Investigations on the biology of ticks, investigations on tick-borne diseases, especially Tick-borne-encephalitis-virus-infection and Lyme-Borreliosis, species determination by matrix-assisted laser desorption/ionisation mass spectrometry.
TBE and importance of TBE-virus diagnostics in animals
Tick-borne encephalitis (TBE) is the most important viral tick-borne zoonosis in Europe. In Germany, approximately 250 autochthonous human clinical cases are registered per year with strong annual fluctuations. In veterinary medicine, clinical cases of TBE are seldom, but have been described for example in dogs, horses, sheep, goats and monkeys, in some cases with a severe clinical course. However, TBE-virus (TBEV) is excreted in milk of goats, sheep and cattle and can be ingested orally by consumption of non-pasteurized milk or by cheese produced from raw milk and can cause the so-called alimentary TBE. Compared with a TBE infection via tick bite this way of infection is very seldom. Nevertheless, it should be borne in mind as in the last years alimentary TBE infections have occurred in Estonia, Austria and Hungary, recently also in Germany.
In addition, data of TBEV diagnostics in animals could be a helpful tool to investigate TBEV epidemiologically. In contrast to Borrelia burgdorferi s.l., that is ubiquitously endemic in all areas in Germany where Ixodes ricinus occurs, TBEV circulates in geographically strictly limited natural foci that can range in size from large to very small and are distributed in a patchy pattern with the main focus in Southern Germany. These foci can newly arise, exist in some regions over a long period of time or expire in other regions. So far, the reasons are unclear and the investigation of animals (pathogen detection via quantitative real-time RT-PCR (RT-qPCR) as well as serological investigations of TBEV-antibody titres) can increase the understanding of the epidemiology of TBE as the most important viral tick-borne disease in Central Europe.
Detection of TBEV
TBEV can be detected in ticks (in Germany: Ixodes ricinus), in tissue samples for pathological investigations, in sera or cerebrospinal fluid. Negative results should be interpreted with care, especially in areas with a TBE history, because of the very low TBE virus prevalence in ticks (even in TBE risk areas lower than 10%) and the patchy pattern of TBEV natural foci. It is strongly recommended to examine a larger quantity of ticks.
Several RT-qPCR-assays for the detection of TBEV-RNA have been published. Two independent duplex assays were developed and optimized at the FLI. The two TBEV assays were located in the 3`- and 5`- non-translated region of the TBEV genome, respectively. The assays were combined with different internal controls. Thus, successful extraction of RNA from ticks (16S) was checked simultaneously with pathogen detection and in the second assay a partial inhibition of the amplification process was excluded.
Detection of TBEV antibody titres in animal sera
The number of serological methods for veterinary use is very limited. In our lab a two-step method was used successfully: At first all sera were screened by an all species ELISA, then all positive sera were confirmed by a serum neutralization test (SNT).
This method allows using a commercially available ELISA test for all species. There is no need to develop animal species specific tests. Furthermore, ELISA screening replaces testing of all sera in laborious SNT. However, non-specific positive ELISA results are possible due to protein G based conjugate in the all species ELISA. Therefore it is highly recommended to confirm all positive results by SNT.
For the SNT, the low pathogenic cross-reactive strain Langat can be used instead of viraemic TBEV strains, as it allows working in a lab with a lower biosafety level than TBEV (biosafety level 2 instead of biosafety level 3) However, in most cases SNT will be performed in specialized labs.
The method was proven successfully in our investigations examining 3590 sheep sera and 3793 goat sera collected in several districts of Baden-Württemberg, Bavaria, Thuringia, North Rhine-Westphalia, Lower Saxony, Schleswig-Holstein and Mecklenburg-West Pomerania. Sheep and goats as sentinels provide new insights into TBEV epidemiology. Sera of these animal species are well suited for this purpose because they are collected for other purposes in all federal states and are therefore generally available, well documented and of high quality. The grazing areas of these species are well-known so that collecting ticks in areas with high seroprevalences will increase the chances for a direct detection of TBE virus in spite of the patchy pattern of natural foci and the low virus prevalence in ticks. This has been confirmed by our own investigations of sheep flocks and horse herds, where it was possible to detect TBEV positive ticks in the surroundings of the seropositive animals. It is likely that serological screening of goat and sheep flocks and other grazing animals prior to the investigation of ticks will become an integral part of the diagnosis and risk assessment of this important zoonosis in the future. Our previous investigations of 18,000 ticks resulting in only 7 TBEV-positive ticks illustrate that non-targeted screening of tick populations even in TBE risk areas is time-consuming and expensive with low chances of success. This could be improved substantially by screening of animal sera prior to tick collection as described above.
