Investigations on the vaccination against the Bluetongue Virus serotypes 4 and 8
in sheep
Johanna Christine Hilke
Hannover 2019
University of Veterinary Medicine Hannover
Investigations on the vaccination against the Bluetongue Virus serotypes 4 and 8
in sheep
Inaugural-Dissertation to obtain the academic degree Doctor medicinae veterinariae
(Dr. med. vet.)
submitted by
Johanna Christine Hilke Starnberg
Hannover 2019
Academic supervision: Prof. Dr. med. vet. Martin Ganter DipECSRHM Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Service
University of Veterinary Medicine Hannover, Foundation
1. Referee: Prof. Dr. med. vet. Martin Ganter
2. Referee: Prof. Dr. med. vet. Martin H. Groschup
Day of the oral examination: 30th October 2019
The publication of the manuscript „Presence of Antibodies against Bluetongue Virus (BTV) in Sheep 5 to 7.5 Years after Vaccination with Inactivated BTV-8 Vaccines” was supported by Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation within the funding programme Open Access Publishing.
To the shepherds.
Parts of this work have already been published in the following journals:
Presence of Antibodies against Bluetongue Virus (BTV) in Sheep 5 to 7.5 Years after Vaccination with Inactivated BTV-8 Vaccines
Hilke, Strobel, Woelke, Stoeter, Voigt, Moeller, Bastian, Ganter Viruses 2019, 11, 533; doi: 10.3390/v11060533
A Comparison of Different Vaccination Schemes Used in Sheep Combining Inactivated Bluetongue Vaccines Against Serotypes 4 and 8
Hilke, Strobel, Woelke, Stoeter, Voigt, Grimm, Meilwes, Punsmann, Blaha, Salditt, Rohn, Bastian, Ganter
Vaccine 2019, 37, 5844–5853; doi: 10.1016/j.vaccine.2019.08.011
Furthermore, the following parts have already been published:
Blauzungenkrankheit – es bleibt spannend!
J. Hilke
Oral presentation
5. Triesdorfer Schafgesundheitstag 2018, 15.03.2018, Triesdorf, Germany
Blauzungenkrankheit- Untersuchungen zur Impfung gegen Serotyp 4 und 8 beim Schaf
J. Hilke, H. Strobel, M. Bastian, M. Stöter, K. Voigt, H. Axt, U. Domes, D. Spengler, I.
Blaha, A. Salditt, M. Ganter Oral presentation
Tagung der DVG Fachgruppe „Krankheiten kleiner Wiederkäuer“, 13.-15.06.2018, Landquart, Switzerland
Antibody Persistence in Sheep 5- 7.5 Years after Vaccination with Inactivated Bluetongue Virus Serotype 8 Vaccines
J. Hilke, H. Strobel, S. Woelke, M. Stoeter, K. Voigt, M. Bastian, M. Ganter Poster
Ontario Small Ruminant Veterinary Conference, 17.-19.6.2019, Guelph, Canada
TABLE OF CONTENT
Introduction ... 1
Manuscript I ... 6
Manuscript II ...15
General discussion ...26
Summary ...32
Zusammenfassung ...34
References ...37
Acknowledgements ...47
List of abbreviations
% percentage
< less than
> greater than
≤ less or equal
≥ greater or equal
°C degree centigrade µl microlitre
Ab antibodies
afv after first vaccination
BT Bluetongue
BTV Bluetongue Virus CTL cytotoxic t lymphocytes ds double-stranded
e. g. exempli gratia
EFSA European Food Safety Agency
ELISA enzyme-linked immunosorbent assay FLI Friedrich-Loeffler-Institute
logELISA log transformed ELISA results MLV modified live virus
nAb neutralising antibodies PCR polymerase chain reaction PN percentage negativity RNA ribonucleic acid
SNT serum neutralisation test Th2 cells t helper two cells
VP structural viral protein
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Introduction
Bluetongue (BT) is a notifiable disease of ruminants caused by the Bluetongue Virus (BTV). It is an arthropod-borne virus (genus Orbivirus within the family Reoviridae) with currently 28 characterised serotypes (Verwoerd et al., 1970; Belbis et al., 2017;
Bumbarov et al., 2019) and several putative new serotypes (Wright, 2014; Sun et al., 2016; Savini et al., 2017; Lorusso et al., 2018; Marcacci et al., 2018).
The virus consists of 10 segments double-stranded (ds) RNA encoding for seven structural proteins (VP) and four to five non-structural proteins (Huismans and Van Dijk, 1990; Roy, 2008; Hardy et al., 2015). The outer capsid is composed of VP2 and VP5, with VP2 being the major and VP5 the minor determinant of BTV serotypes (Mertens et al., 1989). Both proteins are the most variable of the VPs. Significant variations were found in the genome of VP2 between strains of the same serotype, allowing a classification according to their origin into eastern and western topotypes (Maan et al., 2007). Essentially, VP2 is responsible for eliciting serotype-specific neutralising antibodies (nAb), which are detected by serum neutralisation tests (SNT). The intermediate layer consists of VP7. The inner subcore is formed by VP3, which houses VP1, VP4, VP6 and the dsRNA (Roy, 2008). VP7 is the immuno- dominant antigen, which allows BTV identification in polymerase chain reaction (PCR) (Anthony et al., 2007) and induces BTV-specific non-nAb when infecting animals, which are used for serotype unspecific serological diagnostics by enzyme- linked immunosorbent assays (ELISA) (Mertens et al., 2009).
The virus is primarily transmitted by hematophagous Culicoides spp. midges (Du Toit, 1944; Wilson and Mellor, 2009). Furthermore there is evidence for at least some strains or serotypes being transmitted transplacentally (De Clercq et al., 2008; Backx et al., 2009; Spedicato et al., 2019), horizontally (Batten et al., 2014; Bréard et al., 2018), iatrogen (Darpel et al., 2016) or orally (Menzies et al., 2008; Backx et al., 2009; Mayo et al., 2010).
