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Feral swine, Sus scrofa L., have become a nuisance to landowners across the United States by damaging agriculture, property, and ecosystems. Additionally, these animals have been found to host various ixodid ticks including Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor variabilis (Say), and Ixodes scapularis Say, which can maintain and transmit several rickettsial pathogens to livestock, wildlife, and humans. Though previous research has identified the maintenance cycle of several rickettsial pathogens in ticks and native wildlife, little is known about the role S. scrofa plays in supporting ixodid ticks and the pathogens these ticks could be harboring. This study sought to identify rickettsial agents (Rickettsiales: Anaplasmataceae and Rickettsiaceae) in ticks collected from S. scrofa obtained in Florida and South Carolina. Overall, ticks from four species (A. americanum, D. variabilis, I. scapularis, and A. maculatum) totaling 258 collected individuals were obtained from S. scrofa (n = 45). We found an Ehrlichia chaffeensis Anderson et al. infection prevalence in A. americanum of 2.7% and 2.9% in Florida and South Carolina, respectively. A Rickettsia parkeri Lackman et al. prevalence of 100% and 33% was found in A. maculatum from Florida and South Carolina, respectively. Additionally, a 0.9% infection prevalence of R. parkeri was identified in A. americanum collected in South Carolina. A 1.9% Ehrlichia ewingii Anderson et al. infection prevalence was documented in collected A. americanum in South Carolina. Further studies are warranted to better understand the role S. scrofa plays in the natural maintenance of rickettsial agents in various regions of the United States.

The Asian longhorned tick, Haemaphysalis longicornis, an invasive species associated with human pathogens, has spread rapidly across the eastern USA. Questing H. longicornis ticks recovered from active surveillance conducted from 1 May to 6 September, 2019 throughout Pennsylvania were tested for rickettsial pathogens. Of 265 ticks tested by PCR for pathogens, 4 (1.5%) were positive for Anaplasma phagocytophilum. Sequence analysis of the 16S rRNA gene confirmed two positives as A. phagocytophilum-human agent variant. This is the first reported detection of A. phagocytophilum-human pathogenic strain DNA in exotic H. longicornis collected in the USA.

