Ticks (Acari: Ixodidae) and Associated Pathoge Collected From Domestic Animals and Vegetation in Stann Creek District, Southeastern Belize, Central America.

Data on the prevalence and distribution of ticks and tick-borne diseases in Belize are lacking. Ticks (n = 564) collected from dogs, horses, and vegetation in two villages in Stann Creek District in southeastern Belize in 2018, were molecularly identified and screened for tick-borne nonviral human pathogens. The identity of 417 ticks was molecularly confirmed by DNA barcoding as Rhipicephalus sanguineus (Latreille) (66.43%), Amblyomma ovale Koch (15.59%), Dermacentor nitens Neumann (11.51%), Amblyomma sp. ADB0528 (3.6%), and the remainder being small records (2.87%) of Amblyomma coelebs Neumann, Amblyomma imitator Kohls, Amblyomma tapirellum Dunn, Amblyomma auricularium Conil, and Amblyomma maculatum Koch. Individual tick extracts were screened for the presence of Rickettsia spp., Babesia spp., Babesia microti, Borrelia spp., Ehrlichia spp., and Anaplasma spp. using available conventional polymerase chain reaction (PCR) assays. Rickettsia parkeri strain Atlantic Rainforest was identified in five specimens of A. ovale, and one other unidentified tick, all collected from dogs. Another unidentified tick-also collected from a dog-tested positive for an undefined but previously detected Ehrlichia sp. With the exception of D. nitens, all eight other tick species identified in this study were collected on dogs, suggesting that dogs could be usefully employed as sentinel animals for tick surveillance in Belize.

Molecular Characterization of Bartonella Species Discovered in Ectoparasites Collected from Domestic Animals, Cuzco, Peru.

Rickettsiae and bartonellae are Gram-negative bacteria that can cause zoonotic and human diseases and are vectored by hematophagous arthropods. In the Americas, rickettsioses and bartonelloses have reemerged as significant public health threats. Bartonella species have been identified as causing zoonotic infections responsible for a variety of clinical syndromes in humans and animals. The aim of this study was to investigate the distribution, prevalence, and molecular heterogeneity of Rickettsia spp. and Bartonella spp. among ectoparasites collected from domestic animals in 14 farming communities in the Andes Mountains of Cuzco, Peru. A total of 222 domestic animals representing 8 different species (sheep, donkeys, goats, cattle, pigs, llamas, guinea pigs, and horses) were sampled. Nine species of ectoparasites (n = 1,697) collected from 122 animals were identified resulting in 1,657 chewing lice, 39 ticks, and 1 flea. DNA was individually extracted from a random sample of 600 (35.4%) considering variability of ectoparasite species, hosts, and sample location elevation. All 600 samples were negative for rickettsial DNA by a genus-specific molecular assay. A subset of 173 (28.8%) samples were selected based on variability of arthropods species, host, and location for Bartonella testing. Ninety-one (52.6%) of these samples including Melophagus ovinus (90/110) and Bovicola bovis (1/7) were positive for Bartonella by a genus-specific molecular assay. Five Bartonella genes of seven DNA samples from M. ovinus were analyzed by the multilocus sequence typing for characterization. We identified five identical Bartonella melophagi specimens and two specimens with Bartonella species related to B. melophagi from the seven M. ovinus. The Bartonella agents detected were widely distributed and frequent in multiple studied locations.

Human seroepidemiology of Rickettsia and Orientia species in Chile - A cross-sectional study in five regions.

In recent years, the spectrum and epidemiology of human rickettsioses has become an emerging topic in Chile. This survey aimed to assess the seroprevalence of spotted fever group rickettsiae (SFGR), typhus group rickettsiae (TGR), and scrub typhus group orientiae (STGO) in northern, central, and southern Chile. We performed a cross-sectional study of healthy adults in rural and urban settings of five regions. Participants were chosen by double stratified random sampling in urban and by convenience in rural locations (n = 1302). Serum specimens were analyzed for group-specific IgG antibodies against SFGR, TGR, and STGO by enzyme-linked immunosorbent assays (ELISAs). Overall seroprevalences to SFGR, TGR, and STGO were 5.3 %, 1.2 %, and 0.4 %, respectively. Prevalences showed geographical differences. Statistical analyses revealed an association of older age with seropositivity to SFGR and to TGR and of rural setting and male gender with seropositivity to SFGR. The study indicates that SFGR, TGR, and STGO are endemic in Chile. The very low STGO seroprevalence might indicate an insufficient sensitivity of serological tests using Asian O. tsutsugamushi strains as ELISA antigens for the detection of antibodies against Chilean Orientia species.