Sheep farmers in South Africa had already known BT in the beginning of the 19th century, presumably after the first introduction of Merino sheep (Erasmus and
Potgieter, 2009). The first scientific descriptions were given by Hutcheon in 1880 and 1902 (Verwoerd, 2012; Chand, 2015) and Spreull in 1905 (Spreull, 1905).
Historically, BT was seen as an African disease until its spread since the middle of the 20th century to all continents except Antarctica. Subsequently, the disease was considered as distributed enzootically in tropical, subtropical and Mediterranean areas, limited by the geographical distribution of the different vector’s habitats, which form distinct episystems (Tabachnick, 2004, 2010; Erasmus and Potgieter, 2009;
Maclachlan, 2011; Ganter, 2014). Due to multiple reasons, e. g. climate change (Purse et al., 2005), mutations in the virus’ genome (Coetzee et al., 2012) and global trade (Mintiens et al., 2008), new Culicoides subspecies became vector-competent and hence enlarging the geographic occurrence and possible enzootic area of BT (Meiswinkel et al., 2007; Hoffmann et al., 2009; Foxi et al., 2016).
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The virus causes asymptomatic, severe and even fatal disease in ruminants, depending on the strain and species involved. Sheep are considered the most susceptible species, whereas cattle, goats, South American camelids and wild ruminants are classically seen as subclinical infected virus reservoir (Nevill, 1978;
Maclachlan and Gard, 2009; Falconi et al., 2011; Schulz et al., 2012).
Insect vectors become infected when feeding blood on BTV viraemic ruminants. The virus infects midgut cells first, disseminates throughout the insect body, reaching and replicating in the salivary glands. This process takes 7 to 9 days at 25°C and is enhanced at higher temperatures and ceases at degrees below 10-15°C (Mellor et al., 2009; Carpenter et al., 2011). If at this time, the infected insect bites the host, midge saliva, together with infectious BTV will be deposited subdermally. It has been shown, that a single bite is capable to reliably infect a susceptible ruminant (Baylis et al., 2008). An infected midge remains infectious for its entire life. That is to say an adult midge lives at moderate temperatures for 10-20 days, shortened at higher temperatures and prolonged at colder temperatures with a maximum of 3 months.
Several other factors e.g. humidity, wind and rainfall affect the survival of the adults (Mellor et al., 2000; Goffredo et al., 2004; Wilson et al., 2008; Purse et al., 2015). So far there is no evidence for transovarial virus transmission within the investigated Culicoides species (Nunamaker et al., 1990; Fu et al., 1999; White, 2005; Mayo et al., 2014; Osborne et al., 2015).
After the insect’s bite, primary infection comprises dermal fibroblasts, mononuclear phagocytes, dendritic cells, lymphocytes and endothelial cells (Maclachlan and Gard, 2009; Darpel et al., 2012). Then BTV reaches the regional lymph nodes draining the site of BTV inoculation, first replication occurs there in phagocytes and dendritic cells (Hemati et al., 2009). Afterwards BTV disseminates via blood and/or lymph and replicates in endothelial cells and mononuclear phagocytes of different organs and finally leads to generalized viraemia (Barratt-Boyes and MacLachlan, 1994; Darpel et al., 2009).
The virus associates with all blood cells and is able to persist within invaginations of the erythrocyte cell membrane (Maclachlan and Gard, 2009). Apparently it is hereby protected from immune clearance, thus leading to a prolonged cell-associated
viraemia for up to 35-60 days (Barratt-Boyes and MacLachlan, 1994). Non-infectious viral structures have been proven for up to 222 days after infection (Bonneau et al., 2002). Their role as well as the role of persistently infected γδ T-cells (Takamatsu, 2003) and the contribution of the skin as a possible major site of replication (Darpel et al., 2012) is not yet fully understood.
The main pathogenic mechanism of BTV are injuries to microvasculature in target tissue. This is caused by direct damage resulting from virus-induced apoptosis and necrosis as well from vasoactive mediators produced by thrombocytes, dendritic cells, macrophages and infected endothelial cells, which result in increased vascular permeability. Furthermore, BTV infection evokes the production of pro-inflammatory
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mediators by dendritic cells and macrophages, leading to haemorrhagic fever-like syndrome (Schwartz-Cornil et al., 2008; Rao et al., 2017).
Clinical signs may include one or more of the following symptoms: hyperaemia, fever, mucosal erosions, ulcers and/or haemorrhage of the face and oral cavity, nasal
discharge, salivation and lacrimation, facial oedema, lameness caused by coronitis and respiratory distress (Spreull, 1905; Maclachlan and Gard, 2009; Rao et al., 2017). If infection develops in a chronical way, animals show extreme weakness, fatigue and torticollis, as well as effects of high fever, such as temporary infertility and wool-break or -loss (Parsonson, 1990; Maclachlan et al., 2009). Infection during early pregnancy may cause abortion, fetal death or birth of malformed and/or
underdeveloped neonates (Maclachlan and Osburn, 2017). The severity of the symptoms is affected by several virus, host and environmental factors (Caporale et al., 2014; Coetzee and Venter, 2015; Noaman and Arzani, 2017).
BTV is known to generate only low to no cross-protection between serotypes (Schwartz-Cornil et al., 2008). Inactivated vaccines are commonly thought to
stimulate an immune response dominated by CD4+ Th2 cells which in their turn act as stimulators for Ab-producing B cells (Tizard, 2013). Umeshappa (Umeshappa et al., 2010) proved stimulation of the cellular immune response after vaccination with inactivated BTV. Therefore both parts of the immune system are assumed to
participate in the protective effect of vaccination with inactivated BTV. On the cellular side of the immune reaction, cytotoxic T-cells (CTLs) are assumed to be involved in cross-serotype protection (Jeggo et al., 1985; Umeshappa et al., 2010; Wäckerlin et al., 2010; Breard et al., 2015a). On the humoral side of the immune reaction, pan- BTV species-specific, non-neutralising Abs against VP7 are detected by ELISA, but their role in protective immunity is unclear (Darpel et al., 2009). Additionally,
serotype-specific nAb are directed against VP2 and to a smaller extent against VP5 (Schwartz-Cornil et al., 2008).
