Experimental infection of dogs with<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu stricto using<Emphasis Type="Italic"> Ixodes scapularis</Emphasis> ticks artificially infected by capillary feeding

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.     

Wild ruminants in the area of the North-Western Poland as potential reservoir hosts of <Emphasis Type="Italic">Bartonella schoenbuchensis</Emphasis> and <Emphasis Type="Italic">B. bovis</Emphasis>

The aim of this work was to examine if the game species from the north-western Poland, roe deer (Capreolus capreolus), red deer (Cervus elaphus) and wild boar (Sus scrofa), may be reservoir hosts of bacteria from the genus Bartonella, and whether the sheep tick (Ixodes ricinus) is their vector. To this end, the prevalence of Bartonella DNA in the tissues of these game species was measured, just as in sheep ticks (I. ricinus) infesting them, and ticks collected from plants in the hunting area. The prevalence of Bartonella DNA was 39% (23/59) in roe deer and 35% (7/20) in red deer. No Bartonella DNA was detected in any of the 21 wild boars. The presence of Bartonella DNAwas detected in 1.9% of ticks infesting roe deer (2/103), while no pathogen DNA was found in the 20 ticks infesting the red deer and the 3 ticks infesting wild boars, or the 200 ticks collected from plants. Amplicons of two different lengths were obtained; 198 bp, characteristic for B. bovis, and 317 bp, characteristic for B. schoenbuchensis, which were confirmed later by sequencing. The examined ruminants are probably the reservoir hosts of B. schoenbuchensis and B. bovis in the biotope of the Puszcza Wkrzańska Forest, and wild boars do not participate in the Bartonella propagation in the environment. I. ricinus is unlikely to be the main vector of Bartonella species detected in the examined roe deer and red deer; probably other bloodsucking arthropods, parasitizing wild ruminants, play this role.     

Characterization of Borrelia burgdorferi sensu lato strains isolated from Ixodes ricinus in Mecklenburg-Vorpommern,Germany

Epidemiological studies in Mecklenburg-Vorpommern have shown a high prevalence ofBorrelia burgdorferi-infected ticks. A total of 17B. burgdorferi sensu lato strains were isolated from ticks and investigated by Western blots (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). Except for one, all strains could be classified using this system. The majority of strains belonged to theB. garinii-associated OspA serotypes 3, 5 and 6. Three isolates were classified as OspA serotype 2 (B. afzelii).B. burgdorferi sensu stricto strains (Ospa serotype 1) as well asB. garinii-associated OspA serotype 4 were not present.     

An<Emphasis Type="Italic"> ospA</Emphasis>-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato species and OspA types

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3–7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.     

Effect of proteins from the spirochete <Emphasis Type="Italic">Borrelia burgdorferi sensu lato</Emphasis> on myelinated nerve excitability

We studied the effect of spirochete Borrelia burgdorferi sensu lato cell membrane proteins on excitability of myelinated nerve fiber. It was found that cell surface proteins of spirochetes B. burgdorferi s. s. bind to Ranvier nodes of the axon and to Schwann cells. Binding of B. burgdorferi s. s. and B. garinii to the nerve fiber modulates the amplitude and conduction velocity of the action potential, while B. afzelii had no effect on these parameters. The decrease in the spike amplitude and conduction velocity during sorption of B. burgdorferi s. s. or cell wall proteins was accompanied by desorption of membrane-bound calcium. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 42–45, January, 2007     

Comparison of different Borrelia burgdorferi sensu lato strains for detection of immune response in patients with erythema migrans

