Serum Complement Sensitivity as a Key Factor in Lyme Disease Ecology

The sensitivity of Borrelia burgdorferi sensu lato to animal sera was analyzed. Complement-mediated borreliacidal effects were observed with particular combinations of host serum and Borrelia genospecies. The species-specific pattern of viability and/or lysis is highly consistent with the pattern of reservoir competence of hosts for B. burgdorferi sensu lato, suggesting a key role of complement in the global ecology of Lyme borreliosis.     

Lyme borreliosis caused by diverse genospecies of Borrelia burgdorferi sensu lato in northeastern China

The variety of Borrelia burgdorferi sensu lato (B. burgdorferi) genospecies leads to distinction in clinical manifestations of Lyme borreliosis (LB). There are reports of LB clinical characteristics in China, where the B. burgdorferi genospecies in ticks and animal hosts are different from those in Europe and North America. During May to September in 2010 and 2011, all patients who had erythema migrans (EM, more than 5 cm in diameter) after a recent tick-bite, and sought medical care at Mudanjiang Forestry Central Hospital, Heilongjiang Province of northeastern China, were enrolled in the study. Specific PCR was used to determine the B. burgdorferi genospecies in the disseminated patients. Of 265 EM patients, B. burgdorferi DNA was detected in blood specimens from 15 of 55 disseminated patients. Sequence analyses of 5S–23S rRNA, flagellin, ospC, 16S rRNA and ospA genes revealed that 11 patients were infected with Borrelia garinii, three with Borrelia afzelii and one with Borrelia valaisiana-related genospecies. Among 15 patients, 40%, 13.3% and 13.3% manifested pruritus, pain and ulceration, respectively. Systemic symptoms, arthralgia or a swollen joint and lymphadenopathy were observed in 26.7%, 13.3% and 6.7% patients, respectively. In northeastern China, three genospecies of LB patients were detected. The B. burgdorferi genospecies identified in this study was predominantly B. garinii. A case infected with B. valaisiana-related genospecies was reported for the first time.     

Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences.

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from the Autonomous Province of Trento, Italy

Sequences of the variable intergenic spacer region 5S (rrfA) 23S (rrlB) rRNA were used to identify Borrelia genospecies present in Ixodes ricinus nymphs collected from the Lamar Lakes area of the Province of Trento, Italy (overall prevalence=6.3%). Four genospecies were identified, one for the first time in this Province (B. valaisiana), and three which have been noted previously (B. afzelii, B. garinii, and B. burgdorferi s.s.). In order to compare the genetic variability of these genospecies in Trento with that at a European level, our 21 sequences (15 new haplotypes) and all appropriate European Borrelia sequences registered in GenBank (up to the end of 2004) were subjected to a phylogenetic analysis (for a total of 73 sequences and 43 haplotypes). Clusters of sequences representing the five main European genospecies (afzelii, garinii, burgdorferi s.s., valaisiana, lusitaniae) are well-supported. At least two other groups of haplotypes (genospecies) are suggested by our analysis; moreover, divergent evolution may be occurring in several genospecies. The maximum uncorrected pairwise differences between sequences within genospecies ranges from 1.5% (B. burgdorferi s.s.), to 2.3% (B. garinii and B. valaisiana) to 4.7% (B. afzelii), and are not correlated with geographical distribution. Within the Province of Trento, these values for the same genospecies are 1.5%, 2.3%, 0.9%, 1.9%, respectively. These high mutation rates within genospecies suggest that the sequencing of haplotypes should continue if we are to fully understand and monitor the evolution and epidemiology of Borrelia.     

