Serological investigation
Samples and statistical methods
The aim of the study was to determine regional BoHV-2 seroprevalences with a statistical certainty of 95% and an accuracy of at least 5%. According to the data provided by Böttcher, Boje [26], it was assumed that Swabia was more affected than other administrative regions of Bavaria. For this reason, a putative prevalence of about 10% for Swabia and 5% for Upper Bavaria, Lower Bavaria, and North Bavaria (Upper Palatinate, Upper, Middle, and Lower Franconina) and infinite cattle populations were taken as a basis in a first calculation step. Sample sizes of at least 139 farms from Swabia and 73 farms from the other regions resulted [38]. In a second step, the distribution of the farms to be selected was adapted to the respective regional cattle densities.
Out of the large pool of blood samples from healthy animals that were sent in in 2017, 2018, and in the beginning of 2019 to the Bavarian Health and Food Safety Authority, 6801 sera stemming from 406 farms were randomly chosen and included in this study. Following the sampling scheme explained above, finally 141 farms (2615 sera) from Swabia, 82 farms (1200 sera) from Upper Bavaria, 78 farms (1070 sera) from Lower Bavaria, and in summary 105 farms (1916 sera) from the four administrative regions of North Bavaria, i.e. Upper Palatinate, Upper Franconia, Middle Franconia, and Lower Franconia, were included.
In order to better describe seroprevalences, confidence intervals (CI) that should cover the true seroprevalence with a certainty of 95%, were used. To check for the relationship between BoHV-2 seroprevalence and sample origin on the one hand and between BoHV-2 seroprevalence and BoHV-1 test results on the other hand, the chi-squared test was applied. A quantification of these relationships was made by odds ratios. Values of p < 0.05 were regarded as statistically significant.
ELISAs detecting BoHV-1 or BoHV-2 antibodies
To detect antibodies against BoHV-2 an indirect ELISA (ID Screen® BHV-2 Indirect; ID VET) was used. A purified French BoHV-2 isolate serves as antigen [39]. According to the manufacturer’s instructions, sera are classified as positive, when the sample to positive control-ratio (S/P%) is greater or equal 110%, as negative, when the S/P% is less or equal 90%, and as doubtful, when the S/P% lies in between (90% < S/P% < 110%). Using these cut-off values, a diagnostic sensitivity of 92.0% or 96.6% and a respective diagnostic specificity at 100.0% or 98.2% has been determined depending on whether or not doubtful results are considered positive (ID VET: Internal validation report ID Screen® BHV-2 Indirect; available upon request). The underlying data largely bases on the results of a study analyzing a large number (n = 424) of preselected samples in the ELISA and the BoHV-2 specific serum neutralization test (SNT) as a gold standard [40]. To increase the sensitivity in this study an additional range of sample/positive control ratios (S/P%) was considered. Ratios between 50 and 90%, hence lower still than the test systems range of “doubtful” results (90% < S/P% < 110%), were defined as “increased”. Samples with “doubtful” results (n = 20) were retested by BoHV-2 specific SNT. “Increased” results (n = 62) were controlled accordingly for all sera that were suitable for this test system (n = 61). One sample had to be excluded as it caused toxic reactions in cell culture. Furthermore, to ensure the comparability of these low level antibody results between ELISA tests, an additional weak positive reference serum (“MRI-BHV-2”, BoHV-2 positive, freeze dried serum, IDVET) was used in every assay.
In Germany, a variety of commercially available ELISA test kits are registered for the official testing of cattle sera for antibodies against BoHV-1. Three different BoHV-1 antibody ELISA systems were applied in a hierarchical order: To begin with, an indirect virus ELISA, hereafter called Trachitest (IDEXX Trachitest Serum Screening, IDEXX Europe B.V.), was used. According to the manufacturer’s instructions samples have to be retested when reactivity exceeds 35% but does not yet reach positive values (≥45%). However, to increase the sensitivity within the framework of the present study, this cut-off value was reduced to 25%. In case of a reactivity above this value the test was repeated and additionally samples were analyzed in a second ELISA, a competitive gB-ELISA (IDEXX IBR gB X3, IDEXX Europe B.V. or cattletype BHV1 gB Ab, Indical). In case of positive or suspect results in the gB-ELISA, according to the test specifications, the third ELISA, a competitive gE-ELISA (IDEXX IBR gE, IDEXX Europe B.V.) was used. Finally, considering the results of the three ELISAs, sera were categorized using the following nomenclature: “BoHV-1 ELISA negative” (Trachitest negative), “Trachitest-positive” (Trachitest positive, gB- ELISA negative), “non-negative” (Trachitest and gB-ELISA positive or suspect, gE-ELISA negative), and “BoHV-1 antibody positive” (all assays, especially gE-ELISA, positive). The latter definition is in accordance with German BoHV-1 regulations, determining procedures to protect cattle against BoHV-1 infections [37].
Serum neutralization test (SNT)
BoHV-1-strain “Schönböken” (RVB-0073) and BoHV-2-strain “Riems 8/85” (RVB-0064), both obtained from the Collection of Viruses in Veterinary Medicine, Friedrich-Loeffler-Institute, Insel Riems, Germany, were propagated on KOP-R cells, a diploid bovine esophageal cell line (RIE244, Collection of Cell Lines in Veterinary Medicine (CCLV), Friedrich-Loeffler-Institute, Insel Riems, Germany). Standard procedures were followed. Briefly, KOP-R cells were cultivated for about 24 h in 96-well tissue culture plates at 37 °C/5% CO2 in MEM with Earle’s salts and L-glutamine (Gibco™ by life technologies, USA) supplemented by 10% gamma irradiated fetal bovine serum (FBS; Biowest, South America), 1% non-essential amino acids and 1% pyruvate.
