Prevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in questing <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks in relation to the density of wild cervids

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.

Methods

For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp 2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The Osp A gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.

Results

Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ² = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ² = 24.04, p < 0.0001); Strøm vs. Tjore (χ² = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ² = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ² = 16.27, p = 0.0001); Strøm vs. Tjore (χ² = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ² = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ² = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ² = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.

Conclusion

The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.

     

Immunochemical analysis of lipopolysaccharide-like component extracted from<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato

Immunoelectrophoresis and its modifications were applied to analysis of a lipopolysaccharide-like component (LPS-LC) extracted from Borrelia garinii strains K24 and K48 isolated from Ixodes ricinus and Borrelia burgdorferi sensu stricto strain B31. A modification of the hot phenol-water method was used for isolation of LPS. Immunoelectrophoresis (IE) and crossed immunoelectrophoresis (CIE) of LPS-LC with polyclonal rabbit antisera revealed a pattern and properties partially similar to LPS from other Gram-negative bacteria. B. garinii LPS-LC formed in CIE a diffuse band extending from the start to the anode. Similarly, the shape and position of the band in IE did not show major differences from LPS of other Gram-negative bacteria. The LPS-LC extracted from the three genomic groups of B. burgdorferi sensu lato were found to have similar immunochemical properties irrespective of their genotype origin.     

Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Serological and molecular detection of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasmataceae</Emphasis> in rodents

The presence of Anaplasma spp. and Borrelia burgdorferi sensu lato in rodents from Eastern Slovakia were followed by serological and molecular methods. The seroprevalence for Borrelia was detected in 16.6 %, for Anaplasmataceae (APT) in 13.2 % and co-occurrence of Borrelia and APT in 7.5 %. Out of 110 ear biopsies of rodents, 5 were B. afzelii-positive. Five biopsies tested positive with the Ehr521-Ehr747 primers amplifying all the members of the family APT. A. phagocytophilum was detected in 1.8 %, 2.7 % were infected with Anaplasma-like organisms. Co-occurrence of Borrelia and Anaplasma in ear biopsies was found in 1.8 %. The circulation of both Borrelia and Anaplasma in the region of Eastern Slovakia was confirmed.     

Identification of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in patients with erythema migrans

Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.     

Prevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in rodents from Gansu,northwestern China

Background  

Lyme disease is a multi-organ infection disease caused by Borrelia burgdorferi sensu lato. Lyme disease was first documented in north-east China in 1986. Since then more than 20 provinces in China were confirmed the existence of nature foci of Lyme disease. In the present study, a molecular epidemiological survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato in rodents from Gansu Province for the first time.     

Exposed and hidden lectin-binding epitopes at the surface of<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.     

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.     

Invasion of the Lyme Disease Vector <Emphasis Type="Italic">Ixodes scapularis</Emphasis>: Implications for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> Endemicity

Lyme disease risk is increasing in the United States due in part to the spread of blacklegged ticks Ixodes scapularis, the principal vector of the spirochetal pathogen Borrelia burgdorferi. A 5-year study was undertaken to investigate hypothesized coinvasion of I. scapularis and B. burgdorferi in Lower Michigan. We tracked the spatial and temporal dynamics of the tick and spirochete using mammal, bird, and vegetation drag sampling at eight field sites along coastal and inland transects originating in a zone of recent I. scapularis establishment. We document northward invasion of these ticks along Michigan’s west coast during the study period; this pattern was most evident in ticks removed from rodents. B. burgdorferi infection prevalences in I. scapularis sampled from vegetation in the invasion zone were 9.3% and 36.6% in nymphs and adults, respectively, with the majority of infection (95.1%) found at the most endemic site. There was no evidence of I. scapularis invasion along the inland transect; however, low-prevalence B. burgdorferi infection was detected in other tick species and in wildlife at inland sites, and at northern coastal sites in years before the arrival of I. scapularis. These infections suggest that cryptic B. burgdorferi transmission by other vector-competent tick species is occurring in the absence of I. scapularis. Other Borrelia spirochetes, including those that group with B. miyamotoi and B. andersonii, were present at a low prevalence within invading ticks and local wildlife. Reports of Lyme disease have increased significantly in the invasion zone in recent years. This rapid blacklegged tick invasion—measurable within 5 years—in combination with cryptic pathogen maintenance suggests a complex ecology of Lyme disease emergence in which wildlife sentinels can provide an early warning of disease emergence.     