Genetic analysis of the outer surface protein C gene of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) isolated from rodents in Taiwan

The outer surface protein C gene (ospC) of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) was analysed for the first time in Taiwan. The genetic identities of these Taiwan isolates (TWKM1-7) were determined by restriction fragment length polymorphism (RFLP) analysis and sequence similarities of the PCR-amplified ospC gene amplicons. After cleavage by nuclease Dral, differential fragment patterns of PCR-amplified ospC DNA in relation to different genospecies of Lyme disease spirochaetes were observed and all of these Taiwan isolates were genetically affiliated to the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis on the sequence similarity of these Taiwan isolates revealed a highly homogeneous genotype, ranging from 99.3% to 100%, within the genospecies of B. burgdorferi sensu stricto and was distinguished from other genospecies of Borrelia isolates. The sequence similarity analysis also revealed the high sequence variability of the ospC gene among Borrelia strains that belong to the same genospecies but were isolated from different biological and geographical sources. Thus, these results provide the first investigation on the genetic identity of the ospC gene of these Taiwan isolates and show that these Taiwan isolates were closely related genetically to the genospecies of B. burgdorferi sensu stricto.     

Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany

From March to October 2003, a total of 2,518 host-seeking Ixodes ricinus ticks (1,944 nymphs, 264 females, 310 males) were collected by blanket dragging at 45 sites all over the city area of Bonn, western Germany, to be checked for Borrelia burgdorferi infection. The collection sites included 20 private gardens, nine public recreational parks, the boundaries of 14 sylvatic suburban areas and two footpaths between suburban farmed fields. Generally, numbers of specimens collected along sylvatic suburban areas and at urban sites with dense tree populations were significantly higher than at the other collection sites. Out of 1,394 specimens (865 nymphs, 241 females, 288 males) that were randomly chosen for Borrelia analysis by a simple PCR, 250 (17.9 %) were found to be infected with B. burgdorferi sensu lato. While the infection prevalences varied significantly between females (26.6%), males (12.5%) and nymphs (17.3%), there were no striking differences between sylvatic and unwooded sites. A total of 92.8% of the ticks Borrelia-positive by the simple PCR were also positive in a diagnostic nested PCR. Using genospecies-specific oligonucleotide probes, single Borrelia genospecies infections (91.4%) could be assigned to B. afzelii (39.5%), B. garinii (27.9%), B. burgdorferi sensu stricto (15.6%) and B. valaisiana (8.6%) by DNA hybridization. Various combinations of double infections were observed in 4.3% of the infected ticks. Another 4.3% of the Borrelia infections were untypeable. The B. burgdorferi genospecies distribution in the city area was shown to be variable from site to site and, even more, it was distinct from rural collection sites near Bonn. This is ascribed to a different spectrum of reservoir hosts. Taking into account the infection prevalences of host-seeking ticks in the forested surroundings of Bonn, our study demonstrates that the risk of acquiring Lyme disease after a tick bite in urban/suburban areas is comparably as high as in woodlands outside of the city.     

Identification and characterization of 31 isolates of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> (Spirochaetales,Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.     

Prevalence of avian-associated Borrelia burgdorferi s.l. genospecies in Ixodes ricinus ticks collected from blackbirds (Turdus merula) and song thrushes (T. philomelos)

Between May and September of 2002, forest passerine birds and immature Ixodes ricinus ticks infesting them were surveyed for infection with Borrelia burgdorferi s.l. in a sylvatic habitat in west-central Poland. A total of 738 feeding I. ricinus ticks were recovered from 114 birds belonging to 9 species. The majority of ticks (65.7%) were collected from two thrush species, the blackbird (Turdus merula) and the song thrush (T. philomelos). Since only the two Turdus species proved to be parasitized by Borrelia-infected ticks, this study focused mainly on these birds. Immature ticks were removed from 53 of the 54 captured thrushes. A total of 304 partially or fully engorged ticks (110 larvae and 194 nymphs), and 53 bird-derived blood samples were tested for borreliae. B. burgdorferi s.l. DNA was detected by PCR in 11% of the larval ticks and in 7.2% of the nymphs. The PCR-positive ticks were found on 8 of 53 tick-infested thrushes. B. garinii and B. valaisiana accounted for 88.5% and 11.5% of the infections detected in Borrelia-positive ticks, respectively. Only one out of 53 of Turdus blood samples were PCR-positive for borreliae. The blood-positive thrush was infected with B. burgdorferi s.s. B. afzelii was the most frequently identified genospecies in PCR-positive questing I. ricinus ticks, followed by B. valaisiana (89% and 11%, respectively). The absence of B. afzelii infection in ticks feeding on passerine birds, despite its strong predominance in local questing nymph populations, indicates that avian hosts are not reservoirs of this genospecies and consequently that the rodent-adapted B. afzelii is negatively selected in ticks feeding on these hosts. These results validate the concept of some avian-associated genospecies within the B. burgdorferi s.l. complex and demonstrate that thrush species may support the circulation of B. garinii and B. valaisiana under natural conditions.     