Identifying febrile patients requiring antibacterial treatment is challenging, particularly in low-resource settings. In South-East Asia, C-reactive protein (CRP) has been demonstrated to be highly sensitive and moderately specific in detecting bacterial infections and to safely reduce unnecessary antibacterial prescriptions in primary care. As evidence is scant in sub-Saharan Africa, we assessed the sensitivity of CRP in identifying serious bacterial infections in Tanzania.Samples were obtained from inpatients and outpatients in a prospective febrile illness study at two hospitals in Moshi, Tanzania, 2011-2014. Bacterial bloodstream infections (BSI) were established by blood culture, and bacterial zoonotic infections were defined by ≥4 fold rise in antibody titre between acute and convalescent sera. The sensitivity of CRP in identifying bacterial infections was estimated using thresholds of 10, 20 and 40 mg/l. Specificity was not assessed because determining false-positive CRP results was limited by the lack of diagnostic testing to confirm non-bacterial aetiologies and because ascertaining true-negative cases was limited by the imperfect sensitivity of the diagnostic tests used to identify bacterial infections.Among 235 febrile outpatients and 569 febrile inpatients evaluated, 31 (3.9%) had a bacterial BSI and 61 (7.6%) had a bacterial zoonosis. Median (interquartile range) CRP values were 173 (80-315) mg/l in bacterial BSI, and 108 (31-208) mg/l in bacterial zoonoses. The sensitivity (95% confidence intervals) of CRP was 97% (83%-99%), 94% (79%-98%) and 90% (74%-97%) for identifying bacterial BSI, and 87% (76%-93%), 82% (71%-90%) and 72% (60%-82%) for bacterial zoonoses, using thresholds of 10, 20 and 40 mg/l, respectively.C-reactive protein was moderately sensitive for bacterial zoonoses and highly sensitive for identifying BSIs. Based on these results, operational studies are warranted to assess the safety and clinical utility of CRP for the management of non-malaria febrile illness at first-level health facilities in sub-Saharan Africa.Identifier les patients fébriles nécessitant un traitement antibactérien est un défi, en particulier dans les milieux à faibles ressources. En Asie du Sud-Est, il a été démontré que la protéine C-réactive (CRP) est très sensible et modérément spécifique dans la détection des infections bactériennes et qu'elle réduit en toute sécurité les prescriptions antibactériennes inutiles dans les soins primaires. Comme les données sont rares en Afrique subsaharienne (ASS), nous avons évalué la sensibilité de la CRP dans l'identification des infections bactériennes sévères en Tanzanie. MÉTHODES: Des échantillons ont été obtenus auprès de patients hospitalisés et ambulatoires dans une étude prospective sur les maladies fébriles dans deux hôpitaux à Moshi, en Tanzanie de 2011 à 2014. Les infections bactériennes du sang (IBS) ont été identifiées par la culture du sang et les infections bactériennes zoonotiques ont été définies par une élevation ≥ 4 fois le titre des anticorps entre les sérums en aiguë et en convalescence. La sensibilité de la CRP dans l'identification des infections bactériennes a été estimée en utilisant des seuils de 10, 20 et 40 mg/L. La spécificité n'a pas été évaluée parce que la détermination des résultats faux positifs de la CRP était limitée par le manque de tests de diagnostic pour confirmer les étiologies non bactériennes et parce que la confirmation des vrais cas négatifs était limitée par la sensibilité imparfaite des tests de diagnostic utilisés pour identifier les infections bactériennes. RÉSULTATS: Sur 235 patients ambulatoires fébriles et 569 patients hospitalisés fébriles évalués, 31 (3.9%) avaient une IBS et 61 (7.6%) avaient une zoonose bactérienne. Les valeurs médianes (intervalle interquartile) de la CRP étaient de 173 (80-315) mg/L dans les IBS et de 108 (31-208) mg/L dans les zoonoses bactériennes. La sensibilité (intervalles de confiance à 95%) de la CRP était de 97% (83-99%), 94% (79-98%), 90% (74-97%) pour identifier les IBS et 87% (76-93% ), 82% (71-90%), 72% (60-82%) pour les zoonoses bactériennes, en utilisant des seuils de 10, 20 et 40 mg/L respectivement.La CRP était modérément sensible pour les zoonoses bactériennes et hautement sensible pour l'identification des IBS. Sur la base de ces résultats, des études opérationnelles sont justifiées pour évaluer la sécurité et l'utilité clinique de la CRP pour la prise en charge des maladies fébriles non paludiques dans les établissements de santé de premier niveau en ASS.

Crimean-Congo hemorrhagic fever (CCHF), Q fever, and Lyme disease are endemic to southern Kazakhstan, but population-based serosurveys are lacking. We assessed risk factors and seroprevalence of these zoonoses and conducted surveys for CCHF-related knowledge, attitudes, and practices in the Zhambyl region of Kazakhstan. Weighted seroprevalence for CCHF among all participants was 1.2%, increasing to 3.4% in villages with a known history of CCHF circulation. Weighted seroprevalence was 2.4% for Lyme disease and 1.3% for Q fever. We found evidence of CCHF virus circulation in areas not known to harbor the virus. We noted that activities that put persons at high risk for zoonotic or tickborne disease also were risk factors for seropositivity. However, recognition of the role of livestock in disease transmission and use of personal protective equipment when performing high-risk activities were low among participants.

The American dog tick, Dermacentor variabilis Say, is the principal vector of spotted fever group rickettsiae that cause illness in people. Because the composition and structure of microbial communities in D. variabilis are poorly understood, we conducted high-throughput amplicon sequencing of the 16S rRNA gene to characterize the bacterial communities of adult D. variabilis ticks collected from field populations in North Carolina. Sequence analyses were performed using QIIME 2 with the DADA2 plugin and taxonomic assignments using the Greengenes database. After quality filtering and rarefaction, the bacterial DNA sequences were assigned to 432 operational taxonomic units (OTUs) and were dominated by a single OTU classified as Francisella spp. Subsequent cloning, sequencing and phylogenic analysis of nearly full length 16S rRNA gene fragments revealed that most Francisella were similar to Francisella-like endosymbionts (FLEs) of D. variabilis reported previously. One cloned Francisella, however, was genetically distinct and more related to the FLE of D. occidentalis. The core microbiome of D. variabilis was comprised of Francisella spp., Sphingomonas spp., Delftia spp., and Hymenobacter spp. The taxonomic resolution and detection of less abundant bacterial taxa, including Rickettsia, are discussed. Alpha diversity metrics revealed lower bacterial community diversity in females. Beta-diversity also distinguished the composition of female bacterial communities from those of males. These findings advance current knowledge of the microbial ecology and structure and composition of D. variabilis microbiome and sets the foundation for further studies to determine the influence of microbiota on vector susceptibility to pathogens.

