Co-exposure to Anaplasma spp., Coxiella burnetii and tick-borne encephalitis virus in sheep in southern Germany
Acta Veterinaria Scandinavica volume 65, Article number: 6 (2023)
The intracellular bacteria Anaplasma spp. and Coxiella burnetii and the tick-borne encephalitis virus (TBEV) are tick-transmitted pathogens circulating in the southern German sheep population. Knowledge of interaction among Anaplasma spp., C. burnetii and TBEV in sheep is lacking, but together they might promote and reinforce disease progression. The current study aimed to identify co-exposure of sheep to Anaplasma spp., C. burnetii and TBEV. For this purpose, 1,406 serum samples from 36 sheep flocks located in both southern German federal states, Baden-Wuerttemberg and Bavaria, were analysed by ELISAs to determine the antibody levels of the three pathogens. Inconclusive and positive results from the TBEV ELISA were additionally confirmed by a serum neutralisation assay. The proportion of sheep with antibodies against Anaplasma spp. (47.2%), C. burnetii (3.7%) and TBEV (4.7%) differed significantly. Significantly more flocks with Anaplasma spp. seropositive sheep (91.7%) were detected than flocks with antibodies against TBEV (58.3%) and C. burnetii (41.7%), but there was no significant difference between the number of flocks which contained TBEV and C. burnetii seropositive sheep. Seropositivity against at least two pathogens was detected in 4.7% of sheep from 20 flocks. Most co-exposed sheep had antibodies against Anaplasma spp./TBEV (n = 36), followed by Anaplasma spp./C. burnetii (n = 27) and Anaplasma spp./C. burnetii/TBEV (n = 2). Only one sheep showed an immune response against C. burnetii and TBEV. Flocks with sheep being positive against more than one pathogen were widely distributed throughout southern Germany. The descriptive analysis revealed no association between the antibody response of the three pathogens at animal level. Taking the flocks as a cluster variable into account, the exposure to TBEV reduced the probability of identifying C. burnetii antibodies in sheep significantly (odds ratio 0.46; 95% confidence interval 0.24–0.85), but the reason for this is unknown. The presence of Anaplasma spp. antibodies did not influence the detection of antibodies against C. burnetii and TBEV. Studies under controlled conditions are necessary to evaluate any possible adverse impact of co-exposure to tick-borne pathogens on sheep health. This can help to clarify rare disease patterns. Research in this field may also support the One Health approach due to the zoonotic potential of Anaplasma spp., C. burnetii and TBEV.
The intracellular bacteria Coxiella burnetii, Anaplasma phagocytophilum and Anaplasma ovis, and the tick-borne encephalitis virus (TBEV, Flaviviridae) are tick-transmitted pathogens and circulate in sheep flocks in the southern German federal states, Baden-Wuerttemberg (BW) and Bavaria (BAV) [1,2,3]. These pathogens also have a zoonotic potential and can cause illness in humans, such as flu-like symptoms and neurological disorders [4,5,6,7]. The main vector of A. phagocytophilum and TBEV is Ixodes ricinus and this tick species is widely distributed throughout Germany [4, 8, 9]. C. burnetii has also been found in I. ricinus , but Dermacentor marginatus is considered to transmit this pathogen to sheep [11, 12]. The existence of D. marginatus is limited to certain areas in southern Germany . Recently, A. ovis was identified in engorged D. marginatus from Bavarian sheep, but this does not prove its vector competence, and solid data about A. ovis vectors are still lacking . The clinical signs of these tick-borne pathogens are diverse in sheep. An infection with C. burnetii can result in reproductive disorders . Haemolytic anaemia is caused by A. ovis, whereas an A. phagocytophilum infection results in tick-borne fever . Moreover, A. phagocytophilum is an immunosuppressive agent and negatively affects the function of neutrophils, resulting in a higher susceptibility to secondary infections . TBEV infection seems to be asymptomatic, but neurological signs in sheep have been reported [2, 15]. Concurrent infections of the louping ill virus (LIV, Flaviviridae) with A. phagocytophilum promote the onset of severe LIV-associated neurological disorders . Furthermore, a dual infection of A. phagocytophilum and TBEV resulted in a significantly higher TBEV antibody response compared to a consecutive infection . However, knowledge of interaction among Anaplasma spp., C. burnetii and TBEV in sheep is lacking, but together they might promote and reinforce disease progression.
The current study aimed to identify to which extent grazing sheep had antibodies against Anaplasma spp., C. burnetii and TBEV. For this purpose, 1,406 serum samples from 36 sheep flocks located in BW and BAV were analysed to detect antibodies against the three tick-borne pathogens. Initially, the blood samples were collected for a Q fever study, and the number of specimens required from each flock to estimate the positivity rate was calculated on the assumption of 3% expected prevalence, 95% confidence interval, 80% power and 5% precision . A maximum of 44 animals per flock were sampled between November 2017 and June 2018. The blood sampling was performed in accordance with high ethical standards and approved by the federal state governments. The locations of the flocks are presented in Fig. 1.
