Abstract

Background

In Europe and some parts of Asia, Tick-borne encephalitis (TBE) is a very important viral tick-borne zoonosis [1], and the TBE virus (TBEV), a flavivirus, occurs in so-called “natural foci”, whose size can range from large to very small. TBEV circulates especially between small mammals as local hosts and ticks as arthropod vectors. A large variety of mammalian species may become infected, and human TBE infections are notifiable in most endemic countries. Despite the reported human cases, symptomatic infections are also known (but very seldom) in dogs [2], horses [3], monkeys [4], and in single cases in sheep, mouflons and goats [5–7].

In addition, seroconversion without any specific clinical signs of a TBEV infection is described in some livestock animal species such as cattle, small ruminants and pigs [8–11]. In previous studies, TBEV-specific antibody titers in both goats and sheep were detected for up to 28months after immunization, and cross reactivity against other related pathogens was very low [12]. Furthermore, grazing animals have been shown to be suitable sentinels for the detection of TBEV foci by serological surveillance [9].

Especially the infection of ruminants is relevant for public health: in goats and sheep, and more rarely also in cattle, TBEV can be excreted via milk for several days after infection. Viraemia was observed between one and 5 days, TBEV was excreted via milk some days longer for two to 7 days in subcutaneously infected sheep [13], in subcutaneously infected cows TBEV could be detected for two to 4 days after infection in the blood and for three to 5 days in the milk [14]. After infection of a goat reported by van Tongeren [15], TBEV was detected in the blood from one to 5 days, from three to 8 days in the milk. Nearly the same results for sheep and a cow were reported by Grešíková and Rehacek after infection via tick bite [16]. Balogh et al. [17] detected TBEV in milk between eight and up to 19days after experimental infection of goats. In the last 20years, human cases of alimentary TBE have been reported from several countries including Hungary, Austria, Estonia, Slovakia [18–21] and very recently also in Germany [22].

The TBEV antibody status of a flock can help evaluate a possible risk of alimentary TBE caused by raw milk from this flock. In this study, (i) 50 goat flocks with 735 animals from the federal states of Bavaria (BY), Baden-Württemberg (BW), Lower Saxony (LS) and Mecklenburg-West Pomerania (MV) were evaluated with regard to TBEV antibodies to estimate the risk of TBEV infection in animals that can cause TBEV excretion via milk for several days.

As described above, knowledge about clinical symptoms, immune response and cross reactivity and longevity of antibody response as well as about the epidemiology of TBEV in animals has increased over the last years. However, some basic questions still remain unanswered or have been incompletely addressed. One relevant parameter is e.g. the longevity of TBEV-specific immunity. In our study, (ii) longevity was observed for more than 6 years. Furthermore, it is unknown whether contact to TBEV causes a long-lasting immunological memory that is reactivated quickly by any further contact to TBEV. Therefore, (iii) this possible recall of immunological information was tested by an additional immunization 5 years after the first TBEV contact.

Results

TBEV-antibody positive samples in the selected goat flocks

In the period from 2013 to 2015, we investigated 735 plasma samples from 50 goat flocks from four different federal states in Germany. The sample panel for the entire three-year period was available only from BY (see Table 1). The serological results are as follows: for MV from overall 205 samples in nine flocks only one goat was positive with a very low TBEV-titer (ND₅₀ 1:10). A similarly low titer (ND₅₀ 1:30) only was detectedin one goat from LS with overall 126 animals from nine flocks investigated and in one goat from BY (ND₅₀ 1:30) with overall 230 animals from 21 flocks tested over 3 years. In contrast, the results from BW: in 2013, three goats out of 69 animals from a total of three herds were tested positive, with two goats from the same herd showing significantly higher titers (ND₅₀ 1:160 and 1:320). The remaining goat had a titer of ND₅₀ 1:20. Similar results could also be seen for 2015 in BW, where a total of 105 samples from eight herds were tested. Here, four positive goats from one herd were determined, which also had very high TBEV titers (ND₅₀ 1:120, 1:120, 1:160 and 1:480), see Table ​Table11.