In the United States, tick-borne diseases are increasing in incidence and cases are reported over an expanding geographical area. Avoiding tick bites is a key strategy in tick-borne disease prevention, and this requires current and accurate information on where humans are at risk for exposure to ticks. Based on a review of published literature and records in the U.S. National Tick Collection and National Ecological Observatory Network databases, we compiled an updated county-level map showing the reported distribution of the American dog tick, Dermacentor variabilis (Say). We show that this vector of the bacterial agents causing Rocky Mountain spotted fever and tularemia is widely distributed, with records derived from 45 states across the contiguous United States. However, within these states, county-level records of established tick populations are limited. Relative to the range of suitable habitat for this tick, our data imply that D. variabilis is currently underreported in the peer-reviewed literature, highlighting a need for improved surveillance and documentation of existing tick records.

The Asian longhorned tick, Haemaphysalis longicornis, is an invasive species, originally from eastern Asia, and was first reported in the USA in New Jersey. It is now reported in several eastern states. In 2018, researchers reported H. longicornis in northwest Arkansas (Benton County). This tick species is a proven vector of livestock and human diseases, which prompted the current survey of ticks in southwest Missouri. A tick drag in Greene County, Missouri, produced 2 H. longicornis nymphs on June 9, 2021. This is the first report of this species for both the state and county.

We identified an established population of the Gulf Coast tick (Amblyomma maculatum Koch) infected with Rickettsia parkeri in Connecticut, representing the northernmost range limit of this medically relevant tick species. Our finding highlights the importance of tick surveillance and public health challenges posed by geographic expansion of tick vectors and their pathogens.

Bourbon virus (BRBV) was first isolated in 2014 from a resident of Bourbon County, Kansas, USA, who died of the infection. In 2015, an ill Payne County, Oklahoma, resident tested positive for antibodies to BRBV, before fully recovering. We retrospectively tested for BRBV in 39,096 ticks from northwestern Missouri, located 240 km from Bourbon County, Kansas. We detected BRBV in 3 pools of Amblyomma americanum (L.) ticks: 1 pool of male adults and 2 pools of nymphs. Detection of BRBV in A. americanum, a species that is aggressive, feeds on humans, and is abundant in Kansas and Oklahoma, supports the premise that A. americanum is a vector of BRBV to humans. BRBV has not been detected in nonhuman vertebrates, and its natural history remains largely unknown.

Portions of northern Mexico are experiencing a re-emergence of Rocky Mountain spotted fever (RMSF), a tickborne disease caused by Rickettsia rickettsii, a member of the spotted fever group of rickettsiae (SFGR). Infection with R. rickettsii can result in serious and life-threatening illness in people and dogs. Canine seroprevalence has been used as a sentinel for human RMSF in previous studies. This study aims to quantify SFGR seroprevalence in canines in three northern Mexican states and identify risk factors associated with seropositivity. A total of 1,136 serum samples and 942 ticks were obtained from dogs participating in government sterilization campaigns and from animal control facilities in 14 Mexican cities in three states. SFGR antibodies were detected using indirect immunofluorescence antibody assays at titre values ≥1/64. Six per cent (69 dogs) showed antibodies to SFGR, with the highest seroprevalence reported in Baja California (12%), Coahuila (4%) and Sonora (4%). Dogs from Baja California had three times higher odds of having SFGR antibodies compared to dogs from Sonora (OR = 3.38, 95% CI, 1.81-6.37). Roughly one quarter (25%) of surveyed dogs were parasitized by ticks (Rhipicephalus sanguineus sensu lato) at the time of sample collection. A portion of collected ticks were tested for rickettsial DNA using polymerase chain reaction. Positive samples were then sequenced, showing evidence of SFGR including R. massiliae, R. parkeri and R. rickettsii. Dogs that spent the majority of time on the street, such as free-roaming or community-owned dogs, showed a greater risk of tick infestation, seropositivity, bearing seropositive ticks, and may play a pivotal role in the spread of SFGR among communities. Estimating the seroprevalence of SFGR in the canine population can help public health campaigns target high-risk communities for interventions to reduce human RMSF cases.

