Pathogenic Genospecies Research Articles

Local Population Structure and Seasonal Variability of Borrelia garinii Genotypes in Ixodes ricinus Ticks, Slovakia.

Lyme disease (LD) is the most common tick-borne human disease in Europe, and Borrelia garinii, which is associated with avian reservoirs, is one of the most genetically diverse and widespread human pathogenic genospecies from the B. burgdorferi sensu lato (s.l.) complex. The clinical manifestations of LD are known to vary between regions and depend on the genetic strain even within Borrelia genospecies. It is thus of importance to explore the genetic diversity of such pathogenic borreliae for the wide range of host and ecological contexts. In this study, multilocus sequence typing (MLST) was employed to investigate the local population structure of B. garinii in Ixodes ricinus ticks. The study took place in a natural wetland in Slovakia, temporally encompassing spring and autumn bird migration periods as well as the breeding period of resident birds. In total, we examined 369 and 255 ticks collected from 78 birds and local vegetation, respectively. B. burgdorferi s.l. was detected in 43.4% (160/369) of ticks recovered from birds and in 26.3% (67/255) of questing ticks, respectively. Considering the ticks from bird hosts, the highest prevalence was found for single infections with B. garinii (22.5%). Infection intensity of B. garinii in bird-feeding ticks was significantly higher than that in questing ticks. We identified ten B. garinii sequence types (STs) occurring exclusively in bird-feeding ticks, two STs occurring exclusively in questing ticks, and one ST (ST 244) occurring in both ticks from birds and questing ticks. Four B. garinii STs were detected for the first time herein. With the exception of ST 93, we detected different STs in spring and summer for bird-feeding ticks. Our results are consistent with previous studies of the low geographic structuring of B. garinii genotypes. However, our study reveals some consistency in local ST occurrence and a geographic signal for one of the clonal complexes.

Borrelia burgdorferi and Anaplasma phagocytophilum Genospecies in Northern California.

The sensu lato (s.l.) complexes of Borrelia burgdorferi and Anaplasma phagocytophilum include pathogenic genospecies each with distinct ecologies in northern California, yet, most work conflates the genospecies of each pathogen into one sensu lato species. Detailed understanding of the differences in geographic distributions and ecology among genospecies is lacking. We aimed to evaluate whether two B. burgdorferi and two A. phagocytophilum genospecies in high-risk locations in coastal northern California were spatially clustered and if presence of a particular genospecies was associated with geographical site, host species, or other demographic or ecological variables. DNA sequencing was performed to differentiate genospecies of Borreliae and Anaplasma from PCR-positive dusky-footed woodrats (Neotoma fuscipes) and sciurids (chipmunks, Tamias spp., and Douglas squirrels, Tamiasciurus douglasii) at four sites in northwestern California. Logistic regression was performed to assess associations of genospecies with the predictor variables host species, host sex, site, season, and year. Spatial clustering was assessed using a Poisson spatial scan statistic in SaTScan. Host species was a significant predictor for Borrelia bissettiae, B. burgdorferi sensu stricto (s.s.), A. phagocytophilum s.s., and the DU1 Anaplasma genospecies. Woodrats were significantly more likely to be PCR-positive for B. bissettiae and A. phagocytophilum DU1 genospecies, while A. phagocytophilum s.s. and B. burgdorferi s.s. were significantly associated with sciurids. We report a single Borrelia lanei in an Allen's chipmunk (Tamias senex) from the Hoopa Valley Tribal Reservation. A significant spatial cluster of A. phagocytophilum s.s. was detected at Hendy Woods State Park in Mendocino County. These results highlight the need to better understand genospecies partitioning according to host species to further assess human risks, aid in future surveillance, and inform targeted research.

Detección de Borrelia burgdorferi sensu lato en perros y sus garrapatas en comunidades rurales de Yucatán, México

