Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

Seasonal pattern of questing ticks and prevalence of pathogenic Rickettsia and Anaplasmataceae in Khao Yai national park, Thailand.

BACKGROUND: Tick-borne diseases (TBD) are considered neglected diseases in Thailand with disease burden likely underestimated. To assess risk for emerging TBD in Thailand, the seasonality of questing tick and pathogen prevalence were studied in Khao Yai National Park, a top tourist destination. METHODS: During 2019, questing ticks around tourist attractions were systematically collected bimonthly and analyzed for Rickettsia and Anaplasmataceae bacterial species by polymerase chain reaction and DNA sequencing. RESULTS: Larvae and nymphs of questing ticks peaked in Khao Yai National Park during the late rainy-winter season, though no specific trends were observed in adult ticks. Winter (November to February) was the highest risk for human tick-bites due to higher numbers of both ticks and visitors. Of the total 5916 ticks analyzed (651 pools), Anaplasma phagocytophilum, Neoehrlichia mikurensis, Ehrlichia ewingii, and Ehrlichia chaffeensis were detected at low rates (≤0.05%). There was a higher prevalence of human rickettsioses (0.2-7%) in ticks surveyed with Rickettsia tamurae, Rickettsia raoultii, and Rickettsia montana the major species. Amblyomma ticks had the highest prevalence of Rickettsia (85%, 35/44 Amblyomma adults), in which only R. tamurae and R. raoultii were found in Amblyomma with mixed species infections common. We report the first detection of R. africae-like and N. mikurensis in Ixodes granulatus adults in Thailand, suggesting I. granulatus as a potential vector for these pathogens. CONCLUSION: This study demonstrated the risk of emerging TBD in Thailand and underscores the need for tick-bite prevention among tourists in Thailand.

The effects of human and rhesus macaque blood meal sources on mosquito reproduction and adult survival under laboratory conditions.

Mass rearing of mosquitoes as required to fulfil research studies is a technically challenging endeavor. Blood meal source has been recognized as a key consideration in mass rearing of mosquitoes that affects colony health and fecundity. Four species of laboratory-colonized mosquitoes from the Department of Entomology, US Army Medical Directorate - Armed Forces Research Institute of Medical Sciences (USAMD-AFRIMS); Anopheles cracens, An. dirus, An. minimus and An. sawadwongporni were fed blood meals from human and rhesus macaque sources using an artificial membrane feeder. The effects of different blood meal sources were evaluated concerning blood-feeding, survival and reproduction (fecundity and hatching rates). Adult survival was monitored at days 7, 14 and 21 post blood-feeding. Although the mosquitoes fed on human blood exhibited higher rates of engorgement, there were no significant differences in blood-feeding rates in An. cracens (P = 0.08) and An. dirus (P = 0.91) between rhesus macaque and human blood sources. Twenty-one days post-feeding, no significant differences were observed in the survival rates of mosquitoes fed on human versus rhesus macaque blood. Except for An. dirus, which had better survival rates with human blood (97.5%) than after feeding on rhesus macaque blood (95.4%). All mosquito species fed on human blood produced significantly more eggs when compared to those fed on rhesus macaque blood. However, there was no statistical difference in hatching rates between blood sources, except for An. dirus, which had better hatching rates with human blood. These results indicate that human and rhesus macaque blood may be a viable alternative for maintaining Anopheles mosquitoes in colony.

Applying next generation sequencing to detect tick-pathogens in Dermacentor nuttalli, Ixodes persulcatus, and Hyalomma asiaticum collected from Mongolia.