Tickborne rickettsial diseases continue to cause severe illness and death in otherwise healthy adults and children, despite the availability of low-cost, effective antibacterial therapy. Recognition early in the clinical course is critical because this is the period when antibacterial therapy is most effective. Early signs and symptoms of these illnesses are nonspecific or mimic other illnesses, which can make diagnosis challenging. Previously undescribed tickborne rickettsial diseases continue to be recognized, and since 2004, three additional agents have been described as causes of human disease in the United States: Rickettsia parkeri, Ehrlichia muris-like agent, and Rickettsia species 364D. This report updates the 2006 CDC recommendations on the diagnosis and management of tickborne rickettsial diseases in the United States and includes information on the practical aspects of epidemiology, clinical assessment, treatment, laboratory diagnosis, and prevention of tickborne rickettsial diseases. The CDC Rickettsial Zoonoses Branch, in consultation with external clinical and academic specialists and public health professionals, developed this report to assist health care providers and public health professionals to 1) recognize key epidemiologic features and clinical manifestations of tickborne rickettsial diseases, 2) recognize that doxycycline is the treatment of choice for suspected tickborne rickettsial diseases in adults and children, 3) understand that early empiric antibacterial therapy can prevent severe disease and death, 4) request the appropriate confirmatory diagnostic tests and understand their usefulness and limitations, and 5) report probable and confirmed cases of tickborne rickettsial diseases to public health authorities.

Tick-borne diseases cause substantial morbidity throughout the United States, and North Carolina has a high incidence of spotted fever rickettsioses and ehrlichiosis, with sporadic cases of Lyme disease. The occupational risk of tick-borne infections among outdoor workers is high, particularly those working on publicly managed lands. This study identified incident tick-borne infections and examined seroconversion risk factors among a cohort of North Carolina outdoor workers. Workers from the North Carolina State Divisions of Forestry, Parks and Recreation, and Wildlife (n = 159) were followed for 2 years in a randomized controlled trial evaluating the effectiveness of long-lasting permethrin-impregnated clothing. Antibody titers against Rickettsia parkeri, Rickettsia rickettsii, "Rickettsia amblyommii," and Ehrlichia chaffeensis were measured at baseline (n = 130), after 1 year (n = 82), and after 2 years (n = 73). Titers against Borrelia burgdorferi were measured at baseline and after 2 years (n = 90). Baseline seroprevalence, defined as indirect immunofluorescence antibody titers of 1/128 or greater, was R. parkeri (24%), R. rickettsii (19%), "R. amblyommii" (12%), and E. chaffeensis (4%). Incident infection was defined as a fourfold increase in titer over a 1-year period. There were 40 total seroconversions to at least one pathogen, including R. parkeri (n = 19), "R. amblyommii" (n = 14), R. rickettsii (n = 9), and E. chaffeensis (n = 8). There were no subjects whose sera were reactive to B. burgdorferi C6 antigen. Thirty-eight of the 40 incident infections were subclinical. The overall risk of infection by any pathogen during the study period was 0.26, and the risk among the NC Division of Forest Resources workers was 1.73 times that of workers in other divisions (95% confidence interval [CI]: 1.02, 2.92). The risk of infection was lower in subjects wearing permethrin-impregnated clothing, but not significantly (risk ratio = 0.81; 95% CI: 0.47, 1.39). In summary, outdoor workers in North Carolina are at high risk of incident tick-borne infections, most of which appear to be asymptomatic.

