Detection of <Emphasis Type="Italic">Rickettsia</Emphasis> spp. in ticks parasitizing toads (<Emphasis Type="Italic">Rhinella marina</Emphasis>) in the northern Brazilian Amazon

This study evaluated rickettsial infection in ticks collected on toads from the northern Brazilian Amazon (Amapá state), where to our knowledge there are neither records of ticks from amphibians nor rickettsial infections in ticks. During 2016–2017, a total of 22 free-living toads were captured and identified as Rhinella marina. Overall, 12 (54.5%) toads were parasitized by a total of 97 ticks (6 males, 39 females, 31 nymphs, 21 larvae), giving a mean intensity of 8.1 ticks per infested toad. Two tick species were morphologically identified: Amblyomma rotundatum Koch (31 females, 14 nymphs), and Amblyomma dissimile Koch (6 males, 8 females, 17 nymphs). The 21 larvae were morphologically denoted as Amblyomma sp. Five toads were co-infested by A. rotundatum and A. dissimile. Morphological identifications were confirmed by nucleotide sequencing of fragments of the mitochondrial genes 16S rDNA, 12S rDNA and/or COX1. A total of 54 ticks were analyzed for the presence of rickettsial DNA. Eleven (9 females and 2 nymphs) out of 14 A. rotundatum ticks contained Rickettsia bellii. None of the 25 specimens of A. dissimile (6 males, 6 females, 13 nymphs) contained amplifiable rickettsial DNA. From 15 Amblyomma sp. larvae, a pool of 10 individuals contained Rickettsia sp. strain Colombianensi. Sequencing of the 16S rDNA amplicon derived from the positive pool yielded a sequence of A. dissimile. We detected Rickettsia sp. strain Colombianensi for the first time in Brazil. Prior records of this agent were restricted to Colombia and Honduras. In addition, we report the presence of A. rotundatum for the first time in the state of Amapá, where the only other record of A. dissimile was registered over 20 years ago.     

Phylogenetic insights on Mediterranean and Afrotropical <Emphasis Type="Italic">Rhipicephalus</Emphasis> species (Acari: Ixodida) based on mitochondrial DNA

This study was performed to determine the tick species that infest cattle and humans throughout an altitudinal gradient in the Yungas Biogeographic Province of Argentina. The presence of tick-borne bacteria of the genera Rickettsia, Ehrlichia and Borrelia in the collected ticks was also evaluated. Samples of ticks parasitizing cattle and humans were carried out in different seasons. Questing ticks (adults and nymphs) were collected from vegetation and analyzed to detect the presence of Rickettsia, Ehrlichia and Borrelia by a battery of different PCRs. Five species of hard ticks were found parasitizing cattle: Amblyomma sculptum, Amblyomma tonelliae, Amblyomma hadanii, Haemaphysalis juxtakochi and Ixodes pararicinus. Amblyomma sculptum (immature and adults), A. tonelliae (immature and adults), A. hadanii (larvae) and one nymph of I. pararicinus were found attached to humans. Rickettsia amblyommatis was detected in one nymph of A. hadanii. DNA of a Borrelia genospecies belonging to the B. burgdorferi s.l. complex (phylogenetically related to haplotypes previously reported in Ixodes aragaoi from Uruguay and I. pararicinus from Argentina) was detected in adults of I. pararicinus. Amblyomma sculptum and I. pararicinus appear to be the tick species more frequent on cattle in the YBP from Argentina, and A. sculptum and A. tonelliae, were the main ticks found attached to humans. The medical importance of the bacteria of the genus Rickettsia and Borrelia detected in this work remains unknown.     