In Mexico, the distribution and the presence of pathogenic genospecies of B. burgdorferi in dogs and their ticks has not been extensively investigated. The study of canine borreliosis is acquiring greater relevance, since the dog is considered to be an important sentinel for pathogens pertaining to the complex Borrelia burgdorferi sensu lato; in addition, dogs could be playing a key role in the spread of ticks from forested areas into the domestic environment. This study aimed to detect and estimate the prevalence of B. burgdorferi s.l. in dogs and their ticks in two rural communities of Yucatán, Mexico. In each community, 50 houses were visited, where 144 blood samples from dogs were studied by puncture of the saphenous vein, as well as the collection of their ticks. To detect the presence of B. burgdorferi s.l. in these samples, the conserved gene flaB, p66 and ospC were PCR amplified. A total of 144 dog blood samples, and 846 of ticks were obtained from the examined animals. Considering tick species, Rhipicephalus sanguineus sensu lato (786 / 846) was common, while Ixodes affinis (33 / 846), and Amblyomma mixtum (27 / 846) resulted less frequent. As per gene conservation, the prevalence of B. burgdorferi in canine blood was 17.3 % (25 / 144) to flaB, 12.50 % (18 / 144) for p66 and 1.38 % (2 / 144) for the ospC gene. Within the analyzed ticks, R. sanguineus s.l. had a prevalence of 0.89 %, A. mixtum 5.88 % and I. affinis 15.15 %, being this last species the one that presented higher prevalence. Two dogs and their ticks I. affinis were positive to the flaB gene. Only a tick R. sanguineus s.l. was positive to the gene p66 and no tick species was positive the ospC gene. This study confirmed the existence of B. burgdorferi s.l. in dogs and their ticks in rural communities of Yucatán, Mexico. The detection of Borrelia in dogs may be an important criterion for the evaluation of the risk of borreliosis in humans, since the dog can be used as an epidemiological indicator for the identification of new outbreaks of this disease.

Lyme Disease in the Horse – A current literature study considering methods of diagnosis and treatment, as well as preventive measures

Lyme borreliosis is caused by bacteria belonging to the Borrelia burgdorferi sensu lato complex, which belongs to the family Spirochaetaceae. The disease was described in humans for the first time in 1977 near the town Lyme (Connecticut, USA), from which the name of the disease is derived. In Europe and Asia, there are several pathogenic genospecies (Borrelia burgdorferi sensu stricto, B. bavariensis, B. garinii and B. afzelii), which can cause Lyme disease. Borrelia have a low tenacity, and are therefore dependent on hosts for survival. Borrelia are generally extracellular pathogens, which reproduce in a multiannual enzootic cycle in ticks (Ixodes spp.) and mammals. A manifestation of Lyme borreliosis is always dependent on its transmission by the carrier ticks. During the life cycle of ticks in Europe, its prevalence rate in the population nearly doubles. Epidemiological data regarding the prevalence of the disease in horses in Germany are not yet available. A manifestation of Lyme borreliosis occurs rarely in horses, and the clinical signs of the disease are not pathognomonic. The diagnosis of Lyme borreliosis should be confirmed only if the following criteria apply to the given patient: The horse has experienced tick exposure, shows manifestation of clinical signs and detection of the infectious organism or specific host antibody response was proved. Important differential diagnoses have been eliminated. Clinical signs might progress or recur despite antibiotic treatment. Due to the possible persistence of the pathogen, the prevention of the disease should be granted highest priority. Prophylactic measures available include the earliest possible mechanical removal of ticks, the use of repellents and vaccination.

Diversifying forest communities may change Lyme disease risk: extra dimension to the dilution effect in Europe.

Lyme disease is caused by bacteria of the Borrelia burgdorferi genospecies complex and transmitted by Ixodid ticks. In North America only one pathogenic genospecies occurs, in Europe there are several. According to the dilution effect hypothesis (DEH), formulated in North America, nymphal infection prevalence (NIP) decreases with increasing host diversity since host species differ in transmission potential. We analysed Borrelia infection in nymphs from 94 forest stands in Belgium, which are part of a diversification gradient with a supposedly related increasing host diversity: from pine stands without to oak stands with a shrub layer. We expected changing tree species and forest structure to increase host diversity and decrease NIP. In contrast with the DEH, NIP did not differ between different forest types. Genospecies diversity however, and presumably also host diversity, was higher in oak than in pine stands. Infected nymphs tended to harbour Borrelia afzelii infection more often in pine stands while Borrelia garinii and Borrelia burgdorferi ss. infection appeared to be more prevalent in oak stands. This has important health consequences, since the latter two cause more severe disease manifestations. We show that the DEH must be nuanced for Europe and should consider the response of multiple pathogenic genospecies.

A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America

Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and animals comprising strains that cause clinical disease in people, dogs and horses (the pathogenic A. phagocytophilum “genospecies”) and more distantly related strains. A rodent-adapted genospecies named DU1, found primarily in woodrats, is unable to infect horses. We developed a real-time PCR (RT-PCR) assay, which targets an 85 base pair region of the ank gene and is specific for the pathogenic genospecies of A. phagocytophilum from North America. Thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents and ticks for which the ank gene had previously been sequenced and had been identified as either the pathogenic genospecies or DU1 were used for validation. All nine samples with the pathogenic genospecies tested positive using the new RT-PCR and all 21 samples with the DU1 genospecies tested negative. Two strains from which the whole genome has been sequenced and are known to be pathogenic (A. phagocytophilum, strain HZ isolated from a human from New York and strain MRK isolated from a horse from California) both tested positive using the new RT-PCR assay. We also tested blood from 20 horses and six dogs that were RT-PCR-positive using a previously validated RT-PCR protocol: all were PCR-positive using the new assay as well. The assay has high efficiency and reproducibility and there was no cross-reaction with related and tick-borne bacteria tested, although the assay cross-reacts with the ungulate-adapted genospecies Ap-Variant 1. This new RT-PCR assay will aid in future research on the ecology and epidemiology of A. phagocytophilum by allowing researchers to easily identify the pathogenic genospecies in reservoir hosts and vectors.