Table 1

Origin and serological results of the goat samples

	2013	2014	2015	total
MV flocks	9	–	–	9
MV samples	205	–	–	205
MV TBEV pos.	1	–	–	1
MV SNT titer (ND50)	1:10
LS flocks	2	7	–	9
LS samples	77	49	–	126
LS TBEV pos.	1	0	–	1
LS SNT titer (ND50)	1:30
BW flocks	3	–	8	11
BW samples	69	–	105	174
BW TBEV pos.	3a	–	4b	7
BW SNT titer (ND50)	1:20, 1:160, 1:320		2×1:120, 1:160, 1:480
BY flocks	12	8	1	21
BY samples	115	105	10	230
BY TBEV pos.	0	1	0	1
BY SNT titer (ND50)		1:30

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^atwo positive samples from the same flock

^ball four positive samples from the same flock

MV Mecklenburg-West Pomerania, LS Lower Saxony, BW Baden-Württemberg, BY Bavaria

Before starting, positive sera and plasmas from goats were tested for their suitability by TBEV-VNT. No deviations between sera and plasmas were seen, and both starting materials could be used equally well (data not shown).

Longevity of TBEV-specific antibody titers in immunized animals

In the previous study, titers increased in the vaccinated sheep and goats until 18weeks after the first immunization and decreased over the following weeks to reach lower but still positive titers 28months after vaccination [12]. Here in the following months, the titers further decreased but were detectable in goats for the whole observation period (6 years and 10 months). Overall, one sheep was tested positive up to 3 years and 6 months, the other one up to 4 years and 7 months.

TBEV antibody titers of these four multiple vaccinated animals and of four additional single vaccinated animals without previous contact to TBEV are shown in Table 2. VNT titers detected in sera of the animals immunized years before (multiple vaccinated) after one, two and 3 months ranged between 1:5 and 1:160. Generally, VNT titers in goat sera were higher when compared to titers detected in the sheep sera. In addition, the two goat sera revealed reactive TBEV antibody titers already 1 week after booster immunization. In contrast, three of the four naïve animals developed only low antibody titers (up to 1:15) after immunization, while one sheep did not display a positive TBEV antibody titer at all.

Table 2

Boost of TBEV-antibody titers (VNT) in pre-immunized goats and sheep^a

time point after last immunization	multiple vaccinated				single vaccinated
	goat 1	goat 2	sheep 1	sheep 2	goat 3	goat 4	sheep 3	sheep 4
1week	1:40	1:10	<1:5	<1:5	<1:5	<1:5	<1:5	<1:5
1month	1:113	1:80	1:160	1:160	1:15	1:7.5	1:10	<1:5
2months	1:80	1:28.2	1:15	1:160	1:15	<1:5	<1:5	<1:5
3months	1:28.2	1:80	1:5	1:28.2	1:7,5	1:5	<1:5	<1:5

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^aVNT titers of all animals <1:5 before immunization

All results were obtained either with blood samples collected for other usages (see ethics statement) or with only eight animals kept in one flock at the FLI. At present animal samples cannot be replaced completely for these investigations. In the study the number of animals was minimized and all procedures (only subcutaneous immunization and collection of blood samples) were refined.

Discussion

Our field study for the evaluation of goat flocks in Germany showed a TBEV seropositivity in single animal samples only. Most of the identified TBEV-reactive goats were found in Baden-Württemberg (11 flocks with 174 tested animals) with eight TBEV antibody-positive sera (4.6%). In Bavaria, Mecklenburg-West Pomerania and Lower Saxony only a single TBEV antibody positive sample could be detected (0.43, 0.49 and 0.78% of all tested samples, respectively). These results clearly show that even in grazing animals in the selected regions, contact to TBEV via tick bites is seldom and therefore the general risk of alimentary TBEV infection for humans is low. However, within a spatial TBEV natural focus the local risk of an alimentary TBEV infection can be remarkably higher. Since these foci can be very small, it is possible that only one ruminant flock is involved and that this particular flock has a high risk of TBEV infections. One single flock in Baden-Württemberg exhibited e.g. four of the eight TBEV antibody-positive samples. This is in accordance with our own findings in a previous study with 3590 sera from sheep and 3793 sera from goats in 2003 and 2006–2009 in seven federal states of Germany with in general low seroprevalences for the whole state. However, in single flocks in the same village seroprevalences between 0 and 43% were seen which confirmed the patchy pattern of TBEV [9]. Nearly the same differences between 2% (Mureş County) and 27.73% (Bihor County) seropositive sheep were found in Romania with a higher prevalence than in our investigated samples of 15.2% of all tested 519 samples in Romania [23]. A low overall seroprevalence was seen in Lithuanian domestic ruminants of 1.7% in eight out of 18 checked regions with high regional differences. Sheep from one region had the highest rate of 16% [24].

In a TBEV-positive case, it is highly recommended to test all milk and milk products from the affected flock for TBEV and to conduct further epidemiological investigations including anamnestic information about the purchase of animals from other regions with a known TBEV risk. Unfortunately, such information was not available for this study. A nationwide database of small ruminants, equally structured as for bovines, could help in this context to provide more background information for the veterinary authorities. A clearly worthwhile recommendation with regard to such a flock is to sell only pasteurized milk and milk products to avoid any cases of alimentary TBEV.