Antibodies against Anaplasma spp., C. burnetii and TBEV were determined by three different commercial ELISAs in accordance with the manufacturers’ instructions and described in detail elsewhere [1, 17, 18]. An inhibition of ≥ 30% was assessed as positive for the Anaplasma spp. assay (Anaplasma Antibody Test Kit, cELISA v2, VMRD, Inc., Pullman, WA, USA), but this ELISA does not differentiate between antibodies against A. phagocytophilum and A. ovis. A sensitivity of 91.9% and a specificity of 86.9% were assumed according to Shabana et al. . A S/P (%) > 40 for the C. burnetii ELISA was considered positive (Q Fever Antibody Test Kit, IDEXX Switzerland AG, Liebefeld, Switzerland), in accordance with the sensitivity and specificity of 100% each stated by the manufacturer. Regarding the TBEV ELISA (Immunozym® FSME IgG all Species, PROGEN Biotechnik GmbH, Heidelberg, Germany), the manufacturer specified samples with > 126 Vienna Units (VIEU)/mL as positive; values between 63 and 126 VIEU/mL were classified as inconclusive. A sensitivity of 97% and a specificity of 99% were assumed in accordance with the product information. The inconclusive and positive samples were confirmed with a serum neutralisation assay as recently described, and antibody titres of ≥ 1:40 were counted as positive .
The test results and their agreement were evaluated in descriptive tables. To determine the true prevalence at animal and flock level, the apparent prevalence was corrected for misclassification probabilities (sensitivity and specificity of the diagnostic tests) using the Rogan-Gladen estimator . The prevalence of antibodies against more than one pathogen in the same individual or flock was also adjusted by correcting the test accuracies for parallel testing . In addition, the proportion of positive antibody results of the three pathogens at animal and flock level was compared by Fisher’s exact test. Subsequently, a logistic regression that considered the antibody result of one pathogen as the outcome and the other pathogen as the risk factor as well as the flocks as a cluster variable, was performed for the binary test results at animal level. The results of the two antibody tests were analysed in a logistic regression model to detect a significant association between pathogen exposure. Odds ratios were calculated to determine the strength and direction of a possible association. The association of the test results at flock level was analysed using Fisher’s exact test. A P-value of < 0.05 was considered significant. For all calculations, the statistical software SAS (SAS Institute Inc., Cary, NC, USA) was used.
There was a significant difference among the results of true seroprevalence between Anaplasma spp., C. burnetii and TBEV at animal level (p < 0.05). Most sheep had antibodies against Anaplasma spp. (47.2%), followed by TBEV (4.7%) and C. burnetii (3.7%). Significantly more flocks with Anaplasma spp. seropositive sheep were detected (n = 33; 91.7%) compared to flocks being seropositive for TBEV (n = 21; 58.3%) and C. burnetii (n = 15; 41.7%) (p < 0.05), but there was no significant different between flocks which contained TBEV and C. burnetii seropositive sheep. Seropositivity against at least two pathogens was detected in 66 (4.7%) sheep from 20 flocks (55.6%). The flocks with co-exposed sheep were widely distributed throughout southern Germany. Details of sheep and flocks with antibodies against more than one of the pathogens are presented in Table 1; Fig. 1, respectively. The descriptive analysis revealed no association of the three pathogens at animal level (Table 2). Taking the flocks as a cluster variable into account, there was a significant association between presence of C. burnetii and TBEV antibodies. The exposure to TBEV reduced the probability of identifying C. burnetii antibodies in sheep by half (Table 3). The presence of Anaplasma spp. antibodies did not influence the antibody detection of C. burnetii and TBEV. Based on current knowledge, A. ovis appeared only locally in one sheep flock from northern Bavaria, but this flock did not participate in the current study . Therefore, most Anaplasma spp. antibodies were possibly induced by A. phagocythophilum due to the wide dissemination in the German sheep population [3, 22, 23]. This was taken into account while interpreting the presented findings.
