Molecular Identification of Aedes, Armigeres, and Culex Mosquitoes (Diptera: Culicidae) Using Mitochondrial Cytochrome Oxidase Subunit I Genes in Myanmar.

PURPOSE: Mosquitoes are important vectors that carry disease-causing agents that can affect humans and animals. DNA barcoding is a complementary identification which can be used to validate morphological characterization of mosquito species. The objectives of this study were to identify the mitochondrial sequence of the COI gene and to construct a molecular phylogeny based on the genetic divergence of the mosquito species studied. METHODS: In this study, DNA extraction and the amplification of the mitochondrial cytochrome oxidase subunit I genes (COI) were performed on pooled mosquito samples collected in Nay Pyi Taw area, Myanmar. RESULTS: Fragments of the COI gene showed 99-100% identity with sequences of Aedes aegypti, Armigeres subalbatus, Culex pipiens complex, and Cx. quinquefasciatus, respectively, deposited in GenBank. This study categorized two haplotypes from each Ar. subalbatus and Cx. pipiens complex COI gene sequence, as well as three haplotypes from Cx. quinquefasciatus COI gene sequences. The highest haplotype diversity and nucleotide diversity were observed in the Ar. subalbatus population (Hd = 1.0000; π = 0.0033), followed by the Cx. pipiens complex and Cx. quinquefasciatus populations. CONCLUSION: This study provides useful information on the molecular identification and genetic diversity of mosquito vectors with medical and veterinary significance, which may assist in the improvement of mosquito control programs.

Comparative mitogenomics elucidates the population genetic structure of Amblyomma testudinarium in Japan and a closely related Amblyomma species in Myanmar.

Ticks are the second most important vector capable of transmitting diseases affecting the health of both humans and animals. Amblyomma testudinarium Koch 1844 (Acari: Ixodidae), is a hard tick species having a wide geographic distribution in Asia. In this study, we analyzed the composition of A. testudinarium whole mitogenomes from various geographical regions in Japan and investigated the population structure, demographic patterns, and phylogeographic relationship with other ixodid species. In addition, we characterized a potentially novel tick species closely related to A. testudinarium from Myanmar. Phylogeographic inference and evolutionary dynamics based on the 15 mitochondrial coding genes supported that A. testudinarium population in Japan is resolved into a star-like haplogroup and suggested a distinct population structure of A. testudinarium from Amami island in Kyushu region. Correlation analysis using Mantel test statistics showed that no significant correlation was observed between the genetic and geographic distances calculated between the A. testudinarium population from different localities in Japan. Finally, demographic analyses, including mismatch analysis and Tajima's D test, suggested a possibility of recent population expansion occurred within Japanese haplogroup after a bottleneck event. Although A. testudinarium has been considered widespread and common in East and Southeast Asia, the current study suggested that potentially several cryptic Amblyomma spp. closely related to A. testudinarium are present in Asia.

First molecular detection of Hemolivia and Hepatozoon parasites in reptile-associated ticks on Iriomote Island, Japan.

Hepatozoon and Hemolivia are members of the haemogregarines and are reported in reptiles and reptile-associated ticks. However, no studies have reported on Hepatozoon and Hemolivia in Japanese reptile-associated ticks. This study aimed to molecularly identify and to characterize Hepatozoon and Hemolivia in Japanese reptile-associated ticks, Amblyomma geoemydae (Cantor, 1847) and Amblyomma nitidum (Hirst & Hirst, 1910). A total of 41 and 75 DNA samples from A. geoemydae and A. nitidum ticks, respectively, were used for screening of Hepatozoon and Hemolivia with polymerase chain reaction targeting 18S rDNA. As a result, Hemolivia and Hepatozoon were detected in two A. geoemydae and one A. nitidum, respectively. The sequences of Hemolivia spp. showed a 99.5% (1,050/1,055 bp) identity with Hemolivia parvula (KR069083), and the Hemolivia spp. were located in the same clade as H. parvula in the phylogenetic tree. The sequences of Hepatozoon sp. showed a 98.4% (1,521/1,545 bp) identity with Hepatozoon colubri (MN723844), and the Hepatozoon sp. was distinct from validated Hepatozoon species in the tree. Our findings highlight the first molecular record of Hemolivia in Japan and present the first detection of Hepatozoon in A. nitidum. Further investigations on these tick-borne protozoa are required to understand their life cycle and pathogenicity.