Molecular Description of a Novel Orientia Species Causing Scrub Typhus in Chile.

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%-3.5% for rrs and 11.2%-11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloé Archipelago where the pathogen was identified.

Identification of trombiculid mites (Acari: Trombiculidae) on rodents from Chiloé Island and molecular evidence of infection with Orientia species.

BACKGROUND: Scrub typhus is an emerging vector-borne zoonosis, caused by Orientia spp. and transmitted by larvae of trombiculid mites, called chiggers. It mainly occurs within a region of the Asia-Pacific called the tsutsugamushi triangle, where rodents are known as the most relevant hosts for the trombiculid vector. However, the reservoir(s) and vector(s) of the scrub typhus outside Asia-Pacific are unknown. The disease has recently been discovered on and is considered endemic for Chiloé Island in southern Chile. The aim of the present work was to detect and determine the prevalence of chiggers on different rodent species captured in probable sites for the transmission of orientiae responsible for scrub typhus on Chiloé Island in southern Chile and to molecularly examine collected chiggers for the presence of Orientia DNA. METHODOLOGY/PRINCIPAL FINDINGS: During the austral summer 2018, rodents were live-trapped in six sites and examined for chigger infestation. All study sites were rural areas on Chiloé Island, previously identified as probable localities where human cases acquired the scrub typhus. During a total of 4,713 trap-nights, 244 rodents of seven species were captured: the most abundant was Abrothrix olivacea. Chiggers were detected on all seven rodent species with a 55% prevalence rate. Chiggers showed low host specificity and varied according to site specific host abundance. Three genera of trombiculids were identified. Herpetacarus was the most abundant genus (93%), prevalent in five of the six sites. Infestation rates showed site specific differences, which were statistically significant using a GLM model with binomial errors. Molecular analyses proved that 21 of 133 (15.8%) mite pools were positive for Orientia species, all of them belonged to the genus Herpetacarus. CONCLUSIONS/SIGNIFICANCE: This study firstly reports the presence of different rodent-associated chigger mites positive for Orientia sp., in a region endemic for scrub typhus in southern Chile. Herpetacarus and two other genera of mites were found with high infestation rates of rodents in sites previously identified as probable exposure of scrub typhus cases. A substantial percentage of mite pools were positive for Orientia DNA, suggesting that chigger mites serve as vectors and reservoirs of this emerging zoonosis in South America.

Scrub Typhus in Continental Chile, 2016-20181.

Endemic scrub typhus was recently detected on Chiloé Island in southern Chile. We report a series of cases, acquired over a wide geographical range in continental Chile during 2016-2018, demonstrating that this emerging rickettsial infection is also found on the mainland of South America.

Imported scrub typhus: first case in South America and review of the literature.

BACKGROUND: Scrub typhus is a neglected vector-borne zoonosis causing life-threatening illnesses, endemic in the Asian-Pacific region and, as recently discovered, in southern Chile. Scrub typhus is rarely reported in travelers, most probably due to the lack of clinical experience and diagnostic tests in non-endemic countries. We report the first case of imported scrub typhus in South America. CASE PRESENTATION: A 62-year-old tourist from South Korea presented severely ill with fever, rash, and eschar in Santiago, Chile. Laboratory exams showed thrombocytopenia and elevated inflammation parameters, hepatic enzymes, and LDH. With the clinical suspicion of scrub typhus, empirical treatment with doxycycline was initiated and the patient recovered rapidly and without complications. The diagnosis was confirmed by IgM serology and by real-time PCR, which demonstrated infection with Orientia tsutsugamushi (Kawasaki clade). CONCLUSIONS: Only due to the emerging clinical experience with endemic South American scrub typhus and the recent implementation of appropriate diagnostic techniques in Chile, were we able to firstly identify and adequately manage a severe case of imported scrub typhus in South America. Physicians attending febrile travelers need to be aware of this rickettsiosis, since it requires prompt treatment with doxycycline to avoid complications.

Canine seroprevalence to Orientia species in southern Chile: A cross-sectional survey on the Chiloé Island.