The aim of the present study was to establish which combination of serological method and Borrelia strain used as an antigen would provide the most appropriate demonstration of borrelial infection in patients with eythema migrans residing in Slovenia. Four different strains were chosen as antigens: two strains of B. afzelii and two strains of B. garinii which differed in their expression of the outer proteins OspA, OspB and OspC. Each individual strain was used as antigen in immunofluorescence test (IFT), enzyme-linked immunosorbent assay (EIA) with whole borrelial cells, and EIA with ultrasonicated borrelial cells. With these 12 different tests, 100 samples were examined for the presence of specific IgM and IgG antibodies: 50 sera of blood donors and 50 sera of patients with erythema migrans. The latter were further subdivided into skin culture-positive and -negative subgroups. A commercial Western blot (WB) test was performed for 26 sera of the control group and 25 sera of patients with erythema migrans. The four different methods had distinct specificity and sensitivity. The most specific approaches were IFT (100% for IgM and 90–92% for IgG) and the WB test (100% for IgM and 73% for IgG), followed by EIA with whole borrelial cells (80–98% for IgM and 76–84% for IgG) and EIA with ultrasonicated borrelial cells (76–94% for IgM and 72–80% for IgG). The sensitivity levels of all these tests were low. The most sensitive were EIA tests with whole borrelial cells (28–36% for IgM and 32–42% for IgG) followed by EIA with ultrasonicated borrelial cells (22–32% for IgM and 24–36% for IgG), the WB test (16% for IgM and 32% for IgG) and IFT (0–2% for IgM and 14–20% for IgG). The following methods gave significant differences between patients and negative controls in detecting IgM antibodies: EIA with whole borrelial cells with both B. afzelii antigens and with antigen B. garinii that expressed OspA and OspC, EIA with ultrasonicated borrelial cells with antigen B. afzelii that expressed OspA, OspB and OspC. In detecting IgG antibodies, significant differences were observed between EIA with whole borrelial cells and with antigen B. afzelii that expressed OspA and OspB. Borreliae were isolated from the skin of 34/50 (68%) patients with erythema migrans: two strains failed to grow, while 26/32 (81%) strains were identified as B. afzelii, 5/32 (16%) as B. garinii and 1/32 (3%) as B. burgdorferi sensu stricto. No statistically significant differences in serologic test results between culture-positive and -negative patients with erythema migrans were found.     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

C-<Emphasis Type="Italic">erb</Emphasis>B-2 oncogene and <Emphasis Type="Italic">P21</Emphasis><Superscript><Emphasis Type="Italic">WAF/CIP1</Emphasis></Superscript> tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas

To evaluate the use of c-erbB-2 oncogene and p21 ^WAF1/CIP1 suppressor gene products as the prognosis markers for canine mammary tumors, expression of these gene products were examined immunohistochemically using tumor tissues and clinical data from 96 dogs with malignant mammary tumors. Semiquantitative data was compared with histopathological grades, proliferating cell nuclear antigen (PCNA)-positive index, and clinicopathological matters. The expression c-erbB-2 protein was found in the cellular membrane and cytoplasm of neoplastic epithelial cells, and the positive index had no significant relation to the histopathological features and PCNA-positive index, except for the individual age of affected dogs (P < 0.05). The product of p21 ^WAF1/CIP1 was mostly found in cytoplasm and occasionally in the nucleus of neoplastic cells. The quantitative data had significant association to the malignancy grade and size of tumors (P < 0.05). However, that had no significant relationship to the PCNA-positive index. The present study concluded that both gene products could not apply as the direct markers to evaluate the prognosis of canine mammary tumors. The detection of c-erbB-2 product may be partly beneficial to the differential diagnosis of epithelial type of mammary cancer. The use of p21 ^WAF1/CIP1 product in prognosis of canine mammary cancer needs further investigation.     

A frameshift mutation of the chloroplast <Emphasis Type="Italic">mat</Emphasis>K coding region is associated with chlorophyll deficiency in the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> virescent mutant <Emphasis Type="Italic">Wogon</Emphasis>-Sugi

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> harboring <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> increases cytopathogenicity in vitro

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis. In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.     