Natural history of highly pathogenic avian influenza H5N1

The ecology of highly pathogenic avian influenza (HPAI) H5N1 has significantly changed from sporadic outbreaks in terrestrial poultry to persistent circulation in terrestrial and aquatic poultry and potentially in wild waterfowl. A novel genotype of HPAI H5N1 arose in 1996 in Southern China and through ongoing mutation, reassortment, and natural selection, has diverged into distinct lineages and expanded into multiple reservoir hosts. The evolution of Goose/Guangdong-lineage highly pathogenic H5N1 viruses is ongoing: while stable interactions exist with some reservoir hosts, these viruses are continuing to evolve and adapt to others, and pose an un-calculable risk to sporadic hosts, including humans.     

Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.

Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.     

中国莱姆病螺旋体的核糖体基因分型研究

目的 莱姆病螺旋体的基因型和临床表现、疫苗菌株和抗原菌株的选择存在密切的关系，所以对中国菌株进行分子流行病学研究，可为莱姆病的防治提供科学依据。方法 5S－23S rRNA基因间隔区RFLP分析和16＋23S rRNA基因RFLP分析。结果 中国菌株至少被分为3个基因型（B.burgdorferi sensu stricto、B.garinii和B.afzelii),B.garinii和B.afzelii占优势，B.burgdorferi sensu stricto少见。少数菌株用上述方法尚不能明确其分类地位，需进一步研究，中国很可4能存在世界上独特的新基因型。结论 中国菌株的基因型明显不同于北美菌株，而同欧洲菌株比较接近，5S－23S rRNA基因间隔区RFLP分析方法简便、快速、准确，是理想的基因型分类方法，可作为国内菌株基因型鉴定的常规方法。     

Distribution of Acinetobacter Species on Skin of Healthy Humans

 The distribution of the 19 currently known genospecies of Acinetobacter on human skin, i.e. forehead, forearm and toe webs, was determined. Three selective media were compared for their specificity for all genospecies of Acinetobacter. A minimal-salts agar supplemented with 1% acetate proved to be more efficient than the Leeds medium for the isolation of most genospecies in mixed culture with other bacterial species. Acinetobacter isolates were provisionally identified using biochemical tests and the DNA transformation assay of Juni. Genospecies identification was performed using amplified ribosomal DNA restriction analysis, and duplicate isolates of the same genospecies from individuals were ruled out by random amplified polymorphic DNA analysis. Over 40% of 192 healthy volunteers carried Acinetobacter spp. at one or more body sites, and the frequencies of colonisation were as follows: forearm (51%), forehead (47%) and toe web (34%). Genospecies 8/9 (Acinetobacter lwoffii) was the most common (61%), followed by genospecies 15BJ and 12 (Acinetobacter radioresistens) at 12.5% and 8%, respectively. The Acinetobacter baumannii-Acinetobacter calcoaceticus group (genospecies 1, 2, 3 and 13TU) that predominates in hospital-acquired infections was found in only one individual.     

Serological and genetic examination of some nontypical Streptococcus mutans strains.