BACKGROUND: Scrub typhus is a potentially life-threatening vector-borne infection caused by Orientia species. It occurs mainly in the Asian-Pacific region, where it causes significant morbidity and mortality. Recently, an endemic focus of scrub typhus has been described in South America, on Chiloé Island in southern Chile. Dogs have been used as sentinel hosts to determine the presence and spatial distribution of various vector-borne infections. Their suitability to gain insight into human exposure to Orientia tsutsugamushi has been suggested in studies from Asia. METHODOLOGY: In January 2016, we conducted a cross-sectional study, which included the two main cities on Chiloé Island. Canine blood samples were obtained in households, chosen by double stratified random sampling in urban and by convenience in rural locations. Specimens were tested by ELISA for IgG antibodies against whole-cell antigen preparations from three strains of O. tsutsugamushi. Data were further analyzed for factors associated with seropositivity including spatial clustering. RESULTS: Serum samples from 202 dogs (104 urban, 98 rural) were tested for IgG against O. tsutsugamushi, of which 43 (21.3%) were positive. Seroprevalence rates were higher in rural than in urban settings (p<0.01) and in older compared to younger dogs (p<0.01). Spatial analysis by LISA indicated the presence of four localities of highly grouped cases. CONCLUSIONS: The detected seroprevalence supports the endemicity of scrub typhus in southern Chile and suggests a wide exposure of household dogs to the infected, yet unknown vector(s). The spatial data will be used for future research identifying further human cases as well as the local vector(s)/reservoirs for scrub typhus in southern Chile. The study reinforces that dogs are useful sentinels for Orientia spp. in regions of uncertain endemicity and distribution.

Serologic Evidence of Scrub Typhus in the Peruvian Amazon.

Using a large, passive, febrile surveillance program in Iquitos, Peru, we retrospectively tested human blood specimens for scrub typhus group orientiae by ELISA, immunofluorescence assay, and PCR. Of 1,124 participants, 60 (5.3%) were seropositive, and 1 showed evidence of recent active infection. Our serologic data indicate that scrub typhus is present in the Peruvian Amazon.

Whole-Genome Analysis of Bartonella ancashensis, a Novel Pathogen Causing Verruga Peruana, Rural Ancash Region, Peru.

The genus Bartonella contains >40 species, and an increasing number of these Bartonella species are being implicated in human disease. One such pathogen is Bartonella ancashensis, which was isolated in blood samples from 2 patients living in Caraz, Peru, during a clinical trial of treatment for bartonellosis. Three B. ancashensis strains were analyzed by using whole-genome restriction mapping and high-throughput pyrosequencing. Genome-wide comparative analysis of Bartonella species showed that B. ancashensis has features seen in modern and ancient lineages of Bartonella species and is more related to B. bacilliformis. The divergence between B. ancashensis and B. bacilliformis is much greater than what is seen between known Bartonella genetic lineages. In addition, B. ancashensis contains type IV secretion system proteins, which are not present in B. bacilliformis. Whole-genome analysis indicates that B. ancashensis might represent a distinct Bartonella lineage phylogenetically related to B. bacilliformis.

Rickettsial Disease in the Peruvian Amazon Basin.

Using a large, passive, clinic-based surveillance program in Iquitos, Peru, we characterized the prevalence of rickettsial infections among undifferentiated febrile cases and obtained evidence of pathogen transmission in potential domestic reservoir contacts and their ectoparasites. Blood specimens from humans and animals were assayed for spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR) by ELISA and/or PCR; ectoparasites were screened by PCR. Logistic regression was used to determine associations between patient history, demographic characteristics of participants and symptoms, clinical findings and outcome of rickettsial infection. Of the 2,054 enrolled participants, almost 2% showed evidence of seroconversion or a 4-fold rise in antibody titers specific for rickettsiae between acute and convalescent blood samples. Of 190 fleas (Ctenocephalides felis) and 60 ticks (Rhipicephalus sanguineus) tested, 185 (97.4%) and 3 (5%), respectively, were positive for Rickettsia spp. Candidatus Rickettsia asemboensis was identified in 100% and 33% of the fleas and ticks tested, respectively. Collectively, our serologic data indicates that human pathogenic SFGR are present in the Peruvian Amazon and pose a significant risk of infection to individuals exposed to wild, domestic and peri-domestic animals and their ectoparasites.

Description of Bartonella ancashensis sp. nov., isolated from the blood of two patients with verruga peruana.

Three novel isolates of the genus Bartonella were recovered from the blood of two patients enrolled in a clinical trial for the treatment of chronic stage Bartonella bacilliformis infection (verruga peruana) in Caraz, Ancash, Peru. The isolates were initially characterized by sequencing a fragment of the gltA gene, and found to be disparate from B. bacilliformis. The isolates were further characterized using phenotypic and genotypic methods, and found to be genetically identical to each other for the genes assessed, but distinct from any known species of the genus Bartonella, including the closest relative B. bacilliformis. Other characteristics of the isolates, including their morphology, microscopic and biochemical properties, and growth patterns, were consistent with members of the genus Bartonella. Based on these results, we conclude that these three isolates are members of a novel species of the genus Bartonella for which we propose the name Bartonella ancashensis sp. nov. (type strain 20.00T = ATCC BAA-2694T = DSM 29364T).

