioral or demographic factors were significantly associated with DENV infection. This investigation shows that even trips of short duration to endemic areas present a risk of acquiring dengue.

Environmental investigation following the first human case of babesiosis in Tennessee.

Babesiosis is an emerging tick-borne zoonotic disease in the United States caused by Babesia parasites. In 2009, the first case of babesiosis was documented in Tennessee. Environmental investigation at the reported site of tick exposure included collection of ticks and specimens from eastern cottontail rabbits (Sylvilagus floridanus) and white-tailed deer ( Odocoileus virginianus ) that were tested for piroplasms by molecular and serologic methods. One hundred and sixty-six Ixodes scapularis ticks and biological samples from 8 rabbits and 5 deer were collected. Ixodes scapularis were PCR positive for Babesia odocoilei (n = 7, 4%) and Theileria cervi (n = 24, 14%). Deer were seropositive for B. odocoilei and PCR positive for T. cervi. Rabbits were seropositive for B. odocoilei and Babesia sp. MO1, and 1 rabbit was PCR positive for Babesia sp. MO1. In summary, zoonotic Babesia sp. MO1 infection in rabbits is reported here for the first time in Tennessee as well as infection of deer and I. scapularis ticks with 2 other piroplasms of veterinary importance.

Inadequacy of IgM antibody tests for diagnosis of Rocky Mountain Spotted Fever.

Among 13 suspected Rocky Mountain spotted fever (RMSF) cases identified through an enhanced surveillance program in Tennessee, antibodies to Rickettsia rickettsii were detected in 10 (77%) patients using a standard indirect immunofluorescent antibody (IFA) assay. Immunoglobulin M (IgM) antibodies were observed for 6 of 13 patients (46%) without a corresponding development of IgG, and for 3 of 10 patients (30%) at least 1 year post-onset. However, recent infection with a spotted fever group rickettsiae could not be confirmed for any patient, based on a lack of rising antibody titers in properly timed acute and convalescent serologic specimens, and negative findings by polymerase chain reaction testing. Case definitions used in national surveillance programs lack specificity and may capture cases that do not represent current rickettsial infections. Use of IgM antibodies should be reconsidered as a basis for diagnosis and public health reporting of RMSF and other spotted fever group rickettsiae in the United States.