Investigations on cross-reactions of TBEV antibody-positive sera confirmed the specificity of the ELISA test; cross-reactions were only seen with closely related Louping ill antibody-positive sera. In addition, the test was well suited to elucidate non-specific positive serological results in the West Nile ELISA.
Investigations of antibody titers in goats and sheep revealed that titers persist for several years, albeit at a low level.
Species determination and characterisation of developmental stages of ticks by whole animal matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS)
Classically, ticks and their developmental stages are differentiated by morphological criteria. However, molecular biological methods, such as PCR using mitochondrial 12S and 16S rDNA sequences, have been applied increasingly in tick identification. A very cost-efficient and rapid, yet highly informative tool for tick species determination could be whole animal matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) based on protein spectra which over the past few years has been introduced successfully for the identification and classification of bacteria and also for tissue cultures.
Here, a simple protocol was developed to perform MALDI-MS analysis on extracts from whole ticks. A reference database of spectra was constructed for seven species (I. canisuga, I. hexagonus, I. persulcatus, I. ricinus, I. scapularis, D. reticulatus und R. sanguineus).
Cluster analysis on the basis of MALDI mass spectra indicated that the primary determinant for the mass spectra was the species, followed by the developmental stages, which formed distinct clusters within the given species.
Clusters illustrated phylogenetic correlations between species. With certain limitations, species identification was also possible using “problematic samples” such as body parts (not possible with only legs) and engorged animals. Spectra of developing Ixodes ricinus eggs during 39 days showed dramatic changes with time. Up to now the reason for these changes and their importance is not known, however, suggesting that, beyond its usefulness for species determination, MALDI-MS may have applications in developmental biology of ticks and their supporting and limiting factors.
Upgrading of reference spectra by additional tick species is in progress. Investigations of infected ticks and on supporting and limiting factors for tick development should be done in the future.
- Detection of TBE virus in ticks (realtime RT-qPCR)
- Detection of TBEV-antibody titres in animal sera (after prior consultation)
- Detection of Borrelia-antibody titres in animal sera (after prior consultation)
- Advice on specific diagnostic issues (PCR, sequencing, serology)
- Supply of not commercially available diagnostic reagents
Selected papers of the TBD working group
- Klaus, C., Ziegler, U., Kalthoff, D., Hoffmann, B., Beer, M., 2014. Tick-borne encephalitis virus (TBEV) – findings on cross reactivity and longevity of TBEV antibodies in animal sera. BMC Vet. Res. 10:78.
- Klaus, C., Hörügel, U., Hoffmann, B., Beer, M., 2013. Tick-borne encephalitis virus (TBEV) infection in horses: Clinical and laboratory findings and epidemiological investigations. Vet Microbiol 163, 368-372.
- Karger, A., Kampen, H., Bettin, B., Dautel, H., Ziller, H., Hoffmann, B., Süss, J., Klaus, C., 2012. Species determination and characterization of developmental stages of ticks by whole animal matrix-assisted laser desorption/ionisation mass spectrometry. Ticks Tickborne Dis 3, 78-89
Klaus, C., Beer, M., Saier, R., Schau, U., Moog, U., Hoffmann, B., Diller, R., Süss, J., 2012. Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus - epidemiological studies in areas endemic and non-endemic for TBE virus in Germany. Ticks Tickborne Dis 3, 27-37
- Klaus, C., Beer, M., Saier, R., Schubert, H., Bischoff, S., Süss, J., 2011. Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals. Berl. Münch. Tierärztl. Wochenschr. 124, 443-449
- Klaus, C., Hoffmann, B., Moog, U., Schau, U., Beer, M., Süss, J., 2010. Can goats be used as sentinels for Tick-borne encephalitis (TBE) in non-endemic areas? Experimental studies and epizootological observations. Berl. Münch. Tierärztl. Wochenschr. 123, 441-445
- Klaus, C., Hoffmann, B., Beer, M., Müller, W., Stark, B., Bader, W., Stiasny, K., Heinz, F.X., Süss, J., 2010. Seroprevalence of tick-borne encephalitis (TBE) in naturally exposed monkeys (Macaca sylvanus) and sheep and prevalence of TBE virus in ticks in a TBE endemic area in Germany. Ticks Tickborne Dis 1, 141-144
- Klaus, C., Hoffmann, B., Hering, U., Mielke, B., Sachse, K., Beer, M., Süss, J., 2010. Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) in risk, non-risk, and former risk areas of TBE, and in ticks removed from humans in Germany. Clin Microbiol Infec 16, 238–244