First molecular detection of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in the hard tick <Emphasis Type="Italic">Rhipicephalus haemaphysaloides</Emphasis> in Taiwan

Anaplasma phagocytophilum is transmitted mainly by hard ticks and can cause potentially fatal granulocytic anaplasmosis in humans, but its occurrence in ticks in Taiwan has never been investigated although this pathogen has been detected in Taiwanese rodents before. Ticks collected from small mammals in Hualien, eastern Taiwan, were assayed for Anaplasma infections; infections of Rickettsia and Apicomplexa protozoans were also studied. Of the 270 individually assayed Rhipicephalus haemaphysaloides ticks, A. phagocytophilum was identified in a nymphal tick. Parasites most similar to Anaplasma bovis, Rickettsia rickettsii, Rickettsia sp. TwKM01, and at least seven apicomplexan species (including genera Cryptosporidium, Hepatozoon, and Theileria) were also identified. This study shows that A. phagocytophilum does occur in the hard tick in Taiwan, although whether R. haemaphysaloides can vector this pathogen remains to be determined. This work also reveals a high diversity of tick-borne bacteria and protozoans circulating in a small region and calls for further research on their potential risks for human health.     

Secreted proteins produced by fungi associated with <Emphasis Type="Italic">Botryosphaeria</Emphasis> dieback trigger distinct defense responses in <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Vitis rupestris</Emphasis> cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.     

In vitro efficacy of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> sensu lato against unfed <Emphasis Type="Italic">Amblyomma parvum</Emphasis> (Acari: Ixodidae)

Amblyomma parvum Aragão (Acari: Ixodidae) is a tick species found with wide distribution in the Neotropical region. Even though it is a wildlife-related tick, it is also a frequent parasite of domestic animals, is aggressive to human beings and may harbor pathogenic microorganisms. Therefore, it is a target species for control on domestic animals, particularly those at the rural–wildlife interface. Herein, the efficacy of two isolates (E9 and IBCB 425) of an entomopathogenic fungus, Metarhizium anisopliae sensu lato, already evaluated for ticks that parasitize domestic animals, was tested against unfed A. parvum adults. Both isolates displayed high acaricidal efficacy after immersion in fungal conidial suspensions for 5 min. Isolate E9 killed all ticks by the 7th day post-treatment, and isolate IBCB 425 did so by the 11th day. Tick mortality of 80 and 90% was achieved as early as the 3rd and 4th days, respectively, with both treatments. Thus, if a commercial M. anisopliae s.l. acaricide against domestic animal ticks is developed, it would also be effective against A. parvum.     

Co-feeding as a route for transmission of <Emphasis Type="Italic">Rickettsia conorii israelensis</Emphasis> between <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks

The essential oil from Amyris balsamifera (Rutaceae) and elemol, a principal constituent of the essential oil of Osage orange, Maclura pomifera (Moraceae) were evaluated in in vitro and in vivo laboratory bioassays for repellent activity against host-seeking nymphs of the blacklegged tick, Ixodes scapularis, and the lone star tick, Amblyomma americanum. Both bioassays took advantage of the tendency of these host-seeking ticks to climb slender vertical surfaces. In one bioassay, the central portion of a vertical strip of filter paper was treated with test solution and ticks placed or allowed to crawl onto the untreated lower portion. In the other bioassay, a strip of organdy cloth treated with test solution was doubly wrapped (treatment on outer layer) around the middle phalanx of a forefinger and ticks released on the fingertip. Both amyris oil and elemol were repellent to both species of ticks. Elemol did not differ significantly in effectiveness against A. americanum from the widely used repellent deet. At 2 and 4 h after application to filter paper, 827 μg amyris oil/cm² paper repelled 80 and 55%, respectively, of A. americanum nymphs. Ixodes scapularis was repelled by lower concentrations of amyris oil and elemol than A. americanum.     

Differential diagnosis of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> and <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>: nymphs and larvae

We developed a method for differential diagnosis of nymphs and larvae of sheep (Ixodes ricinus (L.)) and taiga (I. persulcatus Sch.) ticks (Parasitiformes: Ixodidae) which allows to identify live material in the field.     