Thirty-four strains of Streptococcus mutans whose antigenic or genetic positions were unclear or unknown with respect to the serological scheme of Bratthall (1970) and Perch et al. (1974), or the genetic (deoxyribonucleic acid base sequence homology) scheme of Coykendall were analyzed to clarify their relationship to previously well-characterized strains. Strain OMZ175 of the "new" serotype f was genetically homologous with strains of S. mutans subsp. mutans. Strains of the "new" serotype g were homologous with serotype d strains (S. mutans subsp. sobrinus). Strains isolated from wild rats constituted a new genetic group but carried the c antigen. Thus, strains within a "genospecies" (subspecies) of S. mutans may not always carry a unique or characteristic antigen. We suggest that the existence of multiple serotypes within subspecies represents antigenic variation and adaptations to hosts.     

Bacteremia and chorioamnionitis due to cryptic genospecies ofHaemophilus Influenzae biotype I

Nontypable strains ofHaemophilus influenzae are well-known causes of maternal and neonatal infections. Using DNA-DNA hybridization techniques, some of these strains have been shown to belong to a cryptic genospecies ofHaemophilus, which is distantly related toHaemophilus influenzae andHaemophilus hemolyticus. This report describes the first case of sepsis and chorioamnionitis due to Haemophilus influenzae biotype I, which was identified using the RapIDNH system and then confirmed by multilocus enzyme electrophoresis to belong to this cryptic genospecies ofHaemophilus. The electromorph type 92 of the isolate was consistent with that of biotype I of the cryptic genospecies.     

Study of humoral immunity to commensal oral bacteria in human infants demonstrates the presence of secretory immunoglobulin A antibodies reactive with Actinomyces naeslundii genospecies 1 and 2 ribotypes

The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell wall proteins from their own A. naeslundii strains and carbohydrates from standard A. naeslundii genospecies 1 and 2 strains. A. naeslundii genospecies 1 and 2 carbohydrate-reactive SIgA antibodies were not detected in any saliva sample. However, SIgA antibodies reactive with cell wall proteins were present in saliva before these bacteria colonized the mouth. These antibodies could be almost completely removed by absorption with A. odontolyticus, a species known to colonize the human mouth shortly after birth. However, after colonization by A. naeslundii genospecies 1 and 2, specific antibodies were induced that could not be removed by absorption with A. odontolyticus. Cluster analysis of the patterns of reactivity of postcolonization salivary antibodies from each infant with antigens from their own strains showed that not only could these antibodies discriminate among strains but antibodies in saliva samples collected at different times showed different reactivity patterns. Overall, these data suggest that, although much of the salivary SIgA antibodies reactive with A. naeslundii genospecies 1 and 2 are directed against genus-specific or more broadly cross-reactive antigens, species, genospecies, and possibly strain-specific antibodies are induced in response to colonization.     

In vitro susceptibility testing of four antibiotics against Borrelia burgdorferi: a comparison of results for the three genospecies Borrelia afzelii,Borrelia garinii,and Borrelia burgdorferi sensu stricto

MICs and minimal bactericidal concentrations (MBCs) were evaluated for the four antibiotics azithromycin, amoxicillin, ceftriaxone, and doxycycline against the three main genospecies of Borrelia burgdorferi sensu lato. In MBC testing, statistically significant differences between the genospecies could be found in 7 out of 12 comparative evaluations (P < 0.05).     

Borrelia isolates in Northern Colorado identified as Borrelia bissettii

Previous work described Borrelia burgdorferi sensu lato group DN127 as a new genospecies, Borrelia bissettii, and prompted the present study to identify the Borrelia spp. that exist in northern Colorado. To determine the genospecies present, we analyzed two specific intergenic spacer regions located between the 5S and 23S and the 16S and 23S ribosomal genes. Phylogenetic analysis of the derived sequences clearly demonstrated that these isolates, originating from rodents captured in the foothills of northern Colorado, diverged from B. burgdorferi sensu stricto by 5 to 5.5% and were members of the new genospecies B. bissettii.     