Ticks and tick-borne diseases represent major threats to the public health of the Mongolian population, of which an estimated 26% live a traditional nomadic pastoralist lifestyle that puts them at increased risk for exposure. Ticks were collected by dragging and removal from livestock in Khentii, Selenge, Tuv, and Umnugovi aimags (provinces) during March-May 2020. Using next-generation sequencing (NGS) with confirmatory PCR and DNA sequencing, we sought to characterize the microbial species present in Dermacentor nuttalli (n = 98), Hyalomma asiaticum (n = 38), and Ixodes persulcatus (n = 72) tick pools. Rickettsia spp. were detected in 90.4% of tick pools, with Khentii, Selenge, and Tuv tick pools all having 100% pool positivity. Coxiella spp. were detected at an overall pool positivity rate of 60%, while Francisella spp. were detected in 20% of pools and Borrelia spp. detected in 13% of pools. Additional confirmatory testing for Rickettsia-positive pools demonstrated Rickettsia raoultii (n = 105), Candidatus Rickettsia tarasevichiae (n = 65) and R. slovaca/R. sibirica (n = 2), as well as the first report of Candidatus Rickettsia jingxinensis (n = 1) in Mongolia. For Coxiella spp. reads, most samples were identified as a Coxiella endosymbiont (n = 117), although Coxiella burnetii was detected in eight pools collected in Umnugovi. Borrelia species that were identified include Borrelia burgdorferi sensu lato (n = 3), B. garinii (n = 2), B. miyamotoi (n = 16), and B. afzelii (n = 3). All Francisella spp. reads were identified as Francisella endosymbiont species. Our findings emphasize the utility of NGS to provide baseline data across multiple tick-borne pathogen groups, which in turn can be used to inform health policy, determine regions for expanded surveillance, and guide risk mitigation strategies.

Borrelia miyamotoi a neglected tick-borne relapsing fever spirochete in Thailand.

Borrelia miyamotoi is a relapsing fever spirochete that shares the same vector as Lyme disease causing Borrelia. This epidemiological study of B. miyamotoi was conducted in rodent reservoirs, tick vectors and human populations simultaneously. A total of 640 rodents and 43 ticks were collected from Phop Phra district, Tak province, Thailand. The prevalence rate for all Borrelia species was 2.3% and for B. miyamotoi was 1.1% in the rodent population, while the prevalence rate was quite high in ticks collected from rodents with an infection rate of 14.5% (95% CI: 6.3-27.6%). Borrelia miyamotoi was detected in Ixodes granulatus collected from Mus caroli and Berylmys bowersi, and was also detected in several rodent species (Bandicota indica, Mus spp., and Leopoldamys sabanus) that live in a cultivated land, increasing the risk of human exposure. Phylogenetic analysis revealed that the B. miyamotoi isolates detected in rodents and I. granulatus ticks in this study were similar to isolates detected in European countries. Further investigation was conducted to determine the serological reactivity to B. miyamotoi in human samples received from Phop Phra hospital, Tak province and in rodents captured from Phop Phra district using an in-house, direct enzyme-linked immunosorbent assay (ELISA) assay with B. miyamotoi recombinant glycerophosphodiester-phosphodiesterase (rGlpQ) protein as coated antigen. The results showed that 17.9% (15/84) of human patients and 9.0% (41/456) of captured rodents had serological reactivity to B. miyamotoi rGlpQ protein in the study area. While a low level of IgG antibody titers (100-200) was observed in the majority of seroreactive samples, higher titers (400-1,600) were also detected in both humans and rodents. This study provides the first evidence of B. miyamotoi exposure in human and rodent populations in Thailand and the possible roles of local rodent species and Ixodes granulatus tick in its enzootic transmission cycle in nature.

Metagenomic profiles of Dermacentor tick pathogens from across Mongolia, using next generation sequencing.

