Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Group Ticks

We determined prevalence of Rickettsia spp. in 172 ticks of the Amblyomma maculatum group collected from 16 urban sites in Oklahoma City, Oklahoma, USA, during 2017 and 2018. Most ticks (59.3%) were collected from 1 site; 4 (2.3%) were infected with Rickettsia parkeri and 118 (68.6%) with Candidatus Rickettsia andeanae.


Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Group Ticks
Bruce H. Noden R ickettsia parkeri, part of the spotted fever group Rickettsia (SFGR), affects humans throughout much of the southern United States (1). Although R. parkeri in an engorged nymph was reported once in Oklahoma, R. parkeri has not been reported in adult A. maculatum ticks in Oklahoma or Kansas. To date, all test-positive adult ticks in Kansas and Oklahoma have been infected with Candidatus Rickettsia andeanae (2). The absence of R. parkeri in Oklahoma is surprising because it was detected in A. maculatum group ticks recovered from dogs in Arkansas counties bordering eastern Oklahoma (3) and in adult A. maculatum ticks in Texas (4), and A. maculatum ticks have been present in Oklahoma since the 1940s (4). We collected A. maculatum ticks in the Oklahoma City metropolitan area during May-August 2017 and 2018 and tested them for Rickettsia spp.
We selected 16 sites as part of a larger study of tickborne disease epidemiology (Figure). We performed collections during May-August by flagging vegetation or using CO 2 traps (5). We completed identification by using established keys (6).
We tested field-collected ticks for rickettsial DNA by using established PCR protocols (7,8). To limit DNA contamination, we conducted DNA extractions by using site-specific reagents in a separate laboratory. After soaking adult ticks in deionized water for 30 minutes and surface-sterilizing with 70% ethanol, we longitudinally bisected ticks; we used one half for DNA extraction and stored the other half at −80°C. DNA extraction followed established protocols (5). In 2017, we screened all ticks by using assays targeting the gltA and ompA (8) genes and retested positive samples by using an assay targeting the ompB gene (primer pair 120-2788/120-3599) (7). In 2018, we initially screened ticks by using the gltA assay and confirmed the results with an ompB assay.
We sequenced positive ompB amplicons bidirectionally by using an Applied Biosystems 3730 DNA Analyzer (https://www.thermofisher.com) at the Oklahoma State University Core Facility to identify bacterial species. We verified each resulting sequence by using BioEdit 7.2 (https://bioedit.software.informer.com) and aligned bidirectional sequences to create consensus sequences by using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). We compared resulting consensus sequences with Gen-Bank submissions by using default conditions on BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using the highest percentage sequence identity to determine species similarity.
We   We identified A. maculatum group ticks infected with R. parkeri and Candidatus R. andeanae in the Oklahoma City metropolitan area. Oklahoma lies at the western edge of 1 of the highest-incidence areas of SFGR in the United States (1). To date, no human rickettsiosis cases caused by R. parkeri have been reported in Oklahoma, possibly because of treatment based on nonspecific symptoms and the lifting of mandatory reporting to the Centers for Disease Control and Prevention (9). The low prevalence of R. parkeri in Oklahoma ticks differs from other areas of the United States, such as Virginia, where prevalence of R. parkeri is higher in A. maculatum ticks (10). Candidatus R. andeanae prevalence in A. maculatum ticks varies inversely with R. parkeri prevalence in some regions (4). Although Candidatus R. andeanae is not known to cause human illness (4), the high prevalence of Candidatus R. andeanae in Oklahoma ticks might interfere with R. parkeri development, limiting its distribution (2). The potential presence of this human pathogen in the largest metropolitan area in the state, and 1 of the largest in the central United States, necessitates thorough case evaluation of future SFGR cases in this region.
A strovirus is a positive-sense, single-stranded RNA virus first identified in feces of children with gastroenteritis in 1975. Since then, astrovirus has been found in a wide variety of mammals and birds (1). The family Astroviridae comprises 2 genera, Mamastrovirus and Avastrovirus, and classification is based on host origin. Astroviruses cause diarrhea and neurologic diseases in mammals and a spectrum of diseases, including diarrhea, hepatitis, and nephritis, in birds (2). Astrovirus is associated with respiratory disease in humans, cattle, and pigs (3)(4)(5) and has also been found in fecal samples from roe deer with gastrointestinal illness in Denmark (6). Whether astrovirus circulates in other species of deer remains unclear.
In September 2018, the Veterinary Diagnostic Laboratory at Iowa State University (Ames, Iowa, USA) received 5 sets of tissue samples collected from deer of the same farm for identification of the infectious cause of death of 5 male white-tailed deer 8-14 weeks of age. The pen-raised deer experienced pneumonia and sudden death. Postmortem examinations showed pleural fluid in the lungs, pneumonia, and purple-mottled lungs. Histopathologic observations revealed that 3 deer had necrotizing bronchopneumonia, and 2 had interstitial pneumonia.
Although different combinations of the bacterial pathogens Bibersteinia trehalosi, Tureperella pyogenes, Fusobacterium necrophorum, and Pasteurella multocida were found in all cases, an underlying viral cause could not be excluded. Therefore, we used next-generation sequencing, first with pooled lung samples and then with individual lung samples, using Nextera XT DNA Library Preparation Kit with the MiSeq platform and MiSeq Reagent Kit v2 (Illumina, https://www.illumina.com). A bioinformatic analysis indicated the presence of an astrovirus along with the bacteria. The complete genome sequence (6,246 nt) of this astrovirus (WI65268; Gen-Bank accession no. MN087316) was found in the pooled lung tissue sample and 1 lung tissue sample, and partial genomes were found in the other 4 lung samples. A complete-genome comparison revealed that BoAstV/ JPN/Ishikawa24-6/2013 (bovine isolate from Japan) had the highest identity (60.9%) to WI65268. Further nucleotide sequence analysis revealed that WI65268 had a similar genome organization as other astroviruses (Appendix Figure 1, https://wwwnc.cdc.gov/EID/ article/26/2/19-0878-App1.pdf).
Phylogenetic analysis of the complete genome showed that WI65268 is distantly related to other bovine, dromedary, takin, and yak strains (Appendix Figure 2). In phylogenetic analyses of ORF1a and ORF1b protein sequences, WI65268 clustered with

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We report the identification of astrovirus WI65268 in a white-tailed deer with respiratory disease in the United States in 2018. This virus is a recombinant of Kagoshi-ma1-7 and Kagoshima2-3-2 (both bovine astroviruses from Japan) and was characterized as a potential new genotype. Further surveillance of deer might help identify related isolates.