Screening of tick-borne pathogens in argasid ticks in Zambia: Expansion of the geographic distribution of Rickettsia lusitaniae and Rickettsia hoogstraalii and detection of putative novel Anaplasma species.

Ticks (Ixodidae and Argasidae) are important arthropod vectors of various pathogens that cause human and animal infectious diseases. Many previously published studies on tick-borne pathogens focused on those transmitted by ixodid ticks. Although there are increasing reports of viral pathogens associated with argasid ticks, information on bacterial pathogens they transmit is scarce. The aim of this molecular study was to detect and characterize Rickettsia and Anaplasmataceae in three different argasid tick species, Ornithodoros faini, Ornithodoros moubata, and Argas walkerae collected in Zambia. Rickettsia hoogstraalii and Rickettsia lusitaniae were detected in 77 % (77/100) of Ar. walkerae and 10 % (5/50) of O. faini, respectively. All O. moubata pool samples (n = 124) were negative for rickettsial infections. Anaplasmataceae were detected in 63 % (63/100) of Ar. walkerae and in 82.2 % (102/124) of O. moubata pools, but not in O. faini. Phylogenetic analysis based on the concatenated sequences of 16S rRNA and groEL genes revealed that Anaplasma spp. detected in the present study were distinct from previously validated Anaplasma species, indicating that the current knowledge on the diversity and vector range of Anaplasma spp. is incomplete. Our findings highlight new geographical records of R. lusitaniae and R. hoogstraalii and confirm that the wide geographic distribution of these species includes the African continent. The data presented here increase our knowledge on argasid tick-borne bacteria and contribute toward understanding their epidemiology.

Molecular Detection of Spotted Fever Group Rickettsia (Rickettsiales: Rickettsiaceae) in Ticks of Iran.

Ticks are reservoir hosts of pathogenic Rickettsia in humans and domestic animals. Most pathogenic Rickettsia species belong to the spotted fever group (SFG). The present study aimed to determine the tick species infected with Rickettsia based on the genus-specific 23S ribosomal ribonucleic acid (rRNA), 16S rRNA, and citrate synthase (gltA) gene fragments. A total of 61 tick specimens were selected for molecular assay and 12 samples for sequencing. Phylogenetic analysis was conducted using neighbor-joining and Bayesian inference methods. Argas persicus, Haemaphysalis sulcata, Ha. inermis, and Hyalomma asiaticum were infected by spotted fever Rickettsia. The SFG is the main group of Rickettsia that can be detected in the three genera of ticks from Iran.

Rickettsia hoogstraalii and a Rickettsiella from the Bat Tick Argas transgariepinus, in Namibia.

Ectoparasites were collected from Eptesicus hottentotus, the long-tailed serotine bat, caught in Namibia as part of an ecological study. Larvae of Argas transgariepinus, a blood-feeding ectoparasite of bats in Africa, were removed from 3 of 18 bats. We present scanning electron microscope images of unengorged larvae. As with other ectoparasites, this bat tick might transmit pathogens such as Borrelia and Rickettsia to their hosts as has been reported for bat ticks in Europe and North America. We screened 3 pools (25 total) of larvae of A. transgariepinus removed from the long-tailed serotine bat Eptesicus hottentotus caught in Namibia. Two microbes of unknown pathogenicity, including Rickettsia hoogstraalii, a spotted fever group pathogen, and a Rickettsiella sp. were detected by molecular techniques.

Ultrastructural changes induced by the entomopathogenic fungus Beauveria bassiana in the ovary of the tick Argas (Persicargas) persicus (Oken).

The present study was carried out to assess the effects on the ovary of fed female Argas persicus following spraying of the ticks with spores of the fungus Beauveria bassiana suspended in triton X100 at a concentration of 107 conidia/mL. Scanning and transmission electron microscopy observations provided evidence that B. bassiana invaded the ovary, causing extensive morphological damage and deterioration of the developing oocytes. Destruction of the shape and internal organelles of young and previtellogenic oocytes and complete inhibition of vitellogenesis was evident. This histopathological study is the first demonstration of ultrastructural damage in the ovaries of A. persicus after infection with B. bassiana. The data presented confirm that B. bassiana affects the ovary either directly by entering the oocytes and/or indirectly by producing toxins in the haemolymph that interfere with the development of oocytes, thus potentially contributing to the control of this tick in a way that is safe for its host and the environment.