Tick-borne diseases are a major public health concern in Mongolia. Nomadic pastoralists, which make up ~ 26% of Mongolia's population, are at an increased risk of both tick bite exposure and economic loss associated with clinical disease in herds. This study sought to further characterize tick-borne pathogens present in Dermacentor ticks (n = 1,773) sampled in 2019 from 15 of Mongolia's 21 aimags (provinces). The ticks were morphologically identified and sorted into 377 pools which were then screened using Next-Generation Sequencing paired with confirmatory PCR and DNA sequence analysis. Rickettsia spp. were detected in 88.33% of pools, while Anaplasma spp. and Bartonella spp. were detected in 3.18 and 0.79% of pools, respectively. Khentii had the highest infection rate for Rickettsia spp. (76.61%; CI: 34.65-94.79%), while Arkhangai had the highest infection rate for Anaplasma spp. (7.79%; CI:4.04-13.72%). The exclusive detection of Anaplasma spp. in tick pools collected from livestock supports previous work in this area that suggests livestock play a significant role in disease maintenance. The detection of Anaplasma, Bartonella, and Rickettsia demonstrates a heightened risk for infection throughout Mongolia, with this study, to our knowledge, documenting the first detection of Bartonella melophagi in ticks collected in Mongolia. Further research deploying NGS methods is needed to characterize tick-borne pathogens in other endemic tick species found in Mongolia, including Hyalomma asiaticum and Ixodes persulcatus.

Laboratory studies of oviposition site choice of Toxorhynchites splendens: Role of larval holding-rearing water and predator-prey interactions.

Oviposition preference by Toxorhynchites mosquito species is an important aspect of their biological control potential. When ovipositing, the females deposit individual eggs by hovering above the water, indicating that they may be capable of detecting chemical cues from above the water surface. These chemical cues may help the females to determine whether the water is suitable for their larvae. We investigated whether female Tx. splendens has the ability to discriminate against oviposition sites in habitats containing turbid larval/pupal rearing water and predator-prey treatments. In this study, the number of eggs in the test and control cups were compared to assess oviposition preference. According to the results, female Tx. splendens preferred ovipositing in cups with turbid immature water (prey absence) and predator-prey turbid water (prey and predator presences) over ovipositing in filtered water as a control. Results from the third experiment revealed that cups with alive and dead Ae. aegypti larvae proved strongly attractive to Tx. splendens females, while those from conspecific Toxorhynchites larvae caused oviposition deterrence when compared to other treatments in a dual choice test. Our results indicated that both turbid immature water and predator-prey turbid water can efficiently attract Tx. splendens females to deposit eggs. These findings will aid in the development of Toxorhynchites mosquito as a biocontrol agent for the control of Aedes spp. larvae.

US Department of Defense Support of Civilian Vector Control Operations Following Natural Disasters.

The United States Department of Defense (DoD) employs advanced-degreed entomologists as Preventive Medicine and Public Health Officers in the Army, Navy, and Air Force. While the primary objective of military entomologists is service member health and readiness ("force health protection"), military entomology resources can provide support to civil authorities as directed by the President or Secretary of Defense through Department of Defense Directive 3025.18, Defense Support of Civil Authorities (DSCA). The employment of DSCA is complex and involves the consideration of such factors as the proper request process, funding, legality, risk, appropriateness, and readiness. Once approved and mobilized, however, military preventive medicine assets can be of significant help to civil authorities when dealing with emergency vector control. This paper will address some of the policy issues surrounding the use of DSCA, outline the resources available from the individual military services, and provide examples of DoD contingency vector control support to civil authorities.

Potential of Outdoor Ultra-Low-Volume Aerosol and Thermal Fog to Suppress the Dengue Vector, Aedes aegypti, Inside Dwellings.