In 2006, BTV serotype 8 emerged in Central Europe, including Germany. The route of introduction is still unknown (Mintiens et al., 2008). The virus was able to spread rapidly through northern Europe, because indigenous Culicoides species of northern Europe, which until this date were not known to act as efficient vectors for BTV, became vector-competent (Mehlhorn et al., 2009). Furthermore, despite any expectations, the virus was able to overwinter in the northern hemisphere, the mechanism is still unknown (Takamatsu, 2003; Wilson et al., 2008). This epidemic showed two novelties: For the first time cattle were seriously clinically affected (Darpel et al., 2007; Elbers et al., 2008b, 2008a; Conraths et al., 2009). And in addition, transplacental infection by a BT field virus was proven for the first time (De Clercq et al., 2008). Until that date this route of infection was attributed to virus strains adapted on cells or passaged into embryonated chicken eggs like e. g.
modified-live-virus (MLV) vaccine strains (Coetzee et al., 2012; Rasmussen et al., 2013; Savini et al., 2014; Spedicato et al., 2019).
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Because of considerable economic impact due to the morbidity and mortality of livestock as well as movement restrictions (Gethmann et al., 2015), Europe opted for vaccinations as control strategy. MLV-vaccines widely used in South Africa, generate a reliable immunogenic protection after only one vaccination. In areas with no
enzootic BTV they generate several adverse effects like clinical signs of BT and most importantly, viraemia after vaccinations with the risk of natural transmission of MLV strains and novel reassortment (Savini et al., 2008; Coetzee et al., 2014). Out of these reasons, the use of inactivated vaccines was preferred and a vaccination trial for licensing three inactivated vaccines was conducted in Germany. As these proved to be highly efficient and safe (Eschbaumer et al., 2009; Gethmann et al., 2009;
Wäckerlin et al., 2010), the vaccines were initially provisionally licensed and later received a central marketing authorization by the European Medicines Agency.
According to the manufacturer’s instructions, all the vaccines confer immunity for the duration of one year. Following commercial availability of these vaccines, vaccination became mandatory for all domesticated ruminants in 2008 and 2009, followed by voluntary vaccinations from 2010 to 2011. After that time, vaccinations became prohibited and in 2012 Germany was declared free from BTV-8 (Baetza, 2014), like most of the other Northern European countries.
But various serotypes continued to be active in Southern Europe, posing a persistent risk for virus re-introduction.
In September 2015, serotype 8 re-emerged in France after five years of BTV-8 free status (Bréard et al., 2016; Bournez et al., 2018; Sailleau et al., 2018b). Almost simultaneously, the BTV-4 outbreak starting 2014 in Greece and spreading rapidly over the Balkan Peninsula, reached Austria in November 2015 (Anonymous, 2018a).
Due to the geographical proximity to these new outbreaks, the risk of BTV being introduced to Germany was considered probable to high (Mettenleiter, 2015). At this particular time, it was not predictable how clinical disease would appear when animals get infected with co-circulating BTV-4 and BTV-8. Because the rather rapid control of the BTV-8 epidemic in 2006 was attributed to vaccination campaigns (Baetza, 2014), the German Standing Committee on Veterinary Vaccinations
recommended the vaccination of susceptible animals (Bastian, et al., 2016). With the amendment of the German Bluetongue Regulation in 2016 (Anonymous, 2018b), voluntary vaccinations for BTV-4 and -8 using inactivated vaccines were no longer forbidden.
No inactivated bivalent vaccine against BTV-4 and BTV-8 was available in 2015/16.
Vaccination therefore relied on a combination of two monovalent vaccines for these two serotypes. The presence of different BTV serotypes in the same geographic area resulted in simultaneous vaccinations with inactivated monovalent BTV vaccines in different European countries, e.g. BTV-2 and BTV-4 in the Mediterranean Islands, BTV-1 and BTV-8 in France as well as BTV-1 and BTV-4 in Spain, Corsica and Italy.
But no scientific investigation on how to combine two of these vaccines to achieve
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optimal protection has been reported so far. Therefore, no reasonable vaccination recommendation could be stated.
In addition, several studies have shown the presence of BTV Ab after vaccination with inactivated vaccines in cattle for up to eight years (Oura et al., 2012; Batten et al., 2013; Ayrle et al., 2018; Ries et al., 2019) and in sheep for up to two and a half years (Batten et al., 2013). It is postulated that the success of the short mandatory vaccination campaign 2008-2009 might have been supported by the long duration of protective antibodies after infection and especially after vaccination with inactivated BTV vaccines (Eschbaumer et al., 2012; Oura et al., 2012). The question arose whether there would still be detectable antibodies from the previous vaccination campaign after five to seven and a half years in the blood of older sheep. These findings were considered as valuable for further vaccination recommendations as well.
Therefore the aims of this cumulative thesis were the following:
A) The possible detection of antibodies after five to seven and a half years in older animals which have been vaccinated with inactivated BTV-8 vaccine during the vaccination campaign 2008-2011.
B) A comparison of different vaccination schemes and techniques using four commercially available inactivated vaccines against BTV-4 and BTV-8 from different companies to find the most effective immunization scheme for sheep under field conditions with respect to safety, immunogenicity and saving in time.
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Manuscript I
Presence of Antibodies against Bluetongue Virus (BTV) in Sheep 5 to 7.5 Years after Vaccination with Inactivated BTV-8 Vaccines
Johanna Hilke 1,2*, Heinz Strobel 1, Soeren Woelke 3, Melanie Stoeter 2, Katja Voigt 4, Bernd Moeller 5, Max Bastian 3 and Martin Ganter 2
1 Sheep Veterinary Practice Strobel, Am Hopfenberg 8, 89352 Stoffenried, Germany; drheinzstrobel@t- online.de
2 Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany; Melanie.Stoeter@tiho- hannover.de (M.S.); Martin.Ganter@tiho-hannover.de (M.G.)
3 Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Suedufer 10, 17493 Greifswald – Riems, Germany; soeren.woelke@fli.de (S.W.); Max.Bastian@fli.de (M.B.)