PCR detection and genetic characterization of piroplasms from dogs in Myanmar, and a possible role of dogs as reservoirs for Theileria parasites infecting cattle, water buffaloes, and goats.

Canine vector-borne pathogens can act as zoonotic agents in humans; however, it poorly understood whether dogs play a role as reservoirs of vector-borne parasites in livestock animals. Here, we report the unexpected detection of 18S rRNA gene (rDNA) sequences of five ruminant Theileria species from the peripheral blood of dogs in Myanmar, in addition to those of two canine Babesia species. Using novel BTH primers capable of amplifying the 18S rDNA of Babesia, Theileria, and Hepatozoon spp., approximately 1,500 bp nested PCR products were detected in 19% (17/91) of local or imported dog breeds in different regions of Myanmar. Among the sequences of the 17 PCR products, ten were determined as Theileria 18S rDNA, including three as Theileria orientalis, three as Theileria buffeli, two as Theileria cf. velifera, one as Theileria luwenshuni, and one as Theileria sp. Most of these sequences showed higher identities with Theileria sequences determined in previous studies of cattle, water buffaloes, and goats in Myanmar. Six PCR products were identified as Babesia vogeli and one sample was determined as Babesia gibsoni. Furthermore, we obtained approximately 900 bp thrombospondin-related adhesive protein (TRAP) gene fragments from three dog blood DNA samples. Phylogenetic analysis of the TRAP gene showed that B. gibsoni parasites in Myanmar were considerably related to isolates from China, Korea, Japan, and Taiwan, but clearly separated from those from Bangladesh and India. These results provide new insights into a possible role of dogs in maintaining and spreading tick-borne pathogens among livestock and canine populations in Myanmar.

Identification, genetic variation, and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar.

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.

Spiroplasma Infection among Ixodid Ticks Exhibits Species Dependence and Suggests a Vertical Pattern of Transmission.

Members of the genus Spiroplasma are Gram-positive bacteria without cell walls. Some Spiroplasma species can cause disease in arthropods such as bees, whereas others provide their host with resistance to pathogens. Ticks also harbour Spiroplasma, but their role has not been elucidated yet. Here, the infection status and genetic diversity of Spiroplasma in ticks were investigated using samples collected from different geographic regions in Japan. A total of 712 ticks were tested for Spiroplasma infection by PCR targeting 16S rDNA, and Spiroplasma species were genetically characterized based on 16S rDNA, ITS, dnaA, and rpoB gene sequences. A total of 109 samples originating from eight tick species were positive for Spiroplasma infection, with infection rates ranging from 0% to 84% depending on the species. A linear mixed model indicated that tick species was the primary factor associated with Spiroplasma infection. Moreover, certain Spiroplasma alleles that are highly adapted to specific tick species may explain the high infection rates in Ixodes ovatus and Haemaphysalis kitaokai. A comparison of the alleles obtained suggests that horizontal transmission between tick species may not be a frequent event. These findings provide clues to understand the transmission cycle of Spiroplasma species in wild tick populations and their roles in host ticks.

Molecular Prevalence and Identification of Ehrlichia canis and Anaplasma platys from Dogs in Nay Pyi Taw Area, Myanmar.

Ticks are vectors of different types of viruses, protozoans, and other microorganisms, which include Gram-negative prokaryotes of the genera Rickettsiales, Ehrlichia, Anaplasma, and Borrelia. Canine monocytic ehrlichiosis caused by Ehrlichia canis and canine cyclic thrombocytopenia caused by Anaplasma platys are of veterinary importance worldwide. In Myanmar, there is limited information concerning tick-borne pathogens, Ehrlichia and Anaplasma spp., as well as genetic characterization of these species. We performed nested PCR for the gltA gene of the genus Ehrlichia spp. and the 16S rRNA gene of the genus Anaplasma spp. with blood samples from 400 apparently healthy dogs in Nay Pyi Taw area. These amplicon sequences were compared with other sequences from GenBank. Among the 400 blood samples from dogs, 3 (0.75%) were positive for E. canis and 1 (0.25%) was positive for A. platys. The partial sequences of the E. canis gltA and A. platys 16SrRNA genes obtained were highly similar to E. canis and A. platys isolated from different other countries.