Molecular and immunological characterization of the p83/100 protein of various Borrelia burgdorferi sensu lato strains

The complete coding regions of the chromosomally encoded p83/100 protein of four Borrelia garinii strains and one Borrelia burgdorferi sensu stricto strain have been amplified by the polymerase chain reaction (PCR), cloned and sequenced. From alignment studies with the deduced amino acid sequences presented here, and five other published p83/100 sequences, the most heterologous region of the p83/100 molecule was identified to be located between amino acid position 390–540. To study the structure of this heterogeneous region, and internal fragment of the p83/100 genes from 11 additional B. burgdorferi sensu lato strains was amplified by PCR. The PCR products were analyzed by DNA sequencing and restriction enzyme analysis. These internal p83/100 fragments varied in size and sequence. Cluster analysis of internal p83/100 fragments, as well as restriction enzyme analysis, revealed three major groups in accordance with grouping into the three species causing Lyme disease. Strains within the same species (six B. burgdorferi sensu stricto and six B. afzelii strains) showed similar p83/100 partial structures. Nevertheless, nine B. garinii strains showed more sequence variations and could be further divided into two major subgroups. One group is represented by OspA serotype 4 strains, the other more heterogeneous group is represented by OspA serotypes 3, 5, 6 and 7 strains. Phenotypic analysis with four p83/100-specific monoclonal antibodies revealed four distinct reactivity patterns. Antibody L100 1B4 recognized a common epitope of B. burgdorferi sensu stricto and B. afzelii. Antibodies L100 17D3 and L100 18B4 were reactive with an epitope shared by strains of all three species. The broadest reactivity was shown by L100 18B4 which, in constrast to L100 17D3, additionally recognized the relapsing fever borreliae B. turicatae and B. hermsii. L100 8B8 detected a subgroup of the B. burgdorferi sensu stricto strains. Since comparison of the p83/100 molecule with sequences from protein databases showed similarities with characteristics of eukaryotic cell structures, the p83/100 might mimic these structures and may, therefore, be involved in the immune escape mechanism of the pathogenic agent of Lyme disease.     

Stimulation of biofilm formation by insertion of <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> wells within <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> biofilms

Biofilm formation is an important part of the bacterial life cycle. Biofilms provide bacterial resistance to external stresses and protozoan grazing. Biofilm formation by the wild type of B. cenocepacia strain 370 in the presence of the free-living ciliate Tetrahymena pyriformis was studied. T. pyriformis grazed on planktonic bacteria and reduced the planktonic bacterial subpopulation while it noticeably stimulated biofilm formation. When cultivated alone, T. pyriformis did not form visible biofilms. Confocal laser scanning microscopy was used to demonstrate the inclusion and further destruction of protozoan cells within the biofilms formed by the bacteria. The destruction of protozoan cells was accompanied by the exit of bacteria from vacuoles and intracytoplasmic multiplication; changes in the form of protozoan cells; the demolition of internal structures; and the visual exit of the cytoplasmic content from destructing cells. Microcolonies of a characteristic round shape were revealed in the biofilms formed by B. cenocepacia in the presence of T. pyriformis. These structures were absent in the biofilms formed by B. cenocepacia alone. Insertion of protozoan cells within biofilms seems to be a driving force that promotes biofilm proliferation and influences their structure. The mortality of protozoan cells in the biofilms caused a decrease in the T. pyriformis population under conditions advantageous to B. cenocepacia biofilm formation. The mutant B. cenocepacia strain Bcb-1, which is unable to form biofilms, was isolated by plasposon mutagenesis. In contrast to the parental strain, the cocultivation with Bcb-1 bacteria improved the growth of T. pyriformis. A mutation was mapped in the ompR gene. The text was submitted by the authors in English.     

Characterisation of <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis> centromeres and construction of first <Emphasis Type="Italic">Z. rouxii</Emphasis> centromeric vectors

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.     

Evaluation of hematological values in indigenous chickens infected with <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Aegyptianella pullorum</Emphasis>

Plasmodium, Haemoproteus, and leukocytozoon are the most important hematozoa in birds, which have been reported in different areas of the world. The present study was undertaken to find which blood protozoans exist in indigenous chickens in Shiraz, southern Iran and to evaluate hematological parameters in birds infected with hematozoas. Plasmodium and Aegyptianella were the two parasites found in 740 blood samples examined from indigenous chickens of which 29 (3.91%) were positive for Aegyptianella pullorum, 106 (14.32%) for Plasmodium gallinaceum, and 12 (1.62%) for A. pullorum and Plasmodium gallinaceum together. There was no significant difference between hematological parameters of non-infected and naturally infected chickens with Plasmodium gallinaceum, A. pullorum, and both (P > 0.05). Low infection of indigenous chickens with A. pullorum, Plasmodium gallinaceum, and both had no significant effects on hematological parameters (P > 0.05), which is probably due to low parasitemia rate and immunity against these two parasites.     