4 Clinic for Ruminants with Ambulatory and Herd Health Services, Ludwig-Maximilians-University Munich, Sonnenstr. 16, 85764 Oberschleissheim, Germany; k.voigt@med.vetmed.uni-muenchen.de
5 Friedrich-Loeffler-Institute, Institute of Farm Animal Genetics, Hoeltystr. 10, 31535 Neustadt, Germany;
Bernd.Moeller@fli.bund.de
* Correspondence: johanna.hilke@tiho-hannover.de;
Viruses 2019, 11, 533; doi: 10.3390/v11060533 www.mdpi.com/journal/viruses
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Manuscript II
A Comparison of Different Vaccination Schemes Used in Sheep Combining Inactivated Bluetongue Vaccines against Serotypes 4 and 8
Johanna Hilke 1,2*, Heinz Strobel 1, Soeren Woelke 3, Melanie Stoeter 2, Katja Voigt 4, Lucie Grimm 2, Johanna Meilwes 2, Teresa Punsmann 2, Irena Blaha 5, Andreas Salditt 5, Karl Rohn 6, Max Bastian 3 and Martin Ganter 2
1 Sheep Veterinary Practice Strobel, Am Hopfenberg 8, 89352 Stoffenried, Germany
2 Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany
3 Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Suedufer 10, 17493 Greifswald – Riems, Germany
4 Clinic for Ruminants with Ambulatory and Herd Health Services, Ludwig-Maximilians-University Munich, Sonnenstr. 16, 85764 Oberschleissheim, Germany
5 State Veterinary Investigation Centre Aulendorf, Loewenbreitestr. 20, 88326 Aulendorf, Germany
6 Institute for Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Foundation, Buenteweg 2, 30559 Hannover, Germany
* Correspondence: johanna.hilke@tiho-hannover.de;
Vaccine 2019, 37, 5844-5853; doi: 10.1016/j.vaccine.2019.08.011
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General discussion
The aim of this cumulative thesis was to investigate on the one hand whether vaccinations with inactivated BTV-8 vaccines induce antibodies in sheep still detectable after five to seven and a half years. On the other hand, different vaccination schemes and techniques in sheep were compared using four
commercially available inactivated vaccines against BTV-4 and BTV-8 from different companies. Under field conditions, the measurable immunogenicity was compared with respect to safety and saving in time. The findings are supposed to contribute to the development of a reasonable vaccination recommendation.
According to our analyses, the majority of sheep vaccinated annually for at least two consecutive years were BTV seropositive five years after their last vaccination with an inactivated BTV-8 vaccine (14 of 18 sheep). The majority of animals which had received less than two vaccinations showed ELISA or SNT negative results (15 of 18 sheep) five to seven and a half years after the last flock vaccination. However, seven and a half years after only one vaccination, two of five animals showed a positive ELISA and another animal (1 of 5) tested positive for BTV-8 nAbs. Although our study included only a small number of animals, we were able to prove a longer duration of the (n)Ab than expected and presumed by the vaccine manufacturers.
This supports previous studies in cattle and sheep (Oura et al., 2012; Batten et al., 2013; Ayrle et al., 2018; Ries et al., 2019) and suggests to re-evaluate the current vaccination schemes.
All combinations of two inactivated BTV vaccines were well tolerated by the sheep in our study (n=240) with no clinical side effects other than a moderate increase in body temperature immediately after the initial and the booster vaccination, respectively.
This was to be expected according to previous findings (Emidio et al., 2004; Savini et al., 2007; Gethmann et al., 2009; Speiser et al., 2016). We did not observe any other adverse effects following simultaneous vaccinations due to the double amount of aluminium hydroxide as adjuvant which showed diverse side effects in previous studies (Gonzalez et al., 2010; Asín et al., 2018, 2019). This experience is supported by the tolerance of mass vaccinations in the field throughout the last four years in Southern Germany conducted by the author and combining concurrently two monovalent BTV-4 and -8 vaccines.
We could show that a one-shot vaccination induced comparable immunogenicity to a boosted injection with regard to serotype-specific nAb for the measured timeframe of half a year after first vaccination (afv) and with regard to ELISA results for the
measured timeframe of one year afv. The one-shot immunisation resulted in more heterogeneous ELISA-activities than boosted vaccination, supporting a previous study (Wäckerlin et al., 2010), but led to no significant differences between one-shot and boosted groups. We found no relevant significant differences in the measured immunogenic response between the vaccines applied simultaneously at different or at the same injection site. There was no advantage in administering the serotypes in a consecutive vaccination scheme. Moreover, these schemes showed lower
immunogenicity. Whether this effect has to be attributed to the scheme or other factors remains unclear within our study design.
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In summary, we did not find disadvantages in administering two of the tested four monovalent inactivated BTV vaccines as a one-shot application in combination with a twin syringe. This technique allows a time- and cost-effective vaccination of large animal numbers and is supposed thereby to enhance farmers’ compliance and participation in voluntary vaccination programmes.
However, there are different limitations within our study, which demand further research on the following issues.
The first and most fundamental limitation is the lack of a virus challenge, which is considered as the gold-standard to assess BTV vaccine efficacy (Savini et al., 2008), due to the complex immune response evoked by inactivated BTV vaccines, which is not yet fully understood. Due to the design as field trials, it was impossible to perform challenge experiments within our studies. Hence it remains unclear whether low serotype-specific nAb titres measured in setting 2 and 3 of the second study, would reach protective levels in case of infection and would prevent viraemia and clinical disease as already seen (Savini et al., 2007; Eschbaumer et al., 2009).
Secondly, both the humoral and cellular arms of the immune system are supposed to contribute to the protective effect of inactivated BTV vaccines (Lobato et al., 1997; Schwartz-Cornil et al., 2008; Umeshappa et al., 2010;
Wäckerlin et al., 2010). As we investigated only for BTV-group-specific (ELISA) and serotype-specific (SNT) Ab and not for cellular components, the efficacy of the vaccines might be better than assumed based on our results.