Anthropogenic interferences lead to gut microbiome dysbiosis in Asian elephants and may alter adaptation processes to surrounding environments.

Human activities interfere with wild animals and lead to the loss of many animal populations. Therefore, efforts have been made to understand how wildlife can rebound from anthropogenic disturbances. An essential mechanism to adapt to environmental and social changes is the fluctuations in the host gut microbiome. Here we give a comprehensive description of anthropogenically induced microbiome alterations in Asian elephants (n = 30). We detected gut microbial changes due to overseas translocation, captivity and deworming. We found that microbes belonging to Planococcaceae had the highest contribution in the microbiome alterations after translocation, while Clostridiaceae, Spirochaetaceae and Bacteroidia were the most affected after captivity. However, deworming significantly changed the abundance of Flavobacteriaceae, Sphingobacteriaceae, Xanthomonadaceae, Weeksellaceae and Burkholderiaceae. These findings may provide fundamental ideas to help guide the preservation tactics and probiotic replacement therapies of a dysbiosed gut microbiome in Asian elephants. More generally, these results show the severity of anthropogenic activities at the level of gut microbiome, altering the adaptation processes to new environments and the subsequent capability to maintain normal physiological processes in animals.

Bacterial and protozoan pathogens/symbionts in ticks infecting wild grasscutters (Thryonomys swinderianus) in Ghana.

Ticks and tick-borne pathogens constitute a great threat to livestock production and are a potential health hazard to humans. Grasscutters (Thryonomys swinderianus) are widely hunted for meat in Ghana and many other West and Central African countries. However, tick-borne zoonotic risks posed by wild grasscutters have not been assessed. The objective of this study was to investigate bacterial and protozoan pathogens in ticks infecting wild grasscutters. A total of 81 ticks were collected from three hunted grasscutters purchased from Kantamanto, the central bushmeat market in Accra. Ticks were identified as Ixodes aulacodi and Rhipicephalus sp. based on morphological keys, which were further confirmed by sequencing mitochondrial 16S ribosomal DNA (rDNA) and cytochrome oxidase I (COI) genes of specimens. Protozoan infections were tested by PCR amplifying 18S rDNA of Babesia/Theileria/Hepatozoon, while bacterial infections were evaluated by PCRs or real-time PCRs targeting Anaplasmataceae, Borrelia, spotted fever group rickettsiae, chlamydiae and Candidatus Midichloria mitochondrii. The results of PCR screening showed that 35.5% (27 out of 76) of I. aulacodi were positive for parasite infections. Sequencing analysis of the amplified products gave one identical sequence showing similarity with Babesia spp. reported from Africa. The Ca. M. mitochondrii endosymbiont was present in 85.5% (65 out of 76) of I. aulacodi but not in the five Rhipicephalus ticks. Two Anaplasmataceae bacteria genetically related to Ehrlichia muris and Anaplasma phagocytophilum were also detected in two I. aulacodi. None of the ticks were positive for Borrelia spp., spotted fever group rickettsiae and chlamydiae. Since I. aulacodi on wild grasscutters are potential carriers of tick-borne pathogens, some of which could be of zoonotic potential, rigorous tick control and pathogen analyses should be instituted especially when wild caught grasscutters are being used as foundation stock for breeding.

Complete Genome Sequence of Rickettsia asiatica Strain Maytaro1284, a Member of Spotted Fever Group Rickettsiae Isolated from an Ixodes ovatus Tick in Japan.

This is the first report of the complete genome sequence of Rickettsia asiatica strain Maytaro1284, isolated from an Ixodes ovatus tick in Japan. The genome contains a 1,344,324-bp circular chromosome and one plasmid of 74,761 bp. There was no outer membrane protein A (ompA) gene encoded in the genome.

Diversity of spotted fever group rickettsiae and their association with host ticks in Japan.

Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria mainly associated with ticks. In Japan, several hundred cases of Japanese spotted fever, caused by Rickettsia japonica, are reported annually. Other Rickettsia species are also known to exist in ixodid ticks; however, their phylogenetic position and pathogenic potential are poorly understood. We conducted a nationwide cross-sectional survey on questing ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR amplification of the citrate synthase gene (gltA). Conventional PCR and sequencing analyses of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based on the analysis of five additional genes, we characterised five Rickettsia species; R. asiatica, R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R. tarasevichiae and several unclassified SFG rickettsiae. We also found a strong association between rickettsial genotypes and their host tick species, while there was little association between rickettsial genotypes and their geographical origins. These observations suggested that most of the SFG rickettsiae have a limited host range and are maintained in certain tick species in the natural environment.