Molecular detection of avian spirochete Borrelia anserina in Argas persicus ticks in Algeria.

Argasid ticks are one of the most important poultry ectoparasites. They affect poultry directly through blood meal and indirectly through the transmission of pathogens essentially Borrelia anserina, agent of avian borreliosis, one of the most widespread poultry diseases in the world, and is of great economic importance. This study was conducted between April 2014 and March 2015 in the region of Ksar El Boukhari, Algeria, in order to investigate the presence of soft ticks in laying hen farms and to detect B. anserina bacteria using molecular tools. DNA was extracted and screened for the presence of Borrelia spp. DNA by real-time polymerase chain reaction (qPCR). Borrelia spp. screening was performed using primers and probe targeting the 16S rRNA gene. A total of 83 traditional laying hen farms were visited, of which 39 (46.98 %) were found infested with A. persicus tick. Molecular analysis revealed that 2/34 (5.88 %) of ticks were infected by B. anserina. None of the ticks tested were positive for Rickettsia spp., and Coxiella burnetii. These results constitute the first report in Algeria of A. persicus harboring B. anserina.

ARGAS (PERSICARGAS) GIGANTEUS SOFT TICK INFECTION WITH RICKETTSIA HOOGSTRAALI AND RELAPSING FEVER BORRELIA ON WILD AVIAN SPECIES OF THE DESERT SOUTHWEST, USA.

Changing climatic conditions and the northward expansion of ticks and pathogens are of immense importance to human, animal, and environmental health assessment and risk management. From 2014 through 2015, a wildlife rehabilitation center in south-central Arizona, US noted soft ticks (Argasidae) infesting 23 birds, including Cooper's Hawks (Accipiter cooperii), Gray Hawks (Buteo plagiatus), Harris's Hawks (Parabuteo unicinctus), Red-tailed Hawks (Buteo jamaicensis), Great Horned Owls (Bubo virginianus), Common Ravens (Corvus corax), and a Greater Roadrunner (Geococcyx californianus), during the late summer seasonal rainy seasons. The parasites numbered in the hundreds on individual birds. Infested birds were moribund, obtunded, or paralyzed on presentation, with no prior histories of illness or evidence of trauma. Tick and avian blood samples were collected for vector-borne pathogen analysis focusing on the molecular detection of Rickettsia and Borrelia species. Ticks were identified as the neotropical species of soft tick, Argas (Persicargas) giganteus; their occurrence in the southern US on raptors represented an expansion of host range. Pathogen testing of ticks showed that 41% of 54 ticks were infected with Rickettsia hoogstraalii and 23% of 26 ticks were infected with a relapsing fever Borrelia; both agents are associated with uncertain health consequences. Among the blood samples, one was infected with the same Borrelia spp.; this Red-tailed Hawk also had Borrelia-positive ticks. With supportive therapy, the majority of birds, 74% (17/23), recovered and were released or permanent residents. The management of soft tick-infested birds and mitigation of future disease risk will require additional characterizations of these poorly studied soft ticks and their associated pathogens.

Molecular detection of Coxiella (Gammaproteobacteria: Coxiellaceae) in Argas persicus and Alveonasus canestrinii (Acari: Argasidae) from Iran.

BACKGROUND: Coxiella burnetii and non-C. burnetii bacteria or endosymbiotic Coxiella-like were reported in various tick species. We aimed to detect C. burnetii within soft tick species, Argas persicus and Alveonasus canestrinii. METHODS: Argasid ticks were collected from different counties of Lorestan province, west of Iran. Partial fragments of 16S rRNA, IS1111 insertion sequence, com1, htpB, and icd genes related to Coxiella genus were sequenced. RESULTS: A partial 16S rRNA and com1 gene fragment as well as IS1111 was detected in four Ar. persicus and twelve Al. canestrinii pools. Moreover, partial htpB and icd gene was only detected in one pool of Ar. persicus. CONCLUSIONS: Detection of C. burnetii in tick samples was failed due to the occurrence of Coxiella-like endosymbionts and leads to misidentification. Thus, the house-keeping genes should be designated to distinguish C. burnetii within Coxiella-like endosymbionts.