Nevertheless, there are numerous studies showing that nAbs are highly correlated with protection from disease (Jeggo et al., 1984; Lobato et al., 1997; Savini et al., 2008; Oura et al., 2009).
In consequence, the limited amount of SN tests represents a third limitation.
The immunogenic components measured by ELISA and SNT do not necessarily correspond to each other, also seen in two animals of our first study. Nevertheless, in previous challenge studies, individual animals were protected while showing ELISA positive and SNT negative results
(Eschbaumer et al., 2009), ELISA negative results (Wäckerlin et al., 2010) or even negative results in both tests (Stott et al., 1985; Breard et al., 2015a).
Therefore, it was assumed that at least the animals showing either ELISA or SNT positive results would be protected in case of infection. However, further research is needed to confirm this assumption by challenge studies.
Fourthly, as both studies were conducted under field conditions, there are several factors which may have influenced the efficacy of vaccination such as injection technique (angle of injection, needle length, anatomic site), adjuvants and differences between vaccine batches, animal’s body condition, potential pregnancy, health condition, breed, sex and age (Poirier, 1996). Most of these factors were documented in the second paper, but couldn’t be tracked back for five to seven and a half years in the first one. Consequently, differences in the measured immunogenicity could not be attributed solely to the vaccines and their application scheme and technique. On the other hand, as vaccination campaigns for controlling epidemics in animals are not operated in research
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facilities but in the field, our findings reflect the more realistic picture of vaccine efficacy.
Since the start of our studies in 2016, the BTV-8 epidemic has spread from France, but much slower and much less dramatic than expected. Due to the close genetic relationship between strains in 2008 and 2015, it is assumed that BTV-8 circulated at a low level in France since 2008 (Bréard et al., 2016). In accordance, EFSA
concluded after model simulations, that mass vaccinations with a coverage of 95% in susceptible ruminants for five consecutive years would be necessary, followed by sensitive surveillance systems, to eradicate BTV from Europe (EFSA Panel on Animal Health and Welfare, 2017), which was not the case in the 2006 epidemic.
Because of the unpredictable virus dynamics, there was not always sufficient inactivated vaccine available. Instead of mass vaccinations, French Veterinary Authorities established restrictions on livestock movements and vector-free periods were determined in accordance with the EU regulation (Commission Regulation (EC) No. 1266/2007) in 2015. Apparently, this was not a successful control strategy, as the virus spread throughout France and then was detected in two cattle in
Switzerland in October 2017. After another year it was proven in Germany in December 2018 and a few months later (February 2019) in Belgium (Anonymous, 2019a).
The BTV-4 epidemic did not spread further to the north than Austria and ceased there. But presumably the same virus continued to circulate in Italy, spreading from there via Corsica to the mainland of France in 2017 (Sailleau et al., 2018a, 2018b).
Case reports (for all serotypes) reached a maximum in October (with 452 and 935 reported cases in France and Italy, respectively) and in November 2017 (with 399 and 780 reported cases in France and Italy, respectively) (Anonymous, 2019b).
Fortunately, in most cases both serotypes did not induce severe clinical symptoms so that most infections were subclinical. Therefore, most of the reported cases were animals tested for export reasons and it has to be assumed that the real infection rate is much higher (Kundlacz et al., 2019). Whether the rather mild course can be attributed to protecting nAb from previous infection and vaccination (Eschbaumer et al., 2012; Bournez et al., 2018) or to virus alteration (Flannery et al., 2019) remains unclear. The latter assumption could be supported by the observation of a possible new clinical feature: Since December 2018, France reported increasingly cases of newborn calves being blind, weak and dying after a few days, similar to the
symptoms of Schmallenberg. But the examined calves were tested PCR-positive only for BTV. It is supposed that they got infected in-utero, as they were less than one week of age and born in the “vector-free” period (Zientara et al., 2019).
The compulsory vaccination programme against BTV-8 in 2008 in several European countries is seen as a very effective, but also very expensive control measure
(Gethmann et al., 2015a). Furthermore, in human medicine it is a proven fact, that compulsory vaccinations have an adverse effect on willingness to vaccinate (Betsch and Böhm, 2016). Main motivators for BTV-vaccination are obligatory regulations for export animals, prevention of production losses, subsidized vaccination and
recommendation by the veterinarian (Gethmann et al., 2015b; Kundlacz et al., 2019).
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The current European BTV control strategy involves establishment of restriction zones with regulations for trading, trade bans with bilateral exemptions and easing during so called “vector-free” periods (Anonymous, 2019c) and in some countries voluntary vaccinations with more or less subsidies and ongoing surveillance on a rather “insensitive” level as seen by the re-emergence of BTV-8 in France 2015.
There is doubt whether the establishment of vector free periods is a suitable control instrument: some of the indigenous midges seem to be highly adapted to cattle husbandry and may overwinter in cattle manure and stables (Zimmer et al., 2014;
Versteirt et al., 2017). Therefore, it is very likely that the circulating BTV-4 and BTV-8 epidemic will disperse further, which contributes to an areas-wide immunity on
population level. This is desirable as long as infections continue to provoke none to mild clinical symptoms, but it includes the risk of virus alteration. Although research has been increased, BTV evolution has not been thoroughly clarified. It is known that genetic drift and shift, selective sweep and founder effect contribute to the genetic diversity of BTV field strains (Bonneau et al., 2001; Carpi et al., 2010; Coetzee et al., 2012; Nomikou et al., 2015; Jacquot et al., 2019), but underlying selective
mechanisms are still to be investigated. These alterations could affect virulence and transmission potential as well (Coetzee et al., 2012). Consequently, it is
unpredictable to what extent a circulating BTV strain might become more virulent, pathogenic and/or vector competent especially when encountering an
immunologically naive ruminant population and when co-circulating with another serotype.
In the meantime, with regards to vaccination as a control measure, a bivalent inactivated vaccine for BTV-4 and BTV-8 has become available (Syvazul BTV®, laboratorios syva s.a.u., León, Spain). The results of the second study are nevertheless important, as it is not guaranteed that the bivalent vaccine will be available at any time and for an unpredictable amount of demand.