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells.

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.

Human Borreliosis Caused by a New World Relapsing Fever Borrelia-like Organism in the Old World.

BACKGROUND: Relapsing fever is an infectious disease previously neglected in Africa, which imposes a large public health burden in the country. We aimed to investigate and report on a case of relapsing fever borreliosis in Zambia. METHODS: A previously unknown Borrelia species was isolated from the blood of a febrile patient. Investigations of the presumptive vector ticks and natural hosts for the Borrelia species were conducted by culture isolation and/or DNA detection by Borrelia-specific polymerase chain reaction. Using culture isolates from the patient and bat specimens, genetic characterization was performed by multilocus sequence analysis based on the draft genome sequences. RESULTS: The febrile patient was diagnosed with relapsing fever. The isolated Borrelia species was frequently detected in Ornithodoros faini (n = 20/50 [40%]) and bats (n = 64/237 [27%]). Multilocus sequence analysis based on a draft genome sequence revealed that the Borrelia species isolates from the patient and presumptive reservoir host (bats) formed a monophyletic lineage that clustered with relapsing fever borreliae found in the United States. CONCLUSIONS: A febrile illness caused by a Borrelia species that was treatable with erythromycin was identified in Zambia. This is the first study to report on relapsing fever Borrelia in Zambia and suggesting the likely natural reservoir hosts of the isolated Borrelia species. Interestingly, the isolated Borrelia species was more closely related to New World relapsing fever borreliae, despite being detected in the Afrotropic ecozone.

First molecular detection of Theileria luwenshuni from goats in Myanmar.

Tick-borne intracellular protozoan parasites of the Theileria genus infect a wide range of both domestic and wild animals. In the present study, we describe the first PCR detection of Theileria luwenshuni in the blood of goats in Myanmar. Nested PCR targeting the Theileria 18S rRNA gene resulted in seven positive goats in central and northern Myanmar. Nucleotide sequencing of the PCR products revealed that all seven sequences were identical and showed 100% identity with T. luwenshuni sequences in GenBank from goats and sheep in China. Since T. luwenshuni parasites have recently been discovered and shown to have nationwide distribution in China, they might have been introduced into Myanmar via transboundary movement of infected domestic small ruminants and/or wild animals from China.

Tick-borne haemoparasites and Anaplasmataceae in domestic dogs in Zambia.

Tick-borne diseases (TBDs), including emerging and re-emerging infectious diseases, are important threats to human and animal health worldwide. Indeed, the number of reported human and animal infectious cases of novel TBD agents has increased in recent decades. However, TBDs tend to be neglected, especially in resource-limited countries that often have limited diagnostic capacity. The aim of this molecular survey was to detect and characterise tick-borne pathogens (Babesia, Theileria, and Hepatozoon parasites and Anaplasmataceae bacteria) in domestic dogs in Zambia. In total, 247 canine peripheral blood samples were collected in Lusaka, Mazabuka, Monze, and Shangombo. Conventional PCR to detect the selected pathogens was performed using DNA extracted from canine blood. One hundred eleven samples were positive for protozoa and 5 were positive for Anaplasmataceae. Sequencing of thirty-five randomly selected protozoa-positive samples revealed the presence of Babesia rossi, Babesia vogeli, and Hepatozoon canis 18S rDNA. Based on these sequences, a multiplex PCR system was developed to yield PCR products with different amplicons, the size of which depended on the parasite species; thus, each species could be identified without the need for sequence analysis. Approximately 40% of dogs were positive for H. canis. In particular, the positive rate (75.2%) of H. canis infection was significantly higher in Shangombo than in other sampling sites. Multiplex PCR assay detected B. rossi and B. vogeli infections in five and seven dogs, respectively, indicating that this approach is useful for detecting parasites with low prevalence. Sequencing analysis of gltA and groEL genes of Anaplasmataceae revealed that two and one dogs in Lusaka were infected with Anaplasma platys and Ehrlichia canis, respectively. The data indicated that Zambian dogs were infected with multiple tick-borne pathogens such as H. canis, B. rossi, B. vogeli, A. platys, E. canis and uncharacterized Ehrlichia sp. Since some of these parasites are zoonotic, concerted efforts are needed to raise awareness of, and control, these tick-borne pathogens.

