Low Prevalence of Leptospira Carriage in Rodents in Leptospirosis-Endemic Northeastern Thailand.

Leptospirosis is a neglected zoonotic disease affecting mostly the world's tropical regions. The rural people of northeastern Thailand suffer from a large number of leptospirosis infections, and their abundant rice fields are optimal rodent habitats. To evaluate the contribution of diversity and carriage rate of pathogenic Leptospira in rodent reservoirs to leptospirosis incidence, we surveyed rodents, between 2011 and 2012, in four provinces in northeastern Thailand with the highest incidence rates of human leptospirosis cases. We used lipL32 real-time PCR to detect pathogenic Leptospira in rodent kidneys, partial 16S rRNA gene sequencing to classify the infecting Leptospira species, and whole 16S rDNA sequencing to classify species of isolated Leptospira. Overall prevalence of Leptospira infection was 3.6% (18/495). Among infected rodents, Bandicotaindica (14.3%), Rattusexulans (3.6%), and R. rattus (3.2%) had renal carriage. We identified two pathogenic Leptospira species: L. interrogans (n = 15) and L. borgpetersenii (n = 3). In addition, an L. wolffii (LS0914U) isolate was recovered from the urine of B. indica. Leptospira infection was more prevalent in low density rodent populations, such as B. indica. In contrast, there was a lower prevalence of Leptospira infection in high density rodent populations of R. exulans and R. rattus.

Bacterial microbiome of the chigger mite Leptotrombidium imphalum varies by life stage and infection with the scrub typhus pathogen Orientia tsutsugamushi.

Scrub typhus is a mites-borne rickettsiosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. The disease is potentially life threatening and is prevalent in tropical Asia, islands of the western Pacific Ocean and northern Australia where an estimated one million cases occur annually. Orientia tsutsugamushi is transmitted by the bite of larval mites in the genus Leptotrombidium. In the present study, the composition of the microbiome in larvae, deutonymphs and adult males and females from laboratory colonies of L. imphalum that were infected as well as uninfected with O. tsutsugamushi were investigated by high-throughput sequencing of the bacterial 16S rRNA gene. Notably, the bacterial microbiomes of infected adult females were dominated by sequences of O. tsutsugamushi and an unidentified species of Amoebophilaceae, which together comprised 98.2% of bacterial sequences. To improve the taxonomic resolution of the Amoebophilaceae OTU a nearly full length sequence of the 16S rRNA gene was amplified, cloned, and Sanger sequenced. Infected female mites had 89 to 92% nucleotide identity with the Amoebophilaceae family, indicating that the bacterium was likely to be a species of a novel genus. The species composition of bacterial communities varied between mite life stages regardless of their infection status. Uninfected adults exhibited greater species diversity than adults infected with O. tsutsugamushi. In the infected colony, the rate of filial infection with Orientia was less than 100%. Larval and male mites that were PCR-negative for Orientia contained low numbers of sequences of Amoebophilaceae (0.01 and 0.06%, respectively) in their taxonomic profiles, suggesting that a mutualistic relationship exists between the novel species of Amoebophilaceae and O. tsutsugamushi. Our study findings provide the basis for further research to determine the influence of the novel Amoebophilaceae species on the bacterial microbiome and on vector susceptibility to and transovarial transmission of O. tsutsugamushi.

Direct detection of Leishmania from clinical samples.

The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

Vector Competence of Anopheles kleini and Anopheles sinensis (Diptera: Culicidae) From the Republic of Korea to Vivax Malaria-Infected Blood From Patients From Thailand.