A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia -specific PCR targets ( ompA , gltA , and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “ Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia . The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “ Candidatus Rickettsia amblyommii,” R. montanensis , R. felis , and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii .

During 2013, we collected and tested ticks for Heartland virus (HRTV), a recently described human pathogen in the genus Phlebovirus (Bunyaviridae), from six sites in northwestern Missouri. Five sites were properties owned by HRTV patients, and the sixth was a conservation area that yielded virus in ticks during 2012. We collected 39,096 ticks representing five species; however, two species, Amblyomma americanum (L.) (97.6%) and Dermacentor variabilis (Say) (2.3%), accounted for nearly all ticks collected. We detected 60 HRTV-positive tick pools and all were composed of A . americanum : 53 pools of nymphs, six pools of male adults, and one pool of female adults. This is the first record of HRTV in adult ticks. Virus was detected at five properties that yielded A. americanum ticks, including properties owned by four of five patients. Virus was detected at two sites that yielded virus in 2012. Detection of virus in multiple years indicates that the virus persists in ticks within a relatively small geographic area, although infection rates (IR) may vary greatly among sites and between years at a site. IR per 1,000 A. americanum in northwestern Missouri during the April–July 2013 study period were as follows: all adults, IR = 1.13; adult females, IR = 0.33; adult males, IR = 1.90; and nymphs, IR = 1.79. The IR in nymphs, the stage with the largest data set, corresponds to 1/559 infected ticks. Having robust estimates of IR in various stages for A. americanum should lead to more accurate public health messaging and a better understanding of virus transmission.

Rocky Mountain spotted fever (RMSF) is a severe tick-borne rickettsial illness. In the south-western United States and Mexico, RMSF displays unique epidemiologic and ecologic characteristics, including Rhipicephalus sanguineus sensu lato (brown dog tick) as the primary vector. Expansion and spread of the disease from hyperendemic regions of Arizona or Mexico to new areas is a key public health concern. Dogs are thought to play an important role in the emergence and circulation of R. rickettsii in these regions and are often one of earliest indicators of RMSF presence. A canine serosurvey was conducted in 2015 among owned and stray dogs at rabies clinic and animal shelters in three southern Arizona counties where RMSF had not previously been identified. Of the 217 dogs sampled, 11 (5.1%) tested positive for spotted fever group rickettsia (SFGR) IgG antibodies, with seropositivity ranging from 2.9% to 12.2% across the three counties. Large dogs were significantly more likely than small dogs to have positive titres reactive with R. rickettsii; no additional statistically significant relationships were observed between seropositivity of canine age, sex, neuter or ownership status. In addition, 17 (7.8%) dogs had ticks attached at the time of sampling, and stray dogs were significantly more likely to have ticks present than owned dogs (p

The American dog tick, Dermacentor variabilis (Say), is a vector of spotted fever group (SFG) rickettsiae, including Rickettsia rickettsii the causative organism of Rocky Mountain spotted fever (RMSF). In North Carolina, SFG rickettsioses (including RMSF) are a leading cause of tick-borne illness. Knowledge of the infection rate and geographic distribution of D. variabilis ticks infected with Rickettsia spp. provides information on the spatial distribution of public health risk. Accordingly, we extracted genomic DNA from adult D. variabilis collected from field habitats in 32 North Carolina counties from 2009 to 2013. A nested PCR assay of the 23S-5S intergenic spacer (IGS) region of Rickettsia coupled with reverse line blot hybridization (RLBH) with species-specific probes was used to detect and identify rickettsiae to species. Approximately half of the 532 tick DNA samples exhibited a band of the expected size on agarose gels, indicating infection with Rickettsia spp. RLBH analyses showed R. amblyommatis (formerly ‘Candidatus R. amblyommii’), R. parkeri, and R. montanensis were predominant, while other Rickettsia species detected included R. conorii-like, R. massiliae, R. rhipicephali, R. canadensis, R. bellii, and some unknown Rickettsia spp. Some ticks were infected with more than one Rickettsia species. Notably, several Rickettsia-positive ticks harbored R. rickettsii. DNA sequencing was performed on a portion of the 23S-5S IGS amplicons and the results were concordant with RLB assay results. We conclude that Rickettsia spp. are common in D. variabilis in North Carolina. Geographic patterns in the occurrence of Rickettsia-infected D. variabilis ticks across the counties sampled are discussed.