Molecular typing of "Candidatus Bartonella ancashi," a new human pathogen causing verruga peruana.

A recently described clinical isolate, "Candidatus Bartonella ancashi," was obtained from a blood sample of a patient presenting with verruga peruana in the Ancash region of Peru. This sample and a second isolate obtained 60 days later from the same patient were molecularly typed using multilocus sequence typing (MLST) and multispacer sequence typing (MST). The isolates were 100% indistinguishable from each other but phylogenetically distant from Bartonella bacilliformis and considerably divergent from other known Bartonella species, confirming their novelty.

Detection of Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Gulf Coast ticks collected from humans in the United States.

Rickettsia parkeri, a spotted fever group (SFG) rickettsia recently found to be pathogenic to humans, causes an eschar-associated febrile illness. The R. parkeri rickettsiosis, Tidewater spotted fever, has been misdiagnosed as Rocky Mountain spotted fever due to serologic cross reactivity and the lack of specific diagnostic methods. Candidatus Rickettsia andeanae, also a SFG rickettsia, is a recently described agent of unknown pathogenicity originally identified in ticks collected from domestic animals during a fever outbreak investigation in northern Peru. Among 37 Amblyomma maculatum (collected from humans (n=35) and questing (n=2)) obtained from the southern United States during 2000-2009, nine and four A. maculatum nucleic acid preparations were found positive for R. parkeri and Candidatus R. andeanae, respectively, by newly developed genus- and species-specific quantitative real-time polymerase chain reaction assays. In addition Rickettsia felis was found in two A. maculatum nucleic acid preparations.

Endemic scrub typhus-like illness, Chile.

We report a case of scrub typhus in a 54-year-old man who was bitten by several terrestrial leeches during a trip to Chiloé Island in southern Chile in 2006. A molecular sample, identified as related to Orientia tsutsugamushi based on the sequence of the16S rRNA gene, was obtained from a biopsy specimen of the eschar on the patient's leg. Serologic analysis showed immunoglobulin G conversion against O. tsutsugamushi whole cell antigen. This case and its associated molecular analyses suggest that an Orientia-like agent is present in the Western Hemisphere that can produce scrub typhus-like illness. The molecular analysis suggests that the infectious agent is closely related, although not identical, to members of the Orientia sp. from Asia.

Variation of soil fertility and carbon sequestration by planting Hevea brasiliensis in Hainan Island, China.

The development of rubber industry depends on the sustainable management of rubber plantation. To evaluate the environmental effects of planting Hevea brasiliensis on a subsystem of tropical forest ecosystem, the variation of soil fertility and carbon sequestration under rubber plantation within 30-year life period were investigated in Hainan Island. Results showed that (1) with the increase of stand age of rubber plantation, soil fertility decreased all along. From 1954 to 1995, soil organic matter, total N, available K and available P decreased by 48.2%, 54.1%, 56.7% and 64.1%, respectively. (2) If the complete return of litters was considered without additional fertilizer application to the soil of the rubber plantations, the consumption periods for P, N, K, Mg were only 825 years, 329 years, 94 years and 65 years, respectively. To improve soil fertility is essential for rubber plantation development. (3) The C sequestration of rubber trees per hectare accounts for 272.08 t within 30-year life period and 57.91% of them was fixed in litters. In comparison with C sequestration by rain forest (234.305 t/hm2) and by secondary rain forest (150.203 t/hm2), rubber forest has more potentials for C fixation. On the base of above results, the following measures would benefit the maintenance of soil fertility and the development of rubber industry, including applying fertilizer to maintain the balance of soil nutrients, intercropping leguminous plant to improve soil fertility, reducing the collection of litters, optimizing soil properties to improve element P availability such as applying CaCO3. The information gathered from the study can be used as baseline data for the sustainable management of rubber plantation elsewhere.

Phylogenetic analysis of a novel molecular isolate of spotted fever group Rickettsiae from northern Peru: Candidatus Rickettsia andeanae.

Phylogenetic analysis of five rickettsial genes (17-kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the 17-kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from 17-kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

Characterization of spotted fever group rickettsiae in flea and tick specimens from northern Peru.

Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.