Dynamics, co-infections and characteristics of zoonotic tick-borne pathogens in Hokkaido small mammals, Japan.

Many of the emerging infectious diseases originate in wildlife and many of them are caused by vector-borne pathogens. In Japan, zoonotic tick-borne pathogens (TBPs) are frequently detected in both ticks and wildlife. Here, we studied the infection rates of potentially zoonotic species, including Anaplasma, Ehrlichia, Neoehrlichia and Babesia spp., in Hokkaido's most abundant small mammals as they relate to variable extrinsic factors that might affect the infection rates of these pathogens. A total of 412 small mammals including 64 Apodemus argenteus, 219 Apodemus speciosus, 78 Myodes rufocanus, 41 Myodes rutilus, 6 Myodes rex and 4 Sorex unguiculatus were collected from Furano and Shari sites in Hokkaido, Japan, in 2010 and 2011 and were examined by multiplex PCR for TBPs. A reverse line blot hybridization (RLB) was then developed for the specific detection of 13 potentially zoonotic TBPs. A total of 4 TBPs were detected: Anaplasma sp. AP-sd, Ehrlichia muris, Candidatus Neoehrlichia mikurensis and Babesia microti. The infection rates were 4.4% (18/412), 1.2% (5/412), 13.1% (54/412) and 17.2% (71/412), respectively. The infection rates of each of the detected TBPs were significantly correlated with host small mammal species. A total of 22 (two triple and 20 double) co-infection cases were detected (5.3%). The most frequent co-infection cases occurred between Candidatus N. mikurensis and B. microti 68.2% (15/22). Further studies are required to examine human exposure to these zoonotic TBPs in Hokkaido.

Serological Survey and Factors Associated with Toxoplasma gondii Infection in Domestic Goats in Myanmar.

Goat farming is important for the livelihood of millions of rural people because it contributes to food security and creation of assets. However, infection of goats with Toxoplasma gondii could be a source of parasite transmission to humans. The information on T. gondii infection of goat was not reported yet in Myanmar. A total of 119 goat serum samples were collected from three cities in the central region of Myanmar for T. gondii antibody survey. With the occurrence value obtained in this first study, a second one, more complete, with larger number (162) of animals and properties, was carried out and the risk factors and prevalence were determined. In both studies the samples were analyzed by the LAT. Of these, 32 (11.4%) samples were showed to be positive. The infection was associated with the presence of cats at the farm (odds ratio [OR] = 4.66, 95% confidential interval [CI] = 1.03-21.06), farming with different animal species (sheep, cattle, and pigs) (OR = 4.33, 95% CI = 1.57-11.94), and farming without good management practices (OR = 0.23, 95% CI = 0.06-0.83). This is the first T. gondii prevalence study in goats in the country.

Molecular evidence of spotted fever group rickettsiae and Anaplasmataceae from ticks and stray dogs in Bangladesh.

Emerging tick-borne diseases (TBDs) are important foci for human and animal health worldwide. However, these diseases are sometimes over looked, especially in countries with limited resources to perform molecular-based surveys. The aim of this study was to detect and characterize spotted fever group (SFG) rickettsiae and Anaplasmataceae in Bangladesh, which are important tick-borne pathogens for humans and animals worldwide. A total of 50 canine blood samples, 15 ticks collected from dogs, and 154 ticks collected from cattle were screened for the presence of SFG rickettsiae and Anaplasmataceae using molecular-based methods such as PCR and real-time PCR. The sequence analysis of the amplified products detected two different genotypes of SFG rickettsiae in ticks from cattle. The genotype detected in Rhipicephalus microplus was closely related to Rickettsia monacensis, while the genotype detected in Haemaphysalis bispinosa was closely related to Rickettsia sp. found in Korea and Japan. Anaplasma bovis was detected in canine blood and ticks (Rhipicephalus sanguineus and H. bispinosa). Unexpectedly, the partial genome sequence of Wolbachia sp., presumably associated with the nematode Dirofilaria immitis, was identified in canine blood. The present study provides the first molecular evidence of SFG rickettsiae and A. bovis in Bangladesh, indicating the possible emergence of previously unrecognized TBDs in this country.