Tick infestation risk and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> s.l. infection-induced increase in host-finding efficacy of female <Emphasis Type="Italic">Ixodes ricinus</Emphasis> under natural conditions

An investigation of the risk of human tick infestation, together with the prevalence of Borrelia burgdorferi s.l. infection, was conducted in a sylvatic habitat in western Germany to provide data needed for future risk-benefit evaluations of acaricides used for clothing impregnation. Additionally, data were collected on behavioural changes in Borrelia burgdorferi s.l.-infected adult female I. ricinus ticks and the possible impact of such changes on host-finding efficacy. The risk of I. ricinus-infestation was determined by collecting from the protective clothing of volunteers and by dragging in known tick-infested sites in the Kühkopf Mountain area, Koblenz, Germany, from June through October 2006. The overall tick infestation rate per person per hour was 7.4 ± 5.5, with the following sex- and stage-specific differences: males 0.32 ± 0.37, females 1.1 ± 1.2, nymphs 3.6 ± 4.4, larvae 2.4 ± 3.5. Concurrent dragging revealed an average 19.4 ± 16.2 times higher infestation rate as well as a markedly lower infection rate with borreliae in adult I. ricinus ticks when compared to ticks collected from exposed human volunteers. Although the difference in infection rates was statistically significant (P < 0.023) only in adult female ticks, our data indicate that B. burgdorferi s.l. infection may increase host-finding efficacy in adult I. ricinus. The overall exposure risk was 1.0 B. burgdorferi s.l.-infected ticks per person per hour of exposure, or 0.25 ticks per 100 m walking distance in the study area.     

The First Data on the <Emphasis Type="Italic">TROSPA</Emphasis> Gene Structure in <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> and <Emphasis Type="Italic">Ixodes ricinus</Emphasis> Ticks from Russia

For the first time, the sequences of the TROSPA gene from taiga tick and sheep tick from Russia were obtained. Three specimens of sheep tick from Voronezh oblast and three specimens each of taiga tick from Irkutsk oblast and Perm krai were analyzed. At the end of the intron of the TROSPA gene in Ixodes persulcatus, a poly-T region was found. In addition, a fragment of the first exon containing a large number of differences in the nucleotide and amino acid composition both among species and within them was identified.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Multi-criteria Decision Analysis to Model <Emphasis Type="Italic">Ixodes ricinus</Emphasis> Habitat Suitability

Tick-borne diseases present a major threat to both human and livestock health throughout Europe. The risk of infection is directly related to the presence of its vector. Thereby it is important to know their distribution, which is strongly associated with environmental factors: the presence and availability of a suitable habitat, of a suitable climate and of hosts. The present study models the habitat suitability for Ixodes ricinus in Ireland, where data on tick distribution are scarce. Tick habitat suitability was estimated at a coarse scale (10 km) with a multi-criteria decision analysis (MCDA) method according to four different scenarios (depending on the variables used and on the weights granted to each of them). The western part of Ireland and the Wicklow mountains in the East were estimated to be the most suitable areas for I. ricinus in the island. There was a good level of agreement between results from the MCDA and recorded tick presence. The different scenarios did not affect the spatial outputs substantially. The current study suggests that tick habitat suitability can be mapped accurately at a coarse scale in a data-scarce context using knowledge-based methods. It can serve as a guideline for future countrywide sampling that would help to determine local risk of tick presence and refining knowledge on tick habitat suitability in Ireland.     

Plant regeneration of three<Emphasis Type="Italic"> Opuntia</Emphasis> genotypes used as human food

In vitro micropropagation Opuntia cactus plants, whose young stems are consumed as vegetables, is reported. Cladode explants from three Opuntia genotypes, were cultivated in MS medium containing BA and GA₃. Shoots produced were used as secondary explants and BA added at different concentrations to induce shoot development. 0.5 M BA was the best for bud formation. Satisfactory rooting occurred when IBA was added to the medium, and plants were successfully established in soil and adapted to greenhouse conditions. The protocols developed in this work provide a basis for the establishment of a genetic transformation system for Opuntia spp.     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Detection of <Emphasis Type="Italic">Borrelia miyamotoi</Emphasis> in <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

Unfed adult ticks Ixodes persulcatus from five regions of Russia were examined by PCR method in order to analyze distribution and diversity of B. miyamotoi. B. miyamotoi DNA was found in 1.8, 2.9, 4.5, 2.3, and 2.5% of ticks from Leningrad, Sverdlovsk, Novosibirsk, and Irkutsk provinces, and from Khabarovsk Territory, respectively. Molecular typing of B. miyamotoi DNA was based on the partial sequencing of the 16S rRNA, p66, and glpQ genes. A single genetic variant of B. miyamotoi was detected in all the samples of ticks collected from five regions.     

Phenotypic and genotypic analysis of<Emphasis Type="Italic">Borrelia</Emphasis> spp. isolated from<Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks by using electrophoretic chips and real-time polymerase chain reaction

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.