Comparison of different diagnostic assays for the detection of Borrelia burgdorferi-specific antibodies in dogs.

BACKGROUND: Antibody tests are frequently used in the diagnosis of canine Lyme borreliosis, including immunofluorescence assay (IFA) for immunoglobulin G (IgG) and M (IgM) antibodies, kinetic enzyme-linked immunosorbent assay (KELA), and Western blotting (WB). Recently, the SNAP4Dx, an in-house test using C6 technology has become available. OBJECTIVES: The aim of this study was to compare IFA, KELA, and SNAP4Dx assay results, and to determine their sensitivity and specificity when compared with the WB, used as gold standard in this study. METHODS: Two hundred canine sera were tested for the presence of specific antibodies against Borrelia spp. using the above-mentioned tests. RESULTS: The sensitivity and specificity of IFA-IgG was 76.6% (95% confidence interval [CI] 46.87-86.72) and 87.1% (95% CI 80.06-91.90), and 26.3% (95% CI 11.81-48.79) and 81.0% (95% CI 73.64-86.71) for IFA-IgM, respectively. KELA was 100% (95% CI 83.18-100) sensitive and 75.4% (95% CI 67.02-82.09) specific, and the SNAP4Dx was 84.2% (95% CI 62.43-94.48) sensitive and 98.5% (95% CI 94.83-99.60) specific. CONCLUSIONS: Both IFAs had very low sensitivity and specificity and cannot be recommended for screening purposes. In contrast, KELA showed excellent sensitivity, but positive results always need to be confirmed by WB to differentiate the source of antibody formation. The SNAP4Dx had a high sensitivity and specificity, and thus can potentially replace the more labor-intensive WB, at least in untreated dogs.

Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20.

Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum. Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium. In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells.

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST).

BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.

Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model.

OBJECTIVES: Systemic antibiotic treatment of Lyme borreliosis is effective during the early stages of the infection, while chronic manifestations of the disease may remain refractory and difficult to treat. This study was carried out in order to evaluate the potential of topically applied azithromycin to eliminate the spirochaetal organisms in the skin of the freshly bitten host and thereby prevent Lyme borreliosis. METHODS: Laboratory mice were challenged with Borrelia burgdorferi sensu stricto by needle inoculation or via infected ticks as vectors. Then, an azithromycin-containing formulation was applied once daily to the sites of exposure for three consecutive days. In the case of needle inoculation, a 5% azithromycin formulation was applied starting 1 h, 3 days and 5 days after infection. In the case of tick exposure, 4%, 10% and 20% azithromycin formulations were applied, starting directly after the detachment of the engorged ticks. Subsequently, the infection status of the mice was determined. RESULTS: Concentrations of azithromycin in murine skin were >3800-fold higher than the published minimal inhibitory concentration for B. burgdorferi as soon as 3 h after the first application. After needle inoculation, spirochaetes were not detectable in all infected mice after treatment, if the first application started 1 h or even after 3 days post-infection. Furthermore, no borrelial organisms were detected after topical treatment when ticks were used for spirochaete inoculation. CONCLUSIONS: Our data indicate that topical treatment with a formulation containing azithromycin is a promising approach to prevent Lyme borreliosis shortly after a tick bite.

Lyme borreliosis in dogs and cats: background, diagnosis, treatment and prevention of infections with Borrelia burgdorferi sensu stricto.

Lyme borreliosis (LB), synonymous with the often-used term Lyme disease, is an infectious disease caused by the spirochetal bacterium Borrelia burgdorferi. LB is the most frequent vector-borne disease in humans in the Northern Hemisphere. In animals, clinically apparent disease is found primarily in dogs. Severe polyarthritis, fever and lameness in dogs are reported from the main endemic areas of North America: the New England States, and eastern parts of the United States; several cases of LB are also seen in California and the Midwest. Because of the difficulties in finding sufficient indicative clinical signs, additional information (detailed case history, laboratory testing for antibodies) is especially important to make the clinical diagnosis of Lyme borreliosis. This article reviews the etiology, diagnosis, therapy, and prevention of LB.

Borrelia burgdorferi sensu lato species in Europe induce diverse immune responses against C6 peptides in infected mice.

The diversity of Lyme-borreliosis-inducing Borrelia species in Europe set high standards for the use of serodiagnostic test systems in terms of specificity and sensitivity. In the United States, the one-step C6 antibody test system based on the invariable domain IR6 of the VlsE molecule has been established as a successful diagnostic tool for testing canine samples. However, only a limited set of data are available regarding the antigenicity of the C6 peptides in an experimental murine model and sensitivity of the test regarding European Borrelia species. In order to investigate antibody reactions induced by these spirochetes, a total of 142 C3H/HeN mice were inoculated with Borrelia burgdorferi sensu stricto N40, B. garinii PBi, two isolates of B. afzelii, B. spielmanii A14S, B. valaisiana Rio6, B. valaisiana VS116, or B. lusitaniae. Infection of the mice was documented utilizing tissue culture and PCR. The IR6 sequences of B. burgdorferi sensu stricto B31, B. garinii IP90, and two B. afzelii ACAI strains have been used to synthesize and test additional C6 peptides. Compared to the well-established two-tiered test system, the results indicate that single C6 peptides derived from B. burgdorferi sensu stricto and B. garinii can be used in an enzyme-linked immunosorbent assay-based technique to detect murine antibodies induced by either agent. Little is known about the prevalence or pathogenicity of the B. afzelii strains in mammalian hosts, but our experimental data indicate differences in the C6 peptide test sensitivity for the detection of antibodies induced by different strains or isolates of B. afzelii.