The results of the described studies propose that vaccinations with inactivated vaccines induce longer (n)Ab activities than expected and that one-shot
immunization provoke similar immunogenicity to boosted vaccination, even if not recommended by the manufacturer. We therefore propose to confirm these presumptions by long-term studies with more numerous animals and a virus challenge. If reassured, vaccination recommendations should be simplified
substantially to motivate more farmers (cattle and sheep) to vaccinate their livestock.
- One-shot vaccinations of the tested vaccines seem to induce Ab for up to six years in cattle (Ayrle et al., 2018) and for up to seven and a half years in sheep (Hilke et al., 2019b). If two monovalent vaccines are administered at different or the same injection site, Ab are present for a minimum of one year after vaccination (Hilke et al., 2019a).
- Boosted vaccination (either boosted after 3-4 weeks or in the following year) seem to induce Ab for a minimum of four to eight years in cattle (Batten et al., 2013; Bournez et al., 2018; Ries et al., 2019) and for at least up to five years in sheep (Batten et al., 2013; Hilke et al., 2019b).
- Within this period of time the dam most possibly passes Ab via colostrum to their offspring, as proved in cattle (Ayrle et al., 2018).
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- Colostral Ab after vaccination of the dams with inactivated vaccines are detected for a period of at least 84-112 days of age in calves (Vitour et al., 2011) and at least 14 weeks of age in lambs (Oura et al., 2010). After natural BTV infection and booster vaccination of the ewes, colostral Ab were present in their lambs for at least three months (Leemans et al., 2013).
Therefore
Initial whole-flock vaccination should include a booster vaccination either after 3 weeks or up to until one year after the initial vaccination.
Subsequently, vaccination of the replacements, using the same interval, could maintain sufficient flock protection.
Revaccination of the whole flock would depend on age distribution and the replacement rate. At an average replacement rate of 15.5% (10–22%) in Southern Germany (Frohnmayer, 2015), a vaccination coverage of
between 60% and 100% would be achieved in the sixth year. In flocks with less than 15% replacement rate, whole-flock revaccination might be
necessary after five years to ensure sufficient cover to stop virus spread, as well as viremia, and to avoid the occurrence of clinical symptoms.
Fattening lambs who are raised outside during the vector-active period would not be in need of vaccination, if they get slaughtered within 14 weeks after colostrum intake.
Such vaccination schemes could support a high vaccine coverage in the ruminant livestock population and thereby diminish the risk of virus alteration. Governmental or European funding or subsidies are rather suitable for increasing the acceptance of vaccinations than compulsory schemes (Gethmann et al., 2015b; Betsch and Böhm, 2016). The positive effect on vaccination rate in the sheep population of areas with subsidies for BTV vaccinations was observed by the author in Baden Wurttemberg, in contrast to the Bavaria, where no subsidies for BTV vaccinations were provided.
For sheep flocks, there are various vaccinations recommended (Ganter et al., 2018), depending on production system, location and contact with the public. The proposed vaccination scheme for BTV without the need of yearly flock vaccination would also provide logistical facilitation and diminution of possible adverse reactions (Gonzalez et al., 2010; Asín et al., 2019).
With the proposed vaccination scheme the recommended 95% coverage over a period of 5 years (EFSA Panel on Animal Health and Welfare, 2017) are within the realms of possibility. The success of this strategy nevertheless will rely on other factors as well, such as the ongoing climate change, which could contribute to an extended vector activity (Purse et al., 2015) and/or an intensified virus replication in the vector (Carpenter et al., 2011). In either case, control strategy has to include a sensitive monitoring to detect subclinical virus carriers, especially with regard to the circulation of an atypical BTV serotype in southern Germany (Ries, 2019).
However, the main interest should be a flock or population immunity, as it has to be taken into account that vaccination might not induce a measurable immune response in every individual animal as seen in three animals in our second study, due to
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immunogenic reasons (Rendi-Wagner et al., 2006; Tizard, 2013; Stoop et al., 2016) or failures in injections.
Focus should also be laid on any possible heterologous protection, following bivalent inactivated vaccinations, beyond the vaccinated serotypes – such effects have
already been shown by Breard et al. (Breard et al., 2015b). This is of particular interest because of the permanent risk of introducing new BTV serotypes to Europe.
For financial, trading and epidemic reasons, it would be of benefit to be aware of any potential cross-protection against other serotypes following simultaneous vaccination against BTV-4 and -8.
Ongoing research in new vaccines against BTV is promising. There are various field situations, which demand different vaccine requirements, e. g. after a BTV incursion the rapid onset is preferable over a long duration of immunity and minor side effects of a vaccine are acceptable, when a high virulent virus is circulating, but should be avoided if vaccination is preventive (Feenstra and van Rijn, 2017). For all of these situations, the vaccine should protect against disease and viremia, preferably of multiple serotypes and for all ruminant species. Vaccines, which allow differentiating infected from vaccinated animals, would facilitate the monitoring of the disease enormously.
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Summary
“Investigations on the vaccination against the Bluetongue Virus serotypes 4 and 8 in sheep”
Johanna Hilke
Bluetonguevirus (BTV) is the causative agent for Bluetongue disease (BT) in ruminants. BT can cause considerable economic impact due to the morbidity and mortality of ruminant livestock, as well as movement restrictions and control measures. The aim of this cumulative thesis was to investigate on the one hand whether vaccinations with inactivated BTV-8 vaccines induce antibodies in sheep still detectable after five to seven and a half years. On the other hand, different
vaccination schemes and techniques in sheep were compared using four
commercially available inactivated vaccines against BTV-4 and BTV-8 from different companies. Under field conditions, the measurable immunogenicity was compared with respect to safety and saving in time.
Thirty-six female sheep were included in the first study, previously vaccinated against BTV-8 using inactivated vaccines. In Germany, vaccination was compulsory in 2008 and 2009, voluntary in 2010 and early 2011, and was prohibited later in 2011. Due to their age, eighteen sheep had been vaccinated for two or more consecutive years, while a further eighteen animals had only been vaccinated once or not at all. The sheep were blood sampled five (n = 31) to seven and a half years (n = 5) after their last vaccination. All serum samples (n = 36) were tested for BTV group-specific antibody activities by an ELISA. In n = 5 of the animals, the BTV-8 serotype-specific antibody titres were measured by serum neutralization (SN).