Microbial diversity in the tick Argas japonicus (Acari: Argasidae) with a focus on Rickettsia pathogens.

The soft tick Argas japonicus mainly infests birds and can cause human dermatitis; however, no pathogen has been identified from this tick species in China. In the present study, the microbiota in A. japonicus collected from an epidemic community was explored, and some putative Rickettsia pathogens were further characterized. The results obtained indicated that bacteria in A. japonicus were mainly ascribed to the phyla Proteobacteria, Firmicutes and Actinobacteria. At the genus level, the male A. japonicus harboured more diverse bacteria than the females and nymphs. The bacteria Alcaligenes, Pseudomonas, Rickettsia and Staphylococcus were common in nymphs and adults. The abundance of bacteria belonging to the Rickettsia genus in females and males was 7.27% and 10.42%, respectively. Furthermore, the 16S rRNA gene of Rickettsia was amplified and sequenced, and phylogenetic analysis revealed that 13 sequences were clustered with the spotted fever group rickettsiae (Rickettsia heilongjiangensis and Rickettsia japonica) and three were clustered with Rickettsia limoniae, which suggested that the characterized Rickettsia in A. japonicus were novel putative pathogens and also that the residents were at considerable risk for infection by tick-borne pathogens.

Identification of Two Genotypes of Argas persicus and Associated Rickettsia-Specific Genes from Different Regions of Inner Mongolia.

Ticks are important vectors and reservoirs of several zoonotic pathogens. Recently, both known and unknown tick-borne pathogens have emerged and re-emerged, causing unpredictable epidemics. In this study, 211 soft tick samples were collected from Tongliao and Alxa in Inner Mongolia, China. Tick species were identified by morphological and molecular biological analyses. Morphological analysis showed that there was no significant difference in surface features between ticks from the 2 areas. Cloning by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene showed that all ticks belonged to the species Argas persicus. Analysis using Genetyx software indicated that there was a limited degree of diversity between ticks from the 2 areas. Three base changes were detected in the 16S rRNA gene. We constructed phylogenetic trees using MEGA 6.0 software and showed that the ticks from the 2 areas might have evolved independently from species in other geographical areas. To assess the presence of Rickettsia, Streptococcus suis, and Staphylococcus aureus pathogens in tick samples, over 100 16S rRNA sequences belonging to these 3 pathogens were obtained from GenBank. We used CLC Sequence Viewer 7.0 to determine conserved sequences for the design of degenerate primers. Using standard PCR, we detected Rickettsia-specific genes, including 16S rRNA, 17KD, and ompB, in gDNA samples of ticks from Alxa. This study has laid a foundation for future studies on the biodiversity of ticks and for a new pathogen information database of ticks in local areas.

A survey of argasid ticks and tick-associated pathogens in the Peripheral Oases around Tarim Basin and the first record of Argas japonicus in Xinjiang, China.

Argasid ticks (Acari: Argasidae) carry and transmit a variety of pathogens of animals and humans, including viruses, bacteria and parasites. There are several studies reporting ixodid ticks (Acari: Ixodidae) and associated tick-borne pathogens in Xinjiang, China. However, little is known about the argasid ticks and argasid tick-associated pathogens in this area. In this study, a total of 3829 adult argasid ticks infesting livestock were collected at 12 sampling sites of 10 counties in the Peripheral Oases, which carry 90% of the livestock and humans population, around the Tarim Basin (southern Xinjiang) from 2013 to 2016. Tick specimens were identified to two species from different genera by morphology and sequences of mitochondrial 16S rRNA and 12S rRNA were derived to confirm the species designation. The results showed that the dominant argasid ticks infesting livestock in southern Xinjiang were Ornithodoros lahorensis (87.86%, 3364/3829). Ornithodoros lahorensis was distributed widely and were collected from 10 counties of southern Xinjiang. Argas japonicus was collected from Xinjiang for the first time. In addition, we screened these ticks for tick-associated pathogens and showed the presence of DNA sequences of Rickettsia spp. of Spotted fever group and Anaplasma spp. in the argasid ticks. This finding suggests the potential role for Argas japonicus as a vector of pathogens to livestock and humans.

