Yongjin Qiu

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens

Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.

High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers.

The spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted by ticks that cause several tick-borne rickettsioses in humans worldwide. This study was intended to determine the prevalence of SFG rickettsiae in Amblyomma variegatum from 7 districts across Uganda. In addition to sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNA(fMet) was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation allowed simple, accurate, and high-throughput fragment sizing with considerable time and cost savings. Rickettsia genus-specific real-time PCR detected 136 positives out of 140 samples, giving an overall prevalence of 97.1%. Most samples (n=113) had a size combination of 225, 195, and 341 bp for dksA-xerC, mppA-purC, and rpmE-tRNA(fMet), respectively, which was identical to that of R. africae, a causative agent of African tick bite fever. In addition, several samples had size variants in either dksA-xerC or rpmE-tRNA(fMet). Nonetheless, the partial sequences of gltA and ompA genes of samples of all size combinations showed the greatest similarity to R. africae (99.3-100% for gltA and 98.1-100% for ompA). Given these results, it is highly possible that the tested ticks were infected with R. africae or closely related species. This is a first report on molecular genetic detection of R. africae and its high endemicity in Uganda. Clinicians in this country should be aware of this pathogen as a cause of non-malarial febrile illness. This study provided a starting point for the development of Rickettsia species identification based on the sizes of intergenic spacers. The procedure is simple, rapid, and cost-effective to perform; hence it might be particularly well suited for preliminary species identification in epidemiological investigations. The results may be more detailed and reliable when simultaneous sequencing analysis is performed.

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.

First Genetic Detection of Coxiella burnetii in Zambian Livestock

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular gram-negative bacterium. The investigation of C. burnetii infection in Zambian livestock was carried out using molecular detection techniques. A total of 489 cattle and 53 goat blood samples were collected from Chama, Chongwe, Monze, and Petauke districts in Zambia. Molecular screening by polymerase chain reaction was performed using C. burnetii-species-specific primers. In total, 38 cattle and 4 goat samples were positive. The prevalence of C. burnetii differed among the four sites, with Chama (Eastern province) recording the highest, although Monze (Southern province) did not record any case of the bacteria. This study reports the first genetic detection of C. burnetii in Zambia.

Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line

Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated I. scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of I. scapularis-associated virus-1, which was reported in a recent metagenomic study of I. scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks.

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real‐time PCR targeting the 16S ribosomal RNA gene, serotype‐specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.

Molecular detection and characterization of zoonotic Anaplasma species in domestic dogs in Lusaka, Zambia

Although tick-borne pathogens, Anaplasma platys and Anaplasma phagocytophilum are recognized as zoonotic agents associated with appreciable morbidity and mortality in dogs and humans worldwide, there is limited information on these infections in many African countries, including Zambia. The purpose of this study was to detect, identify and phylogenetically characterize Anaplasma species from dogs in Chilanga District in Lusaka Province, Zambia. A total of 301 blood samples were collected from apparently healthy and semi-confined dogs. Initial screening by polymerase chain reaction with specific primers targeting the 16S rRNA gene of Anaplasma species revealed that 9% (27/301) of our samples were positive. Subsequent sequence and phylogenetic analysis of a longer fragment of the 16S rRNA and citrate synthase (gltA) genes of four positive samples showed the presence of A. platys and an Anaplasma species, which was closely related to those detected in dogs in South Africa. This is the first report on molecular identification and characterization of canine-associated zoonotic Anaplasma species in Zambia.

The Unique Phylogenetic Position of a Novel Tick-Borne Phlebovirus Ensures an Ixodid Origin of the Genus Phlebovirus

The emergence of novel tick-borne RNA viruses causing severe illness in humans has complicated the epidemiological landscape of tick-borne diseases, requiring further investigation to safeguard public health. In the present study, we discovered a novel tick-borne phlebovirus from Ixodes persulcatus ticks in Japan. While its viral RNA genome sequences were similar to those of mosquito/sandfly-borne viruses, molecular and biological footprints confirmed that this is a tick-borne virus. The unique evolutionary position of the virus allowed us to estimate the ancestral phlebovirus vector, which was likely a hard tick. Our findings may provide a better understanding of the evolution and emergence of phleboviruses associated with emerging infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Heartland virus disease. ABSTRACT The recent emergence of novel tick-borne RNA viruses has complicated the epidemiological landscape of tick-borne infectious diseases, posing a significant challenge to public health systems that seek to counteract tick-borne diseases. The identification of two novel tick-borne phleboviruses (TBPVs), severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), as causative agents of severe illness in humans has accelerated the investigation and discoveries of novel TBPVs. In the present study, we isolated a novel TBPV designated Mukawa virus (MKWV) from host-questing Ixodes persulcatus females captured in Japan. Genetic characterization revealed that MKWV is a member of the genus Phlebovirus in the family Phenuiviridae. Interestingly, MKWV is genetically distinct from other known TBPVs and shares a most recent common ancestor with mosquito/sandfly-borne (insect-borne) phleboviruses. Despite its genetic similarity to insect-borne phleboviruses, the molecular footprints of its viral proteins and its biological characteristics define MKWV as a tick-borne virus that can be transmitted to mammals. A phylogenetic ancestral-state reconstruction for arthropod vectors of phleboviruses including MKWV based on viral L segment sequences indicated that ticks likely harbored ancestral phleboviruses that evolved into both the tick-borne and MKWV/insect-borne phlebovirus lineages. Overall, our findings suggest that most of the phlebovirus evolution has occurred in hard ticks to generate divergent viruses, which may provide a seminal foundation for understanding the mechanisms underlying the evolution and emergence of pathogenic phleboviruses, such as Rift Valley fever virus and SFTSV/HRTV. IMPORTANCE The emergence of novel tick-borne RNA viruses causing severe illness in humans has complicated the epidemiological landscape of tick-borne diseases, requiring further investigation to safeguard public health. In the present study, we discovered a novel tick-borne phlebovirus from Ixodes persulcatus ticks in Japan. While its viral RNA genome sequences were similar to those of mosquito/sandfly-borne viruses, molecular and biological footprints confirmed that this is a tick-borne virus. The unique evolutionary position of the virus allowed us to estimate the ancestral phlebovirus vector, which was likely a hard tick. Our findings may provide a better understanding of the evolution and emergence of phleboviruses associated with emerging infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Heartland virus disease.

Discovery of Mwinilunga alphavirus: a novel alphavirus in Culex mosquitoes in Zambia

Mosquito-borne alphaviruses are disseminated globally and cause febrile illness in humans and animals. Since the prevalence and diversity of alphaviruses has not been previously investigated in Zambia, reverse transcription PCR was employed as a broad-spectrum approach for the detection of alphaviruses in mosquitoes. From 552 mosquito pools, a novel alphavirus, tentatively named Mwinilunga alphavirus (MWAV), was discovered from a single Culex quinquefasciatus mosquito pool. The full genome of MWAV was subsequently determined, and pairwise comparisons demonstrated that MWAV represented a new alphavirus species. Phylogenetic analyses and a linear discriminant analysis based on the dinucleotide ratios in various virus sequences indicated that MWAV is related to a mosquito-specific alphavirus distinct from other known mosquito-borne alphaviruses due to its inability to replicate in vertebrate cell lines. Further analyses of these novel alphaviruses will help to facilitate a greater understanding of the molecular determinants of host range restriction and the evolutionary relationships of alphaviruses.

First isolation of West Nile virus in Zambia from mosquitoes

Mosquito surveillance studies to identify mosquito-borne flaviviruses have identified West Nile Virus (WNV) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.