Detection of Invasive Borrelia burgdorferi Strains in North-Eastern Piedmont, Italy.

Following reports of human cases of Lyme borreliosis from the Ossola Valley, a mountainous area of Piemonte, north-western Italy, the abundance and altitudinal distribution of ticks, and infection of these vectors with Borrelia burgdorferi sensu lato were evaluated. A total of 1662 host-seeking Ixodes ricinus were collected by dragging from April to September 2011 at locations between 400 and 1450 m above sea level. Additional 104 I. ricinus were collected from 35 hunted wild animals (4 chamois, 8 roe deer, 23 red deer). Tick density, expressed as the number of ticks per 100 m(2), resulted highly variable among different areas, ranging from 0 to 105 larvae and from 0 to 22 nymphs. A sample of 352 ticks (327 from dragging and 25 from wild animals) was screened by a PCR assay targeting a fragment of the 16S rRNA gene of B. burgdorferi s.l. Positive samples were confirmed with a PCR assay specific for the 5S-23S rRNA intergenic spacer region and sequenced. Four genospecies were found: B. afzelii (prevalence 4.0%), B. lusitaniae (4.0%), B. garinii (1.5%) and B. valaisiana (0.3%). Phylogenetic analysis based on the ospC gene showed that most of the Borrelia strains from pathogenic genospecies had the potential for human infection and for invasion of secondary body sites.

Lyme Borreliosis

Lyme Borreliosis

Lyme Borreliosis: From Pathogenesis to Diagnosis and Treatment

Lyme Borreliosis: From Pathogenesis to Diagnosis and Treatment

Acylated cholesteryl galactosides are ubiquitous glycolipid antigens amongBorrelia burgdorferi sensu lato: Figure 1

Lyme disease (LD) is the most common tick-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato, in particular, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. However, other genospecies have been implicated as causative factors of LD as well. Borrelia burgdorferi exhibits numerous immunogenic lipoproteins, but due to strong heterogeneity, the use of these proteins for serodiagnosis and vaccination is hampered. We and others have identified acylated cholesteryl galactosides (ACGal) as a novel glycolipid present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii. ACGal is a strong antigen and the majority of patients display anti-ACGal antibodies in the chronic stages of LD. However, it is unknown whether ACGal is present in other presumably pathogenic B. burgdorferi genospecies. Therefore, we performed an analysis of the total lipid extracts of a wide spectrum of genospecies of B. burgdorferi sensu lato using thin-layer chromatography as well as Western blot and dot-blot assays. We show that ACGal is present in substantial quantities in all B. burgdorferi genospecies tested. Therefore, this molecule might improve the serological detection of rarely pathogenic genospecies, and may be used as a protective vaccine regardless of the prevailing genospecies.

Human PathogenicBorrelia spielmaniisp. nov. Resists Complement-Mediated Killing by Direct Binding of Immune Regulators Factor H and Factor H-Like Protein 1

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.

Distribution and Characterization of <I>Borrelia burgdorferi</I> Isolates from <I>Ixodes scapularis</I> and Presence in Mammalian Hosts in Ontario, Canada

The blacklegged tick, Ixodes scapularis Say (Acari: Ixodidae), has a wide geographical distribution in Ontario, Canada, with a detected range extending at least as far north as the 50th parallel. Our data of 591 adult I. scapularis submissions collected from domestic animals (canines, felines, and equines) and humans during a 10-yr period (1993-2002) discloses a monthly questing activity in Ontario that peaks in May and October. The Lyme disease spirochete Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt & Brenner was detected in 12.9% of I. scapularis adults collected from domestic hosts with no history of out-of-province travel or exposure at a Lyme disease endemic area. Fifty-three isolates of B. burgdorferi were confirmed positive with polymerase chain reaction by targeting the rrf (5S)-rrl (23S) gene. Using DNA sequencing of the ribosomal species-specific rrf (5S) -rrl (23S) intergenic spacer region, all isolates belong to the pathogenic genospecies B. burgdorferi sensu stricto (s.s.). Nucleotide sequence analysis of a 218- to 220-bp amplicon fragment exhibits six cluster patterns and, collectively, these isolates branch into four phylogenetic cluster groups for both untraveled, mammalian hosts and those with travel to the northeastern United States (New Jersey and New York). Four of five geographic regions in Ontario had strain variants consisting of three different genomic cluster groups. Overall, our molecular characterization of B. burgdorferi s.s. shows genetic heterogeneity within Ontario and displays a connecting link to common strains from Lyme disease endemic areas in the northeastern United States. Moreover, our findings of B. burgdorferi in I. scapularis reveal that people and domestic animals may be exposed to Lyme disease vector ticks, which have wide-ranging distribution in eastern and central Canada.