Ixodid ticks of African buffalo (<Emphasis Type="Italic">Syncerus caffer</Emphasis>), impala (<Emphasis Type="Italic">Aepyceros melampus</Emphasis>) and elephant (<Emphasis Type="Italic">Loxodonta africana</Emphasis>) in five protected park estates in the Zambezi valley,Zimbabwe

Wildlife hosts many pathogens of economic importance and is considered as a reservoir of important tick-borne diseases of livestock in southern Africa. The species composition of ticks parasitizing buffalo (Syncerus caffer), impala (Aepyceros melampus) and elephant (Loxodonta africana) was investigated in five protected parks in the Zambezi valley, Zimbabwe. A total of 1104 adult ticks was collected from 75 adult animals comprising five buffaloes, five elephants and five impalas drawn from five protected wildlife parks. Five tick species belonging to two genera were recovered, with Rhipicephalus decoloratus being the most prevalent species in all the three animal groups. Amblyomma hebraeum was only recovered from buffaloes whereas Rhipicephalus zambeziensis was recovered from buffalos and elephants. Significant differences in mean tick species distribution and concentration were observed amongst the wildlife parks and these appeared to be influenced by the number of hosts in each park. The study revealed that buffaloes are the major host of R. decoloratus in the Zambezi valley. The presence of these ixodid ticks within the Zambezi valley may have significant ecological and economic impacts on wildlife conservation, domestic animals and human health.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

Rate of consumption,biological parameters,and population growth capacity of <Emphasis Type="Italic">Orius laevigatus</Emphasis> fed on <Emphasis Type="Italic">Spodoptera exigua</Emphasis>

Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) is already successfully used to control Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in protected crops. In the present work, the predatory capacity of O. laevigatus on the eggs and larvae of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) as well as the population growth capacity of O. laevigatus fed on eggs of the noctuid were determined. Fourth and 5th instar nymphs had a greater consumption of eggs than younger ones. Females consumed more eggs than males. Only 5th instar nymphs and adults preyed on the neonatal larvae of S. exigua. The intrinsic rate of natural increase did not differ between O. laevigatus fed with S. exigua eggs and those offered eggs of the substitute host Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae). Thus, O. laevigatus is a good candidate for the biological control of S. exigua, a cosmopolitan pest of many crops.     

Abundance estimation of <Emphasis Type="Italic">Ixodes</Emphasis> ticks (Acari: Ixodidae) on roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

Despite the importance of roe deer as a host for Ixodes ticks in central Europe, estimates of total tick burden on roe deer are not available to date. We aimed at providing (1) estimates of life stage and sex specific (larvae, nymphs, males and females, hereafter referred to as tick life stages) total Ixodes burden and (2) equations which can be used to predict the total life stage burden by counting the life stage on a selected body area. Within a period of 1½ years, we conducted whole body counts of ticks from 80 hunter-killed roe deer originating from a beech dominated forest area in central Germany. Averaged over the entire study period (winter 2007–summer 2009), the mean tick burden per roe deer was 64.5 (SE ± 10.6). Nymphs were the most numerous tick life stage per roe deer (23.9 ± 3.2), followed by females (21.4 ± 3.5), larvae (10.8 ± 4.2) and males (8.4 ± 1.5). The individual tick burden was highly aggregated (k = 0.46); levels of aggregation were highest in larvae (k = 0.08), followed by males (k = 0.40), females (k = 0.49) and nymphs (k = 0.71). To predict total life stage specific burdens based on counts on selected body parts, we provide linear equations. For estimating larvae abundance on the entire roe deer, counts can be restricted to the front legs. Tick counts restricted to the head are sufficient to estimate total nymph burden and counts on the neck are appropriate for estimating adult ticks (females and males). In order to estimate the combined tick burden, tick counts on the head can be used for extrapolation. The presented linear models are highly significant and explain 84.1, 77.3, 90.5, 91.3, and 65.3% (adjusted R ²) of the observed variance, respectively. Thus, these models offer a robust basis for rapid tick abundance assessment. This can be useful for studies aiming at estimating effects of abiotic and biotic factors on tick abundance, modelling tick population dynamics, modelling tick-borne pathogen transmission dynamics or assessing the efficacy of acaricides.     