Sex determination in platypus and echidna: autosomal location of <Emphasis Type="Italic">SOX3</Emphasis> confirms the absence of <Emphasis Type="Italic">SRY</Emphasis> from monotremes

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.     

Redescription of <Emphasis Type="Italic">Decorataria decorata</Emphasis> (Spirurida,Acuariidae) based on nematodes from <Emphasis Type="Italic">Podiceps cristatus</Emphasis> and <Emphasis Type="Italic">P. grisegena</Emphasis> (Aves,Podicipediformes) from Bulgaria

Decorataria decorata (Cram, 1927) is redescribed on the basis of light-microscopy and SEM observations on specimens collected from the stomach of Podiceps cristatus and P. grisegena from Bulgaria. The SEM study revealed the presence of a porebearing field on each pseudolabium and a pair of spines (one dorsal and one ventral) situated between bases of the cordons. The deirids are spine-like and minute. The light-microscopy examination showed the presence of ornamentation situated under the dorsal surface of caudal alae. The occurrence of D. decorata in Bulgaria is a new geographical record.     

Chromosome elimination in the interspecific hybrid medaka between <Emphasis Type="Italic">Oryzias latipes</Emphasis> and <Emphasis Type="Italic">O. hubbsi</Emphasis>

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes–hubbsi and O. hubbsi–latipes embryos. Whole-mount immunocytochemical analyses using antibodies against α-tubulin, γ-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1α, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.     

Genotyping <Emphasis Type="Italic">Borrelia burgdorferi sensu lato</Emphasis> isolates from <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks in Russia and Ukraine

The four Borrelia burgdorferi sensu lato isolates obtained from I. ricinus ticks collected from the natural loci in Russia and Ukraine that had an unusual RFLP MseI pattern were studied using the sequencing of the rrfA-rrlB spacer and the rrs gene. Isolate Ir-5215 from a tick collected in southern Ukraine represented the recently described genospecies B. spielmanii, which is pathogenic for humans. The three atypical isolates Ir-3519, Ir-4721, and Ir-4812 had 100% identity with the sequence of atypical European B. burgdorferi sensu stricto strains; they constituted a subgroup of B. burgdorferi sensu stricto based on multilocus sequence analysis (MLSA). These data are indicative of the genetic heterogeneity of the current group B. burgdorferi sensu stricto.     

Comparison of growth of Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto in MKP and BSK-II medium

Lyme borreliosis is a tick-borne disease caused by genetically diverse Borrelia strains including B. afzelii, B. garinii, and B. burgdorferi sensu stricto (s.s.). The aim of the present study was to assess and compare the growth of one strain per species of B. afzelii, B. garinii, and B. burgdorferi s.s. in modified Kelly-Pettenkofer (MKP) and Barbour-Stonner-Kelly-II (BSK-II) medium, and to check for the presence of the overgrowth after inoculating the media with a mixture of two different Borrelia species. All three Borrelia strains grew well in both media. In the majority of the experiments the number of B. afzelii cells was higher in MKP than in BSK-II medium while for B. garinii and B. burgdorferi s.s. a tendency for better growth in BSK-II than MKP was established. In a mixture of equivalent amounts of two species, B. burgdorferi s.s. as a rule overgrew the other two species while in the mixture of B. afzelii and B. garinii the latter was a "dominant" strain. Comparing the performance of the two media, B. burgdorferi s.s. usually overgrew either B. afzelii or B. garinii in MKP as well as in BSK-II medium, however, the results were found to be statistically significant only for MKP medium. In the mixture of B. afzelii and B. garinii the latter was the predominant species but significant differences were established only for BSK-II medium. It seems that the overgrowth is predominantly the result of the characteristics of the individual Borrelia species and most probably not a consequence of growth differences in the two culture media. Further work with a larger number of strains is needed to confirm these findings.