According to our analyses, the majority of sheep vaccinated annually for at least two consecutive years were BTV seropositive five years after their last vaccination with an inactivated BTV-8 vaccine (14 of 18 sheep). The majority of animals which had received less than two vaccinations showed ELISA or SN negative results (15 of 18 sheep) five to seven and a half years after the last flock vaccination. However, seven and a half years after only one vaccination, 2/5 animals showed a positive ELISA and another animal (1/5) showed a positive SN result. This study is the first to describe the presence of BTV antibodies in sheep 5 to 7.5 years after vaccination with inactivated BTV-8 vaccines.
The second study included 240 sheep. Eight different vaccination schemes, using four commercially available inactivated Bluetongue vaccines against serotypes 4 and 8 were tested in three different combinations (setting 1-3) under field conditions for their ability to generate a measurable immune response in sheep.
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Animals of setting 1 (groups A-D) were simultaneously vaccinated using either individual injections at different locations (groups A & D) or double injection by a twin- syringe (groups B & C). For both application methods, a one-shot vaccination
(groups C & D) was compared to a boosted vaccination (groups A & B). Sheep of setting 2 (groups E-G) were vaccinated in an alternating, boosted pattern at
fortnightly intervals starting with serotype 4 (groups E & F) or vice versa (group G).
Group H of setting 3 was vaccinated simultaneously and vaccines were injected individually as a one-shot application. Each group consisted of 30 sheep. The immunogenic response was tested in all sheep (n=240) by ELISA
(IDScreen®Bluetongue Competition), while serum neutralisation tests were performed in five to six sheep from each group (n=48).
All vaccine combinations were well tolerated by all sheep. Of all vaccines and
schemes described, the simultaneous double injected boosted vaccination of setting 1 (group B) yielded the highest median serotype-specific titres 26 weeks after the first vaccination (afv) and 100% seropositive animals (ELISA) one year afv. In setting 1, there were no relevant significant differences in the immunogenic response between simultaneously applied vaccines at different sites or at the same injection site.
Importantly, a one-shot vaccination induced comparable immunogenicity to a boosted injection half a year afv. Low serotype-specific neutralising antibody levels were detected in settings 2 and 3 and are attributed to diverse factors, which may have influenced the measured immunogenicity, due to the setting as a field trial.
The results of the described studies propose that vaccinations with inactivated vaccines induce longer (n)Ab activities than expected. Furthermore, the vaccination with two monovalent inactivated BTV vaccines as a one-shot immunization provoke similar immunogenicity as a boosted vaccination, even if not recommended by the manufacturer and if applied at the same or different injection sites. Further long-term research is needed to confirm these findings with more numerous animals in a virus challenge study. If reassured, vaccination recommendations could be simplified substantially to motivate more farmers to vaccinate their ruminant livestock.
Initial whole-flock vaccination could include a booster vaccination either after 3 weeks or up to until one year after the initial vaccination. Subsequently, vaccination of the replacements, using the same interval could maintain sufficient flock protection.
Revaccination of the whole flock would depend on age distribution and the
replacement rate. At an average replacement rate of 15.5% (10–22%) in Southern Germany, a vaccination coverage of between 60% and 100% would be achieved in the sixth year. In flocks with less than 15% replacement rate, whole-flock
revaccination might be necessary after five years to ensure sufficient cover to stop virus spread, as well as viremia, and to avoid the occurrence of clinical symptoms.
The highest possible vaccine coverage of ruminant population is desirable not only to stop the virus from spreading, but to diminish the risk of virus alteration with a
possible increase in virulence and transmission potential. In order to detect subclinical virus spreading, the vaccination campaign has to be combined with a sensitive and continuous monitoring of the domestic and wild ruminant population.
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Zusammenfassung
„Untersuchungen zur Impfung gegen das Blauzungen Virus Serotyp 4 und 8 beim Schaf“
Johanna Hilke
Das Blauzungenvirus (BTV) ist der Erreger der Blauzungenkrankheit (BT) bei Wiederkäuern. BT kann aufgrund der Morbidität und Mortalität von Wiederkäuern sowie von Verbringungseinschränkungen und Kontrollmaßnahmen erhebliche
wirtschaftliche Auswirkungen haben. Ziel dieser kumulativen Arbeit war es, einerseits zu untersuchen, ob Impfungen mit inaktivierten BTV-8 Impfstoffen Antikörper bei Schafen hervorrufen, die nach fünf bis siebeneinhalb Jahren noch nachweisbar sind.
Andererseits wurden verschiedene Impfschemata und -techniken mit vier
zugelassenen inaktivierten Impfstoffen gegen BTV-4 und BTV-8 von verschiedenen Herstellern verglichen. Unter Feldbedingungen wurde die messbare Immunogenität im Hinblick auf Sicherheit und Zeitersparnis beim Schaf verglichen.
In die erste Studie wurden 36 ältere weibliche Schafe einbezogen, die zuvor mit inaktivierten BTV-8 Impfstoffen geimpft wurden. In Deutschland war die Impfung in den Jahren 2008 und 2009 obligatorisch, in den Jahren 2010 und Anfang 2011
freiwillig und wurde später im Jahr 2011 verboten. Aufgrund ihres Alters waren n = 18 Schafe in zwei oder mehr aufeinander folgenden Jahren geimpft worden, während weitere n = 18 Tiere nur einmal oder gar nicht geimpft worden waren. Den Schafen wurde fünf (n = 31) bis siebeneinhalb Jahre (n = 5) nach ihrer letzten Impfung Blutproben entnommen. Alle Serumproben (n = 36) wurden mittels ELISA auf BTV- gruppenspezifische Antikörper-Aktivitäten getestet. Bei n = 5 der Tiere wurden die BTV-8 Serotyp-spezifischen Antikörpertiter mithilfe von Serumneutralisation (SN) ermittelt.