Rickettsia fournieri sp. nov., a novel spotted fever group rickettsia from Argas lagenoplastis ticks in Australia.

Strain AUS118T was isolated from an Argas lagenoplastis tick collected from the nest of a Petrochelidon ariel (fairy martin) in Australia in 2013. Microscopic observation of infected cell cultures indicated this strain had a morphology and intracellular location typical of Rickettsiaspecies. Phylogenetic analysis of this strain based firstly on multi-locus sequence analysis and subsequently on whole genome analysis demonstrated that AUS118T was most closely related to, but divergent from Rickettsia japonica and Rickettsia heilongjiangensis. We therefore propose the creation of a novel species, Rickettsia fournieri sp. nov, with the type strain AUS118T (DSM 28985 and CSUR R501).

Isolation of a divergent strain of Rickettsia japonica from Dew's Australian bat Argasid ticks (Argas (Carios) dewae) in Victoria, Australia.

A divergent strain of Rickettsia japonica was isolated from a Dew's Australian bat argasid tick, Argas (Carios) dewae, collected in southern Victoria, Australia and a full-genome analysis along with sequencing of 5 core gene fragments was undertaken. This isolate was designated Rickettsia japonica str. argasii (ATCC VR-1665, CSUR R179).

Borrelia, Rickettsia, and Ehrlichia species in bat ticks, France, 2010.

Argas vespertilionis, an argasid tick associated with bats and bat habitats in Europe, Africa, and Asia has been reported to bite humans; however, studies investigating the presence of vector-borne pathogens in these ticks are lacking. Using molecular tools, we tested 5 A. vespertilionis ticks collected in 2010 from the floor of a bat-infested attic in southwestern France that had been converted into bedrooms. Rickettsia sp. AvBat, a new genotype of spotted fever group rickettsiae, was detected and cultivated from 3 of the 5 ticks. A new species of the Ehrlichia canis group, Ehrlichia sp. AvBat, was also detected in 3 ticks. Four ticks were infected with Borrelia sp. CPB1, a relapsing fever agent of the Borrelia group that caused fatal borreliosis in a bat in the United Kingdom. Further studies are needed to characterize these new agents and determine if the A. vespertilionis tick is a vector and/or reservoir of these agents.

Candidatus Rickettsia hoogstraalii in Ethiopian Argas persicus ticks.

Ethiopian soft ticks Argas persicus, hard ticks including both Amblyomma variegatum and Rhipicephalus (Boophilus) spp., and fleas were collected from livestock, traditional human dwellings, and cracks and crevices of trees. They were assessed in pools for the presence of Rickettsia using PCR-based methods. The extracted tick DNA was subjected to molecular screening for Rickettsia, which revealed 50.5% of the pooled samples to be positive for Rickettsia spp. These were then subjected to multi-gene analysis using both outer surface proteins and housekeeping genes with proven discriminatory potential. Sequencing of the citrate synthase and outer membrane genes clearly led to the identification of three distinct rickettsial species, Candidatus Rickettsia hoogstraalii in Argas persicus ticks; R. africae in hard tick pools, and R. felis in fleas. Furthermore, we demonstrated the presence of the plasmid-borne small heat-shock protein gene hsp2 in DNA from A. persicus ticks suggesting that Candidatus R. hoogstraalii carried by these ticks possess a plasmid. Unlike chromosomal gene sequences, the hsp2 gene failed to cluster with Candidatus R. hoogstraalii, instead falling into an isolated separate clade, suggesting a different origin for the plasmid.

Borrelia in Ethiopian ticks.