In total, 1,300 each of Anopheles kleini Rueda and Anopheles sinensis Wiedemann sensu stricto (s.s.) females (colonized from the Republic of Korea) and Anopheles dirus Peyton & Harrison (Thai strain) were allowed to feed on blood from Thai malaria patients naturally infected with Plasmodium vivax The overall oocyst infection rates for An. dirus, An. kleini, and An. sinensis s.s. were 77.4, 46.1, and 45.9%, respectively. The mean number of oocysts was significantly higher for An. dirus (82.7) compared with An. kleini (6.1) and An. sinensis s.s. (8.6), whereas the mean number of oocysts for An. kleini and An. sinensis s.s. was similar. The overall sporozoite infection rates for An. dirus, An. kleini, and An. sinensis s.s. dissected on days 14-15, 21, and 28 days post-feed were significantly higher for An. dirus (90.0%) than An. kleini (5.4%), whereas An. kleini sporozoite rates were significantly higher than An. sinensis s.s. (<0.1%). The overall sporozoite indices for positive females with +3 (100-1,000 sporozoites) and +4 (>1,000 sporozoites) salivary gland indices were significantly higher for An. dirus (85.7%), compared with An. kleini (47.1%). Only one An. sinensis s.s. had sporozoites (+2; >10-100 sporozoites). These results indicate that An. kleini is a competent vector of vivax malaria. Although An. sinensis s.s. develops relatively high numbers of oocysts, it is considered a very poor vector of vivax malaria due to a salivary gland barrier.

Transovarial Transmission of Co-Existing Orientia tsutsugamushi Genotypes in Laboratory-Reared Leptotrombidium imphalum.

The co-evolution of Orientia tsutsugamushi and its vector/host Leptotrombidium mites is important for this bacterium to survive and exist in its environment. The data in this study demonstrated that O. tsutsugamushi has adapted to take advantage of the parasitic nature of the host's larval stage and thus increase its chance of transmission to a vertebrate host and potentially to other vector mites by increasing its density at the time of transmission. Our data demonstrated that during the larval stage the density of O. tsutsugamushi was at its highest level compared to other life stages (Kruskal-Wallis, p < 0.0001). We further revealed that the different O. tsutsugamushi 56-kDa type-specific antigen (TSA) genotypes within the mite were maintained and preserved during transovarial transmission from the Leptotrombidium imphalum, lines Li-3 and Li-5. No sequence difference of 56-kDa TSA gene (variable domain I-III, 765 bp) was observed between the UT302-like genotype found in mothers and their offspring (100% identity). However, one or two nonsynonymous mutations in the 56-kDa TSA gene were observed in the Karp-like genotypes found in the F1 offspring with a percent difference ranging from 0.13 to 0.26 for nucleotide sequences and from 0.39 to 0.78 for amino acid sequences. Additionally, the composition of co-existing O. tsutsugamushi genotypes was maintained in L. imphalum lines through transsovarial and transstadial transmission processes; however, the proportion of these genotypes in each stage varied (larva, nymph, adult). These results show some of the key characteristics of O. tsutsugamushi maintenance within and transmission among its vector/host L. imphalum.

The Distribution and Diversity of Bartonella Species in Rodents and Their Ectoparasites across Thailand.