A field study investigated penetration of outdoor ground ultra-low-volume (ULV) aerosol and thermal fog adulticide applications into a dwelling to control the dengue vector Aedes aegypti (L). Four applications of Kontrol 4-4 (4.6% permethrin active ingredient [AI], 4.6% piperonyl butoxide) at the maximum label rate were made at 25-30 m in front of a house at Camp Blanding Joint Training Center, Starke, FL, during summer 2016. The ULV sprayer and thermal fogger nozzles were oriented horizontally, and vehicle travel speeds were 16 and 24 km/h, respectively. All doors and windows of the house were left open. Spray efficacy was assessed using caged female mosquitoes positioned 30 cm above ground, outside and inside of the house. Interior cages were placed in open areas and cryptic sites (i.e., in a closet or cardboard box). A spinner holding 2 rods sized 3 mm × 75 mm was deployed next to each cage (except cryptic sites) to sample droplets and to quantify AI deposition. Thirty minutes after application, cages were removed, slides collected, and mosquitoes transferred to clean cages in the laboratory where mortality was assessed at 24 h posttreatment. The ULV application to the south side of the house produced 100% mortality in outdoor and indoor cages and 24% mortality at cryptic sites. Similarly applied thermal fog resulted in 85% mortality outdoors, 34% indoors, and only 4% in cages at cryptic sites. Application of either method from the west resulted in 19-61% mortality outdoors and 0.5-6.5% indoors. Droplet volume median diameter (Dv0.5) on rods from the ULV application was significantly larger compared with the thermal fogger outdoors, but similar indoors. Outdoors and indoors, the AI deposition from ULV was significantly higher than from thermal fog. Our results show the potential for controlling dengue vectors inside houses with outdoor ground ULV applications in areas where doors and windows are left open for ventilation.

Comparative Analysis of Mosquito Trap Counts In the Peruvian Amazon: Effect of Trap Type and Other Covariates On Counts and Diversity.

Efficient detection of multiple species of adult mosquitoes in various habitats using effective traps is a crucial 1st step in any disease prevention program. Novel trap types that target tropical vectors of human diseases require field testing in the habitat of the vector-disease system in question. This paper analyzes a series of mosquito trapping studies conducted at Mapacocha, San Juan Bautista District, Loreto, Peru, during August-September 2013 and April-May 2014. Six trap configurations were evaluated in forest and rural locations. Adult mosquito counts were analyzed using full Bayesian inference of multilevel generalized linear models and posterior probability point estimates of the difference of means of the combined trap catch by trap type comparisons of all species. Light traps (Centers for Disease Control and Prevention [CDC] incandescent, white light-emitting diode [LED], and ultraviolet LED) caught greater numbers of mosquitoes compared with traps baited with yeast-generated CO2 and Biogents Sentinel™ traps (battery powered traps without light and passive box traps). However, diversity measures (species richness, evenness, and similarity) were consistently nearly equal among trap types. Arbovirus vectors were more common in forest locations, while malaria vectors were more common near human habitations. Location had a significant effect on trap effectiveness and mosquito diversity, with traps from forest locations having greater numbers and greater species richness, compared with traps set near human habitations. The results of this study will inform mosquito surveillance trap choices in remote regions of central South America, including regions with emerging tropical diseases, such and dengue and Zika virus.

Laboratory and semi-field evaluations of two (transfluthrin) spatial repellent devices against Aedes aegypti (L.) (Diptera: Culicidae).

Two transfluthrin-based spatial repellent products (Raid Dual Action Insect Repellent and Home Freshener and Raid Shield (currently not commercially available), SC Johnson, Racine WI) were evaluated for spatial repellent effects against female Aedes aegypti (L.) mosquitoes under laboratory (wind tunnel) and semi-field (outdoor enclosure) conditions. The placement of either product in the wind tunnel significantly reduced host-seeking behaviors. The mean baseline (control) landing counts for the Raid Dual Action and Raid Shield were reduced by 95% and 74% respectively. Mean probing counts for the Raid Dual Action were reduced by 95%, while the probing counts for the Raid Shield were decreased by 69%. Baseline blood-feeding success was significantly reduced for both treatments: Raid Dual Action (100%) and Raid Shield (96%). Semi-field evaluations were conducted in outdoor enclosures at the Navy Entomology Center of Excellence, Jacksonville, Florida. A moderate reduction in mosquito entry into military style tents resulted when either product was placed near the tent opening. The Raid Shield reduced mosquito entry into tents by 88%, while the Dual Action decreased entry by 66%.