We could also show that reactive antibodies against TBEV last very long after immunization, and immunized goats displayed a positive reaction for nearly 7 years. In sheep, this time span was considerably shorter (3 years and 6 months and 4 years and 7 months). In our study highest titers were seen around 13weeks after the first dose (all together four doses) of a vaccine produced for humans and based on TBEV strain Neudoerfl [11]. With three doses of a new vaccine candidate for veterinary use by Salát et al. [25] the highest titers were observed around 42days after first immunization with higher VNT titers like in our study [11]. This vaccine based on TBEV strain Hypr and may cause even better immunity in animals like our vaccine for human use. This should be tested in further investigations.

Interestingly, even after years the small ruminants exhibited a clear immunological recall reaction as demonstrated by the increasing antibody titers in response to administration of one dose of vaccine. This contrasted very well the sero-reactions detected in the four naïve control animals with low or no antibody response after immunization with a single dose of the vaccine.

In this study, the progress of TBEV antibody titers reflects the situation after immunization with an inactivated vaccine, however natural infection may result in relatively similar humoral immune responses, as already shown for horses and dogs [2, 3]. However, the duration of immunity could be different between booster-vaccinated animals and animals after natural TBEV-infection. A fact which should be also taken into consideration.

Conclusions

TBEV-specific antibodies in grazing animals are not widespread, however, in some single flocks a remarkable number of seropositive animals indicate contact of the flock to TBEV. Consequently, a high risk of alimentary TBEV from non-pasteurized milk and milk products must be assumed in these flocks. In these cases epidemiological investigations like testing ticks from the meadows used by the flock for TBEV-RNA, avoiding meadows with TBEV-positive ticks and strict pasteurization of the milk before consumption are highly recommended.

We could also show that immunized goats and sheep had long-lasting TBEV-specific antibody titers, in our study between 3 years and 6 months and 6 years and 10 months. A boost immunization years after the first immunization demonstrated efficient immunological recall reactions.

Finally, it can be concluded that for organic farms selling fresh milk and milk products to private consumers the immunization of milking goats and sheep also could be an alternative to minimize the risk of an alimentary TBEV-infection.

Methods

Acknowledgements

Abbreviations

Authors’ contributions

CK collected sera from immunized animals, evaluated and interpreted data of TBEV, wrote the manuscript; UZ carried out the VNT-TBEV with the goat flock samples, evaluated and interpreted these data and wrote this part of the manuscript; DH developed and carried out VNT; FP and CF collected and prepared the goat plasma from the first project part, evaluated the epidemiological data; MB designed the study and evaluated data; CK, UZ, DH and MB drafted the manuscript. All authors revised the manuscript critically and approved the final version.

Funding

This work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE), grant number 313–06.01-28-1-91-049-15 and funded by the German Center for Infection Research (DZIF) Project Number TTU 01.801. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Blood samples from the free range goat flocks were derived from either obligatory monitoring schemes for other diseases (“Tierseuchenkassen” and “Tiergesundheitsdienste” of the federal states) or from approved research projects of the University of Veterinary Medicine (TiHo) Hannover and the Friedrich-Loeffler-Institut (FLI) (e.g. EU project “EMIDA ERA-NET project Goat-TSE-Free”, project number 2811ERA151). In the case that animal experiments were applied in the original studies, these were approved by the competent authority of the Federal State of Mecklenburg-West Pomerania, Germany, State Institution for Agriculture, Food Safety and Fishing (LALLF 7221.3–2-045/14), on the basis of national and European legislation, namely directive 2010/63/EU on the protection of animals used for scientific purposes. Sample collection was performed under the official supervision of the Federal Ministry of Food and Agriculture (BMEL) and the individual federal states under statement number TV-20140512.

Animal immunization and blood sample collectionwere approved by the competent authority of the Federal State of Thuringia, Germany, Thuringeian State Institution for Food Safety and Consumer Protection (TLLV 04–104/10; BFI-17-102), Bad Langensalza. Special consent of participation was not necessary because these animals were owned by the FLI for the whole study period.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Footnotes

Contributor Information

Christine Klaus, Email: [email protected].

Ute Ziegler, Email: [email protected].

Donata Hoffmann, Email: [email protected].

Franziska Press, Email: [email protected].

Christine Fast, Email: [email protected].

Martin Beer, Email: [email protected].

References