Our study highlights the surveillance of Bartonella species among rodents and their associated ectoparasites (ticks, fleas, lice, and mites) in several regions across Thailand. A total of 619 rodents and 554 pooled ectoparasites (287 mite pools, 62 flea pools, 35 louse pools, and 170 tick pools) were collected from 8 provinces within 4 regions of Thailand. Bandicota indica (279), Rattus rattus (163), and R. exulans (96) were the most prevalent species of rats collected in this study. Real-time PCR assay targeting Bartonella-specific ssrA gene was used for screening and each positive sample was confirmed by PCR using nuoG gene. The prevalence of Bartonella DNA in rodent (around 17%) was recorded in all regions. The highest prevalence of Bartonella species was found in B. savilei and R. rattus with the rate of 35.7% (5/14) and 32.5% (53/163), respectively. High prevalence of Bartonella-positive rodent was also found in B. indica (15.1%, 42/279), and R. norvegicus (12.5%, 5/40). In contrast, the prevalence of Bartonella species in ectoparasites collected from the rats varied significantly according to types of ectoparasites. A high prevalence of Bartonella DNA was found in louse pools (Polyplax spp. and Hoplopleura spp., 57.1%) and flea pools (Xenopsylla cheopis, 25.8%), while a low prevalence was found in pools of mites (Leptotrombidium spp. and Ascoschoengastia spp., 1.7%) and ticks (Haemaphysalis spp., 3.5%). Prevalence of Bartonella DNA in ectoparasites collected from Bartonella-positive rodents (19.4%) was significantly higher comparing to ectoparasites from Bartonella-negative rodents (8.7%). The phylogenetic analysis of 41 gltA sequences of 16 Bartonella isolates from rodent blood and 25 Bartonella-positive ectoparasites revealed a wide range of diversity among Bartonella species with a majority of sequences (61.0%) belonging to Bartonella elizabethae complex (11 rodents, 1 mite pool, and 5 louse pools), while the remaining sequences were identical to B. phoceensis (17.1%, 1 mite pool, 5 louse pools, and 1 tick pool), B. coopersplainensis (19.5%, 5 rodents, 1 louse pool, and 2 tick pools), and one previously unidentified Bartonella species (2.4%, 1 louse pool).

Development of an Orientia tsutsugamushi Lc-1 Murine Intraperitoneal Challenge Model for Scrub Typhus: Determination of Murine Lethal Dose (MuLD50), Tissue Bacterial Loads, and Clinical Outcomes.

Currently, no vaccine has been developed to protect humans from naturally acquired heterologous Orientia tsutsugamushi infections. To enhance the validity of vaccine candidates, we are developing a murine chigger challenge model with the O. tsutsugamushi Lc-1-infected Leptotrombidium chiangraiensis Line-1. To this end, an intraperitoneal (i.p.) murine challenge model using an O. tsutsugamushi Lc-1 isolate was developed for eventual validation of the chigger challenge model. We have determined that the murine lethal dose that kills 50% of the challenged mice (MuLD50) of a liver/spleen homogenate developed from O. tsutsugamushi Lc-1-infected ICR Swiss mice to be 10(-6.9). Employing different inoculum doses of this homogenate, the bacterial load using quantitative real-time PCR (qPCR) was determined to range from 60 to 1.6 × 10(5) genome equivalent copies (GEC)/µL of liver and 33.4 to 2.2 × 10(5) GEC/µL of spleen tissue. The clinical outcomes relative to homogenate dose levels followed a dose-dependent pattern. The successful development and characterization of the O. tsutsugamushi Lc-1 i.p. challenge model will assist in the development and validation of a mouse chigger challenge scrub typhus model.

Factors influencing the feeding response of laboratory-reared Aedes aegypti.

We evaluated the effects of membrane surface area (cm2), female density, and container/cage size on feeding response in laboratory-reared Aedes aegypti. Female density did not affect feeding rates at low surface areas, but higher density did significantly increase feeding as surface area increased. Females in large, cloth cages fed less compared to those in large, plastic cups. The rate of feeding was higher when using live, anesthetized mice versus a membrane feeding system. The AFRIMS Insectary will continue to look for innovative ways to improve our membrane feeding system.

Establishment of Orientia tsutsugamushi Lc-1 (Rickettsiales: Rickettsiaceae) infection in ICR outbred mice (Rodentia: Muridae) by needle challenge.

Orientia tsutsugamushi is a pathogen transmitted by Leptotrombidium that causes scrub typhus. To develop an infection mouse model, a mite-derived isolate of O. tsutsugamushi was established from a laboratory-maintained colony of Leptotrombidium chiangraiensis (O. tsutsugamushi Lc-1). This Lc-1 isolate was initially presented to ICR (CD-1) mice by feeding an infected Lc chigger on the ear of a mouse. Once the Lc-1 was adapted to the ICR mice, quantitative real-time polymerase chain reaction was used to investigate O. tsutsugamushi genomic equivalent copies in tissues and sera. Furthermore, times to onset of the signs of infection are reported in this study. This study provides information useful for future research on this host-pathogen interaction and the associated vaccine efficacy trials.