Estimating reservoir competence of Borrelia burgdorferi hosts: prevalence and infectivity, sensitivity, and specificity

Most vector-borne zoonotic pathogens are transmitted among several host species, but different species vary considerably in their importance to pathogen transmission, at least partially because they vary in their propensity to infect feeding vectors. This propensity is often called realized reservoir competence. Realized reservoir competence is the product of 1) the probability the individual host is infected, i.e., infection prevalence, and 2) the probability that if the host is infected, it will transmit the infection to a feeding vector, or infectivity. Prevalence varies in space and time, whereas infectivity may be a property of the host species. Both prevalence and infectivity are ecologically and epidemiologically important, but measuring them simultaneously is difficult. We present a probabilistic model that separately estimates host infection prevalence and infectivity from data on the infection status of vectors collected from individual hosts, data generally used to measure realized reservoir competence. We then consider how imperfect diagnostic tests (i.e., false negatives and positives) influence these probabilities-estimates of prevalence and infectivity are fairly robust to false negatives, but not to false positives. We thus extend the model to estimate the rate of false positives in order to improve estimates of prevalence and infectivity. We illustrate these methods by reanalyzing data from LoGiudice et al. (2003; Proc. Natl. Acad. Sci. U.S.A. 100: 567-571) on the reservoir competence of ten vertebrate hosts of Borrelia burgdorferi, the agent of Lyme disease. We find that these vertebrate hosts vary both in prevalence and infectivity and that both values are highly, positively correlated among species.     

Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections.

In recent years, reports originating from several areas of the world have identified biotype IV strains of Haemophilus influenzae as a cause of serious urogenital, neonatal, and mother-infant infections. Preliminary analysis of a sample of biotype IV isolates found evidence for a cryptic genospecies of Haemophilus (R. Quentin, A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. J. Gen. Microbiol. 136:1203-1209, 1990). Eighteen biotype IV strains assigned to the cryptic genospecies were further characterized by their rDNA restriction fragment length polymorphism patterns and genomic DNA-DNA hybridization. Isolates of the cryptic genospecies have distinctive rDNA restriction fragment length polymorphisms that differ from those of Haemophilus haemolyticus and H. influenzae. Genomic hybridization studies show that these organisms are allied with H. influenzae and H. haemolyticus and suggest a distant trifurcation of H. influenzae, H. haemolyticus, and the cryptic genospecies.     

Potential of ancestral sylvatic dengue-2 viruses to re-emerge

Dengue viruses (DENV) are the most important arboviral pathogens in tropical and subtropical regions throughout the world. DENV transmission includes both a sylvatic, enzootic cycle between nonhuman primates and arboreal mosquitoes of the genus Aedes, and an urban, endemic/epidemic cycle between Aedes aegypti, a mosquito with larval development in peridomestic water containers, and human reservoir hosts. All 4 serotypes of endemic DENV evolved independently from ancestral sylvatic viruses and have become both ecologically and evolutionarily distinct; this process may have involved adaptation to (i) peridomestic mosquito vectors and/or (ii) human reservoir hosts. To test the latter hypothesis, we assessed the ability of sylvatic and endemic DENV-2 strains, representing major genotypes from Southeast Asia, West Africa and the Americas, to replicate in two surrogate human model hosts: monocyte-derived, human dendritic cells (moDCs), and mice engrafted with human hepatoma cells. Although the various DENV-2 strains showed significant inter-strain variation in mean replication titers in both models, no overall difference between sylvatic and endemic strains was detected in either model. Our findings suggest that emergence of endemic DENV strains from ancestral sylvatic strains may not have required adaptation to replicate more efficiently in human reservoir hosts, implying that the potential for re-emergence of sylvatic dengue strains into the endemic cycle is high. The shared replication profiles of the American endemic and sylvatic strains suggest that American strains have maintained or regained the ancestral phenotype.