Bacteriological analysis of saliva from partially or fully engorged female adult <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> by next-generation sequencing

Tick-borne diseases are a major epidemiological problem worldwide. The aim of this study was to investigate the bacterial composition of saliva obtained from engorged adult Rhipicephalus microplus females. Saliva samples collected from partially or fully engorged adult female ticks were analysed using an ultra-high-throughput Illumina HiSeq 2500 sequencing system. To elucidate the possible routes of bacterial transmission, the bacterial flora from whole ticks were also investigated. Proteobacteria, Firmicutes, and Actinobacteria were the predominant phyla in all samples, and Acinetobacter, Rickettsia, Escherichia and Coxiella were the major genera. Microbial diversity in saliva samples from partially engorged ticks was more complex than that of samples from fully engorged individuals. The comparison of saliva and whole-tick samples suggests that bacteria in saliva also colonize the tick’s body. We believe that some bacterial genera, such as Dermacoccus, Achromia, SMB53, Sutterella, Providencia, Mycoplana, Oscillospira, and Agrobacterium, were found and reported in ticks for the first time. The Coxiella and Rickettsia detected in this study might be tick-borne pathogens, suggesting health risks associated with exposure to R. microplus in humans and animals. These findings may serve as the basis for developing strategies to control ticks and tick-borne diseases.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

qPCR estimates of <Emphasis Type="Italic">Babesia bovis</Emphasis> and <Emphasis Type="Italic">Babesia bigemina</Emphasis> infection levels in beef cattle and <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> larvae

Babesia spp. are tick-transmitted intraerythrocytic apicomplexan parasites that infect wild and domestic animals. Babesia bovis and B. bigemina are endemic and responsible for enormous economic losses to the livestock industry in most of the Brazilian territory, wherein the tick Rhipicephalus microplus is the unique vector. Better understanding of epidemiology and parasite–host interactions may improve the tools for disease control and genetic management for selection of resistant animals. This study aimed to detect, quantify and measure the correlation between B. bigemina and B. bovis infection levels in bovine blood and into tick, by absolute quantification of hemoparasite DNA using qPCR. Blood bovine samples and larvae pools from 10 engorged R. microplus females were collected from each Canchim heifers (5/8 Charolais?+?3/8 zebu, n?=?36). All evaluated samples were positive for both Babesia species tested. Correlations of B. bovis and B. bigemina levels between cattle and tick host were 0.58 and 0.66, respectively. These high positive correlation coefficients indicate that parasitemia load in the bovine may be dependent on or may determine the parasitemia load in the ticks.     

Heritable bacterial endosymbionts in native and invasive populations of <Emphasis Type="Italic">Harmonia axyridis</Emphasis>

Harmonia axyridis Pallas (1773) (Coleoptera: Coccinellidae) is the well-studied system of invasive insect species. Native and invasive parts of the area of H. axyridis are isolated geographically. We studied the species composition and the distribution of bacterial symbionts Spiroplasma and Rickettsia in seven localities of the native area and six localities of the invasive area of H. axyridis. Rickettsia was detected in H. axyridis populations for the first time. We found that the proportion of beetles infected with Rickettsia in native and invasive populations of H. axyridis is about 0.03. Spiroplasma was found only in native populations of H. axyridis. The proportion of infected individuals with Spiroplasma in native populations of H. axyridis is about 0.08. All studied native populations of H. axyridis are infected with Spiroplasma, while all invasive populations are not. We discuss the possible influence of Spiroplasma and Rickettsia in the formation of invasive populations of H. axyridis.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.