Colonization and maintenance of Anopheles kleini and Anopheles sinensis from the Republic of Korea.

Colonization of members of the Anopheles Hyrcanus Group is required to facilitate a better understanding of their roles in the epidemiology, vector competence, transmission potential, and maintenance of vivax malaria in the Republic of Korea (ROK). A total of 158 resting blood-fed wild caught Anopheles species were collected on August 26, 2011 at Tongilchon, near the demilitarized zone, in the ROK and shipped on August 28, 2011 to the Entomology Department, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand. Four of the 5 species of the Anopheles Hyrcanus Group (Anopheles kleini, An. sinensis, An. pullus, and An. belenrae) were identified by polymerase chain reaction. Eggs were obtained from parent populations, and F, progeny provided blood meals using membrane feeding techniques. The mean numbers of eggs/female for An. sinensis and An. kleini were 157 and 146, respectively. Membrane blood feeding rates for An. sinensis (90.6%) and An. kleini (89.0%) were similar for F1-F30 generations. Colonies of An. kleini and An. sinensis were established by force mating techniques and maintained by the Insectary Section, Entomology Department, AFRIMS, to date. This is the 1st report of successful laboratory colonization of An. kleini collected from a vivax malaria high-risk area in the ROK.

Characterization based on the 56-Kda type-specific antigen gene of Orientia tsutsugamushi genotypes isolated from Leptotrombidium mites and the rodent host post-infection.

Abstract. Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector.

Maintenance of mosquito vectors: effects of blood source on feeding, survival, fecundity, and egg hatching rates.

Artificial membrane-feeding techniques have replaced direct feeding on animals for the maintenance of malaria and arbovirus vectors in many laboratories. Membrane feeding facilitates controlled experimentation of pathogen transmission during mosquito feeding. Sheep blood is commonly used due to its availability and low cost. We evaluated the impact of blood source (human, guinea pig, sheep, and hamster via direct feeding) on feeding rates, adult survival, fecundity, hatching rates, and developmental times for five species of laboratory-colonized mosquitoes (Anopheles dirus, An. cracens, An. minimus, An. sawadwongporni, and Ae. aegypti). We found that feeding rates differ among blood sources within mosquito species. Survival, fecundity, and hatching rates were lower in all Anopheles species and Ae. aegypti after membrane feeding on sheep blood. Survival rates seven days post-feeding on sheep blood were significantly lower (P<0.05) for An. dirus (84.2%), An. minimus (67.2%), An. sawadwongporni (51.5%), and An. cracens (35.5%) relative to other blood sources. An. minimus and An. sawadwongporni laid no eggs by seven days post-feeding with sheep blood, while An. dirus and An. cracens produced significantly fewer numbers of eggs and demonstrated significantly lower hatching rates relative to what was observed with the other blood sources. These findings support the conclusion that sheep blood is not a suitable blood source for laboratory rearing of Anopheles spp.

Plasmodium vivax isolates from Cambodia and Thailand show high genetic complexity and distinct patterns of P. vivax multidrug resistance gene 1 (pvmdr1) polymorphisms.

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (HE = 0.86) but closely related (GST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.

Ixodid ticks associated with feral swine in Texas.

Ixodid ticks were collected from feral swine in eight Texas ecoregions from 2008-2011. Sixty-two percent of 806 feral swine were infested with one or more of the following species: Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor albipictus, D. halli, D. variabilis, and Ixodes scapularis. Juvenile and adult feral swine of both sexes were found to serve as host to ixodid ticks. Longitudinal surveys of feral swine at four geographic locations show persistent year-round tick infestations of all gender-age classes for tick species common to their respective geographic locations and ecoregions. Amblyomma americanum, A. cajennense, A. maculatum and D. variabilis were collected from 66% of feral swine harvested through an abatement program in seven ecoregions from March to October in 2009. These results indicate westward geographic expansion of D. variabilis. Summary results show feral swine are competent hosts for ixodid species responsible for the transmission of pathogens and diminished well-being in livestock, wildlife, and humans.

Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites.

Rodents are the natural hosts for Leptotrombidium mites that transmit Orientia tsutsugamushi, the causative agent of scrub typhus, a potentially fatal febrile human disease. Utilizing mite lines that included O. tsutsugamushi infected and non-infected Leptotrombidium species we investigated the varied infection response of outbred mice (ICR) exposed to L. chiangraiensis (Lc), L. imphalum (Li) and L. deliense (Ld). Each of six mite lines (Lc1, Lc5, Li3, Li4, Li7 and Ld) was separately placed in the inner ears of ICR mice either as a single individual (individual feeding, IF) or as a group of 2-4 individuals (pool feeding, PF). The species of infected chigger feeding on mice significantly affected mortality rates of the mice, with mite lines of Lc causing higher mean (±SE) mortality (90.7 ± 3.6 %) than mite lines of Li (62.9 ± 5.6 %) or Ld (53.6 ± 5.8 %). Mouse responses which included time to death, food consumption and total mice weight change depended on mite species and their O. tsutsugamushi genotype, more than on feeding procedure (IF vs. PF) except for mite lines within the Lc. Infected mite lines of Lc were the most virulent infected mites assessed whereas the infected Ld species was the least virulent for the ICR. Mice killed by various mite lines showed enlarged spleens and produced ascites. The results of this investigation of the clinical responses of ICR mice to feeding by various infected mite lines indicated that the different species of infected mites and their O. tsutsugamushi genotype produced different clinical presentations in ICR mice, a scrub typhus mouse model which mimics the natural transmission of O. tsutsugamushi that is critical for understanding scrub typhus disease in terms of natural transmission, host-pathogen-vector interaction and vaccine development.

The genus Hyalomma: VII. Redescription of all parasitic stages of H. (Euhyalomma) dromedarii and H. (E.) schulzei (Acari: Ixodidae).

The ticks, Hyalomma (Euhyalomma) dromedarii Koch, 1844 and Hyalomma (Euhyalomma) schulzei Olenev, 1931, are considered to be the species most closely associated with camels. H. dromedarii can behave as a three-, two-, or one-host species, with the two-host life cycle seemingly most common. Camels are the main hosts of the adults, which also parasitize other domestic animals. Nymphs and larvae can use the same hosts, especially camels, as the adults, but can also parasitize rodents, leporids, hedgehogs, and birds. H. dromedarii is widely distributed in North Africa, the northern regions of West, Central, and East Africa, Arabia, Asia Minor, the Middle East, and Central and South Asia. H. schulzei is a two-host species. Camels are the principal hosts of the adults, with some records from cattle and goats, whereas the immature stages infest hares, burrowing rodents, and hedgehogs. H. schulzei has a more limited geographic distribution in Asia and Egypt than H. dromedarii, and with the exception of southeastern Iran, it is a fairly uncommon tick. Among other features that are fairly similar, males of H. dromedarii can be distinguished from those of H. schulzei by a narrow, subtriangular parma, usually very large subanal shields, and a long dorsal prolongation of the spiracular plates. Males of H. schulzei have a broad and rectangular parma, paramedian festoons that protrude posteriorly, smaller subanal shields, and a very short dorsal prolongation of the spiracular plates. The females of H. dromedarii can be distinguished from those of H. schulzei by a narrow V-shaped genital aperture compared with a very wide, deep, U-shaped genital aperture. Here all the parasitic stages of both species are illustrated and redescribed, and characteristics that distinguish the adults from those of other closely related species are supplied. Data on their hosts, geographic distribution and disease relationships are also provided.