The significant differences among antibody rates of Anaplasma spp., TBEV and C. burnetii at sheep level might correlate with the presence of the pathogens in ticks collected in southern Germany. Up to 8.3% of questing I. ricinus contained A. phagocytophilum , whereas the detection rate of TBEV ranged from 0 to 5.3% , and C. burnetii has been determined only in one engorged D. marginatus so far [12, 26]. Information about natural co-exposure with Anaplasma spp., C. burnetii and TBEV in sheep is extremely rare . Significantly fewer sheep with C. burnetii antibodies were detected in flocks which also had sheep with antibodies against TBEV. Knowledge of interaction between both pathogens is missing, and we can only speculate about the possible reciprocal influence of both pathogens in sheep flocks. This observation needs further investigation in the future. The humoral immune response against Anaplasma spp., C. burnetii and TBEV lasts for several months in sheep [17, 28, 29]. Furthermore, co-infection with various pathogens were described for different tick species, but findings on the coincidental infection with Anaplasma spp., C. burnetii or TBEV are seldom in ticks [30,31,32]. Considering both these circumstances, we assume that natural exposure of sheep occurs through infestation of different ticks infected with one of the above-mentioned agents. In addition, infections with the pathogens might occur consecutively rather than at the same time. This is supported by previous findings of Paulsen et al. , who demonstrated that only a simultaneous infection of A. phagocytophilum and TBEV in lambs resulted in a significantly higher TBEV antibody response, but a consecutive infection had no influence on the antibody response of both pathogens. In the current study, the presence of Anaplasma spp. antibodies also did not influence the TBEV antibody detection at animal and flock level. Nevertheless, the number of lambs suffering from tick-borne encephalitis (TBE) seems to be on the increase in southern Germany , and the immunosuppressive impact of A. phagocytophilum should be investigated in lambs naturally infected with TBEV. Despite the fact that a detrimental influence was not confirmed after experimental infection with TBEV, the artificially infected lambs did not develop clinical signs of TBE . Therefore, the outbreak of TBE in sheep appears to depend on as yet unknown factors.
The authors are aware of the limitations of the present study. The antibody response against the three tick-borne pathogens is the result of a natural exposure in the field in the past. Therefore, it is impossible to determine the exact time of infection and the possibly related clinical impact. Moreover, we cannot rule out that false-seropositive sheep were included in the evaluation because of an estimated specificity of 86.9%  of the Anaplasma spp. ELISA, and a possibly reduced specificity of the C. burnetii ELISA . Only the inconclusive and positive results from the TBEV ELISA were confirmed by a serum neutralisation assay, which is considered as gold standard for TBEV antibody detection . This minimises the risk of sheep being tested false-seropositive for TBEV antibodies. All in all, the low numbers of co-exposures could also be the consequence of imperfect specificity of the diagnostic tests used. In the future, co-infection has to be determined by further tests such as molecular assays to detect pathogen DNA. Nevertheless, our findings contribute to the complex issue of tick-borne pathogens in sheep. Co-exposure to Anaplasma spp., C. burnetii and TBEV seems to be sporadic among grazing sheep flocks in southern Germany. The further spread of TBEV and the emerging onset of A. ovis in Germany might increase cases of co-infection [3, 4]. More targeted investigations are needed to evaluate an adverse impact of co-exposure to tick-borne pathogens on sheep health due to the fact that A. phagocytophilum influences the immune response and disease progression of concurrent flavivirus infections [15, 16]. In addition, sheep flocks can be implemented as sentinels to identify potential new risk areas of emerging zoonotic pathogens such as TBEV . Therefore, further research in this field may also support the One Health approach.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competitive enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay
Louping ill virus
- S/P (%):
Tick-borne encephalitis virus
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The authors would like to thank Annika Wolf for her excellent technical support and Frances Sherwood-Brock for proofreading the manuscript for correct English.
Open Access funding enabled and organized by Projekt DEAL. This study was supported by the Federal Ministry of Education and Research (BMBF) under Grant 01KI1726B and Grant 01KI1719 as part of the Zoonotic Infectious Diseases Research Network, and Lower Saxony State Office for Consumer Protection and Food Safety under Grant P54-34. This Open Access publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) − 491094227 “Open Access Publication Costs” and the University of Veterinary Medicine Hannover, Foundation.
Ethics approval and consent to participate
The study was conducted in accordance with German Animal Welfare Legislation and the EU Directive 2010/63/EU for animal experiments. Blood sample collection was approved by the federal state governments of Baden-Wuerttemberg (AZ 35-9185.82/0351, AZ 35-9185.82/D-18/01, AZ 35- 9185.82/A-1/18, AZ 35/9185.82/Ganter 18.01.2018) and Bavaria (RUF-55.2.2-2532-2-651-5, ROB-55.2- 2532.Vet_03-18-10). All animals were handled in accordance with high ethical standards and national legislation.
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Bauer, B.U., Runge, M., Schneider, M. et al. Co-exposure to Anaplasma spp., Coxiella burnetii and tick-borne encephalitis virus in sheep in southern Germany. Acta Vet Scand 65, 6 (2023). https://doi.org/10.1186/s13028-022-00659-6
- Anaplasma phagocytophilum
- Anaplasma ovis
- Dermacentor marginatus
- Ixodes ricinus
- Tick-borne encephalitis
- Tick-borne fever
- Q fever