Since its recent discovery, Bourbon virus has been isolated from a human and ticks. To assess exposure of potential vertebrate reservoirs, we assayed banked serum and plasma samples from wildlife and domestic animals in Missouri, USA, for Bourbon virus-neutralizing antibodies. We detected high seroprevalence in raccoons (50%) and white-tailed deer (86%).

Human exposure to Bartonella clarridgeiae has been reported only on the basis of antibody detection. We report for the first time an asymptomatic human blood donor infected with B. clarridgeiae , as documented by enrichment blood culture, PCR, and DNA sequencing.

In 1953, investigators at the Rocky Mountain Laboratories in Hamilton, MT, described the isolation of a spotted fever group Rickettsia (SFGR) species from Dermacentor parumapertus ticks collected from black-tailed jackrabbits ( Lepus californicus ) in northern Nevada. Several decades later, investigators characterized this SFGR (designated the parumapertus agent) by using mouse serotyping methods and determined that it represented a distinct rickettsial serotype closely related to Rickettsia parkeri ; nonetheless, the parumapertus agent was not further characterized or studied. To our knowledge, no isolates of the parumapertus agent remain in any rickettsial culture collection, which precludes contemporary phylogenetic placement of this enigmatic SFGR. To rediscover the parumapertus agent, adult-stage D. parumapertus ticks were collected from black-tailed jackrabbits shot or encountered as roadkills in Arizona, Utah, or Texas from 2011 to 2016. A total of 339 ticks were collected and evaluated for infection with Rickettsia species. Of 112 D. parumapertus ticks collected in south Texas, 16 (14.3%) contained partial ompA sequences with the closest identity (99.6%) to Rickettsia sp. strain Atlantic rainforest Aa46, an SFGR that is closely related or identical to an SFGR species that causes a mild rickettsiosis in several states of Brazil. A pure isolate, designated strain Black Gap, was cultivated in Vero E6 cells, and sequence analysis of the rrs , gltA , sca0 , sca5 , and sca4 genes also revealed the closest genetic identity to Rickettsia sp. Atlantic rainforest Aa46. Phylogenetic analysis of the five concatenated rickettsial genes place Rickettsia sp. strain Black Gap and Rickettsia sp. Atlantic rainforest Aa46 with R. parkeri in a distinct and well-supported clade. IMPORTANCE We suggest that Rickettsia sp. Black Gap and Rickettsia sp. Atlantic rainforest Aa46 represent nearly identical strains of R. parkeri and that Rickettsia sp. Black Gap or a very similar strain of R. parkeri represents the parumapertus agent. The close genetic relatedness among these taxa, as well as the response of guinea pigs infected with the Black Gap strain, suggests that R. parkeri Black Gap could cause disease in humans. The identification of this organism could also account, at least in part, for the remarkable differences in severity ascribed to Rocky Mountain spotted fever (RMSF) among various regions of the American West during the early 20th century. We suggest that the wide variation in case fatality rates attributed to RMSF could have occurred by the inadvertent inclusion of cases of milder disease caused by R. parkeri Black Gap.

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.

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.

Heartland virus (HRTV), the first pathogenic Phlebovirus (Family: Bunyaviridae) discovered in the United States, was recently described from two Missouri farmers. In 2012, we collected 56,428 ticks representing three species at 12 sites including both patients' farms. Amblyomma americanum and Dermacentor variabilis accounted for nearly all ticks collected. Ten pools composed of deplete nymphs of A. americanum collected at a patient farm and a nearby conservation area were reverse transcription-polymerase chain reaction positive, and eight pools yielded viable viruses. Sequence data from the nonstructural protein of the Small segment indicates that tick strains and human strains are very similar, ≥ 97.6% sequence identity. This is the first study to isolate HRTV from field-collected arthropods and to implicate ticks as potential vectors. Amblyomma americanum likely becomes infected by feeding on viremic hosts during the larval stage, and transmission to humans occurs during the spring and early summer when nymphs are abundant and actively host seeking.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.