Unseren Analysen zufolge war die Mehrheit der jährlich in mindestens zwei
aufeinander folgenden Jahre geimpften Schafe fünf Jahre nach ihrer letzten Impfung mit einem inaktivierten BTV-8-Impfstoff seropositiv (14/18 Schafen). Die Mehrheit der Tiere, die weniger als zwei Impfungen erhalten hatten, zeigten fünf bis siebeneinhalb Jahre nach der letzten Herdenimpfung ELISA- oder SN-negative Ergebnisse (15/18 Schafen). Allerdings zeigten 2/5 Tieren siebeneinhalb Jahre nach nur einer Impfung einen positiven ELISA und ein weiteres Tier (1/5) ein positives SN-Ergebnis. Diese Studie ist die erste, die das Vorhandensein von BTV-Antikörpern bei Schafen fünf bis siebeneinhalb Jahre nach der Impfung mit inaktivierten BTV-8 Impfstoffen beschreibt.
Die zweite Studie umfasste 240 Schafe. Acht verschiedene Impfschemata mit vier verschiedenen handelsüblichen inaktivierten Blauzungenimpfstoffen gegen die Serotypen 4 und 8 wurden in drei verschiedenen Kombinationen (Setting 1-3) unter Feldbedingungen auf ihre Fähigkeit getestet, eine messbare Immunantwort beim Schaf zu erzeugen.
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Tiere von Setting 1 (Gruppen A-D) wurden gleichzeitig entweder durch einzelne Injektionen an verschiedenen Lokalisationen (Gruppen A & D) oder durch Doppelinjektion mit einer Doppelkanüle (Gruppen B & C) geimpft. Für beide Anwendungsmethoden wurde eine one-shot Impfung (Gruppen C & D) mit einer geboosterten Impfung (Gruppen A & B) verglichen. Schafe des Setting 2 (Gruppen E-G) wurden alternierend und geboostert mit den verschiedenen Serotypen in zweiwöchentlichen Abständen geimpft, beginnend mit Serotyp 4 (Gruppen E & F) oder Serotyp 8 (Gruppe G). Bei Tieren der Gruppe H (Setting 3) wurden die Impfstoffe gleichzeitig an zwei verschiedenen Injektionsorten angewendet. Jede Gruppe bestand aus 30 Schafen. Die Immunreaktion wurde bei allen Schafen (n=240) durch ELISA (IDScreen®Bluetongue Competition) getestet, während Serumneutralisationstests (SNT) bei fünf bis sechs Schafen aus jeder Gruppe durchgeführt wurden (n=48).
Alle Impfstoffkombinationen wurden von allen Schafen gut vertragen. Von allen beschriebenen Impfstoffen und Schemata ergab die gleichzeitige doppelt injizierte geboosterte Impfung des Setting 1 (Gruppe B) 26 Wochen nach der ersten Impfung (afv) die höchsten medianen serotyp-spezifischen Titer und 100% seropositive Tiere (ELISA) ein Jahr afv. In Setting 1 gab es keine relevanten signifikanten Unterschiede in der Immunreaktion zwischen gleichzeitig injizierten Impfstoffen an verschiedenen Stellen oder an derselben Injektionsstelle. Hervorzuheben ist, dass eine einmalige Impfung eine vergleichbare Immunogenität induzierte, wie eine geboosterte Injektion, mindestens für den hier untersuchten Messzeitraum von einem halben bis einem Jahr afv. Niedrige serotyp-spezifische neutralisierende Antikörpertiter wurden in den Settings 2 und 3 nachgewiesen. Da die Studie als Feldversuch durchgeführt wurde, können sie nicht einzelnen, sondern nur einer Summe von Faktoren zugeordnet werden, die die gemessene Immunogenität beeinflusst haben können.
Die Ergebnisse der beschriebenen Studien deuten darauf hin, dass Impfungen mit inaktivierten Impfstoffen längere Antikörper-Aktivitäten induzieren als erwartet.
Darüber hinaus provoziert die Impfung mit zwei monovalenten inaktivierten BTV- Impfstoffen als One-Shot-Immunisierung eine ähnliche Immunogenität wie eine geboosterte Impfung, auch wenn sie vom Hersteller nicht empfohlen wird und an denselben oder verschiedenen Injektionsstellen angewendet wird. Weitere langfristige Untersuchungen sind erforderlich, um diese Ergebnisse mit höherer Tierzahl und experimentellen Belastungsinfektionen zu bestätigen. Wenn die aufgestellten Thesen bestätigt werden, könnten die Impfempfehlungen erheblich vereinfacht werden, um mehr Landwirte zu motivieren, ihre Wiederkäuer zu impfen.
Die Herden-Grundimmunisierung sollte aus einer Impfung sowie einer Auffrischungsimpfung im gleichen oder bis zu einem Jahr nach Erstimpfung bestehen. In den Folgejahren könnte die Impfung im gleichen Intervall auf die Nachzucht beschränkt werden und einen ausreichenden Herdenschutz
gewährleisten.
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Die Wiederholungs-Impfung des gesamten Bestandes würde von der Altersverteilung und der Remontierungsrate abhängen. Bei einer durchschnittlichen Remontierung von 15,5% (10-22%) in Süddeutschland würde im sechsten Jahr eine Impfrate von 60% bis 100% erreicht. Bei Beständen mit einer Remontierung von weniger als 15%
könnte eine Impfung der gesamten Herde nach fünf Jahren nötig sein, damit die Virusausbreitung gestoppt werden kann und Virämie und klinische Symptome vermeiden werden.
Eine möglichst hohe Impfabdeckung der Wiederkäuerpopulation ist nicht nur wünschenswert, um die Ausbreitung des Virus zu stoppen, sondern auch, um das Risiko einer Virusveränderung mit einer möglichen Erhöhung der Virulenz und des Übertragungspotenzials zu verringern. Um eine subklinische Ausbreitung des Virus früh genug erkennen zu können, muss parallel ein kontinuierliches und sensitives Monitoring der Haus- und Wild-Wiederkäuer-Population durchgeführt werden.
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