Two regions (Jimma and Dire Dawa) in Ethiopia were investigated for the presence of soft ticks. Although no Ornithodoros spp. ticks were collected during this survey, published records of their existence in Ethiopia were found. An overwhelming infestation of Argas persicus was revealed in a village located adjacent to Dire Dawa. These ticks primarily were feeding on poultry, but were also biting humans. Furthermore, hard ticks were collected from livestock and companion animals in these regions. Collected ticks were assessed for Borrelia by real-time PCR followed by conventional PCR and sequencing to identify species present. A. persicus ticks were found to carry B. anserina in 3 of 40 (7.5%) A. persicus tick pools, whilst hard tick pools yielded 2 of 16 (12.5%) positive for B. theileri. Collectively, these borrelial species and their tick vectors are likely to have an important economic impact of particular relevance to subsistence farmers in Ethiopia.

Metarhizium anisopliae (Ascomycota: Hypocreales): an effective alternative to chemical acaricides against different developmental stages of fowl tick Argas persicus (Acari: Argasidae).

The fowl bloodsucking tick Argas persicus is of great medical and veterinary importance in tropical and subtropical regions because of its role as vector of certain parasitic, bacterial and viral pathogens. A variety of acaricides are used for the control of tick infestation in poultry, resulting in environmental contamination and the development of resistance. In order to develop an alternative control method, the efficacy of three strains (V245, 685 and 715C) of entomopathogenic fungus Metarhizium anisopliae against different life stages of A. persicus including eggs, larvae, unfed and engorged adult females was evaluated under laboratory conditions. Five concentrations of different strains of M. anisopliae ranging from 10(3) to 10(7)conidia/ml were utilized. The effects of fungal strains on egg hatchability and larva and adult female mortality were significant and dose-dependent compared to the control groups (P<0.05). The mortality rates of larvae ranged from 92% to 100% for two different concentrations (10(3) and 10(4)conidia/ml) of M. anisopliae strains. Treated engorged females were more susceptible than the unfed females reaching mortality rate of 100% at the highest concentration (10(7)conidia/ml) at 18 days post-inoculation. Among strains used in this study, V245 was the most virulent strain regarding the LC(50) values for adult females exposed to fungal conidia. The results demonstrate that the application of M. anisopliae as a biocontrol agent is a promising option in reducing the use of chemical acaricides, resulting in benefits to poultry and the environment.

Avian spirochetosis in chickens following experimental transmission of Borrelia anserina by Argas (Persicargas) miniatus.

This study reports the experimental transmission of Borrelia anserina to domestic chickens by infected Argas (Persicargas) miniatus. Clinical alterations as well as prepatent and patent periods were evaluated. Twenty-seven 67-day-old birds were divided into three groups in a randomized experimental design. The first group was exposed to ticks infected with B. anserina, the second group was exposed to noninfected ticks, and the third group was not exposed to ticks. Blood smears from each bird of groups 1 and 2 were prepared daily and examined for 25 days postexposure (PE). Examination of the blood smears from birds in group 1 revealed large numbers of spirochetes from days 5 to 12 PE. In this group the prepatent and patent periods were 5-7 and 4-7 days, respectively. Birds from group 1 presented ruffled feathers, pale combs, drowsiness, inappetence, loss of weight, and greenish diarrhea after day 6 PE. The current study confirms the viability of experimental transmission of B. anserina to domestic chickens by A. (P.) miniatus.

Molecular evidence for a novel Coxiella from Argas monolakensis (Acari: Argasidae) from Mono Lake, California, USA.

Argasid ticks are vectors of viral and bacterial agents of humans and animals. Recent reports indicate that some ornithophilic argasids harbored rickettsial agents. A Nearctic tick, Argas monolakensis Schwan, Corwin, Brown is ornithophilic and has not previously been examined for rickettsial agents. Thirty adult A. monolakensis were tested by PCR for DNA from Rickettsia or Coxiella. Amplicons from a Coxiella sp. that were divergent from Coxiella burnetii were detected in 16/30 A. monolakensis. These molecular isolates were similar but not identical to C. burnetii, the Coxiella spp. of other ticks, and "Coxiella cheraxi" a pathogen of crayfish.