Molecular analysis of decorin-binding protein A (DbpA) reveals five major groups among European Borrelia burgdorferi sensu lato strains with impact for the development of serological assays and indicates lateral gene transfer of the dbpA gene

The Borrelia (B.) burgdorferi adhesin DbpA (decorin-binding protein A) is a valuable antigen for serodiagnosis of Lyme borreliosis and a promising candidate for a vaccine. To investigate the heterogeneity of DbpA, we aligned DNA sequences of 83 different dbpA genes (37 from the database, where the majority of sequences belong to B. burgdorferi sensu stricto and 46 were newly sequenced). Analysis of 25 sequences from the species B. burgdorferi s.s., 16 from B. afzelii, 40 from B. garinii, and two from the recently described human pathogenic genospecies A14S revealed five distinct DbpA groups. Group I comprises B. burgdorferi s.s. and group II B. afzelii. B. garinii is divided into groups III and IV, whereas A14S strains form group V. Formation of groups is mainly due to insertions of whole sequence sections. Comparison of dbpA sequences with ospC sequences from a subset of 59 strains revealed all kinds of cross-connections indicating processes of lateral gene transfer among strains. The extent of sequence identity within the dbpA genes decreases from the DNA (67%) to the amino acid (AA) level (44%) by about 23%, in contrast ospC sequence identities differed only by about 10%. This might be an indication that DbpA plays an important role in immune escape. Immunoblots using four recombinant DbpAs representing groups I–IV show that DbpA proteins are sensitive and specific antigens and complement one another in their reactivity. Part of the sera showed group-specific reactivity which could also be demonstrated with monoclonal antibodies.

RV002 Report on the activities of the national reference center for Borreliae

The Borrelia (B.) burgdorferi adhesin DbpA (decorin-binding protein A) is a valuable antigen for serodiagnosis of Lyme borreliosis and a promising candidate for a vaccine. To investigate the heterogeneity of DbpA, we aligned DNA sequences of 83 different dbpA genes (37 from the database, where the majority of sequences belong to B. burgdorferi sensu stricto and 46 were newly sequenced). Analysis of 25 sequences from the species B. burgdorferi s.s., 16 from B. afzelii, 40 from B. garinii, and two from the recently described human pathogenic genospecies A14S revealed five distinct DbpA groups. Group I comprises B. burgdorferi s.s. and group II B. afzelii. B. garinii is divided into groups III and IV, whereas A14S strains form group V. Formation of groups is mainly due to insertions of whole sequence sections. Comparison of dbpA sequences with ospC sequences from a subset of 59 strains revealed all kinds of cross-connections indicating processes of lateral gene transfer among strains. The extent of sequence identity within the dbpA genes decreases from the DNA (67%) to the amino acid (AA) level (44%) by about 23%, in contrast ospC sequence identities differed only by about 10%. This might be an indication that DbpA plays an important role in immune escape. Immunoblots using four recombinant DbpAs representing groups I–IV show that DbpA proteins are sensitive and specific antigens and complement one another in their reactivity. Part of the sera showed group-specific reactivity which could also be demonstrated with monoclonal antibodies.

Incidence and identification of mesophilic Aeromonas spp. from retail foods.

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.

Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL.

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.

Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different regions of Poland

Ixodes ricinus ticks, collected in 1996–1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n = 48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferi sensu lato. Spirochetes were detected in 130 adult ticks (22.9 %) and in a minimum of 32 (5.3 %) nymphs. Further identification of 153 B. burgdorferi s. l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2 %). Within this group B. afzelii was found in 38/153 samples (24.9%), followed by B. burgdorferi sensu stricto (n =23/153; 15.0%) and B. garinii (n =22/153; 14.4%). Dual infections with B. burgdorferi s.s. and B. afzelii were detected in 17/121 (14.0%) adults, while both B. burgdorferi s.s./ B. garinii and B. afzelii/B. garinii coinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferi s.s., B. afzelii and B. garinii was noted twice (1.6 %). In general, B. afzelii was found in 72/153 (47.1 %) tick samples and was the predominant species. B. burgdorferi s.s. and B. garinii were detected in a total of 60/153 (39.2%) and 51/153 (33.3 %) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferi s. 1. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.