Heat-inactivated blood sera (30 min, 56 °C) were diluted in log2-steps and incubated with 100 TCID50 of the respective virus in a 1 + 1 ratio at 37 °C and 5% CO2 overnight. The following day the pre-incubated serum-virus-mixtures were transferred to microtiter plates containing KOP-R cells in monolayers at a confluence of about 70–80%. At this point, the FBS content had been reduced to 2%. After incubating at 37 °C/ 5% CO2 for another 72 h, cells were evaluated for virus growth using an inverted microscope. A replicate was considered infected when at least one focus was observed. Vice versa neutralization of infectivity was scored only when neutralization was complete and no cytopathic effect at all could be observed.
PCR-analyses
Samples
Two hundred thirty-nine tissue samples from teat-, udder-, or muzzle lesions (n = 113), corresponding lymph nodes (n = 85), and in some cases trigeminal ganglia (n = 41) from altogether 111 animals (107 bovines, 3 sheep, 1 goat) were included in this study. As BoHV-2 antibodies were clearly more prevalent in South Bavaria, the sampling focused on this region. Practitioners in the field were asked for special attention to udder and teat lesions and to send in samples. Most samples, however, were taken at the pathology department of the Bavarian Health and Food Safety Authority. It was decided to test tissues from all kinds of visible lesions at the udder and the teats, to ensure that no putative disease status – including reactivation – was missed.
Furthermore, lesions at the muzzle and mucosa of the mouth of calves had been reported in the context of BoHV-2 infections, as well as virus isolation not only from these lesions but also from nasal swabs during acute infection and upon experimental reactivation [11]. In consequence, not only such lesions were taken as an indication for analysis, but also case histories of respiratory disease or mucosal reddening. Respective nasal swabs (n = 918) were included with the intention to possibly detect reactivated BoHV-2 infections in the course of stress and clinical disease of any other cause.
PCR
Nucleic acids from tissue samples were either extracted using a magnetic processor (BioSprint96, Qiagen) and the BioSprint96 One-For-All Vet Kit or manually with the QIAamp DNA Mini Kit (Qiagen). Nucleic acids from nasal swabs were purified by means of the QIAamp Viral RNA Mini Kit (Qiagen), which has been thoroughly validated and is routinely used also for the detection of viral DNA in the laboratory. As to date very little sequence information is available on BoHV-2 genomes and as the aim of the study was the detection of putative actual and local strains in Bavaria, a multi PCR-strategy was chosen.
Real time PCR targeting sequences within the glycoprotein B (gB) gene
Published and self-generated sequences of the conserved glycoprotein B gene of different BoHV-2 strains (RVB-0062 and RVB-0064; Collection of Viruses in Veterinary Medicine, Friedrich Loeffler-Institute, Insel Riems, Germany), were used for sequence alignment (Clustal W version 2.1). Two primer and probe combinations, the BoHV2-Mix2.1 qPCR and the BoHV2-Mix2.3 qPCR were applied in parallel. In a total reaction volume of 20 μl (including 5 μl of template DNA) the respective primers (BoHV2-Mix2.1 qPCR: BoHV2-gB-2.1F: 5′-GAG GGC ATC GCC GTA ATC-3′, BoHV2-gB-2.1R: 5′- CAG TCA CGG CCT TGT AGT AC-3′; BoHV2-Mix2.3 qPCR: BoHV2-gB-2.2F: 5′-CAT CGC CGT AAT CTT CAA GGA-3′; BoHV2-gB-2.1R: see above) were added to a final concentration of 0.8 μM and the probe to 0.2 μM (BoHV2-gB-2FAM: 5′-FAM-ACC TCG CGC CGT ACA AGT TTA AGG C-BHQ1–3′). In a Bio-Rad CFX 96™ Real-Time PCR Detection System DNA was amplified basing on the cycling protocol of the PerfeCta MultiPlex qPCR ToughMix (Quanta BioScience). Amplification started with an initial denaturing step of 10 min (95 °C), followed by 40 cycles of denaturation (95 °C, 15 s) and primer annealing and extension (60 °C, 60s).
Conventional PCR targeting sequences within the DNA polymerase gene
The protocol described by Cargnelutti, Weiblen [41] was slightly altered, in as much as the HotStarTaq Polymerase (Qiagen) was used in a total volume of 50 μl, containing 5 μl template DNA and in addition Q-Solution and MgCl2. The cycling protocol differed from the manufacturer’s instructions as annealing was done at 46 °C (60s), as 45 cycles were used, and as the final extension was shortened to 7 min.
PanHerpes PCR targeting the highly conserved sequences within the herpesviral DNA polymerase gene
This PCR protocol has been designed and proven valuable for the detection of a broad range of herpesviral genomes [33]. The HotStarTaq polymerase (Qiagen) was used as described above, otherwise following the procedures published by Ehlers et [33].
For the newly established real time PCR and the conventional PCRs targeting the DNA-polymerase or the gB gene, specificity was tested using the following putatively contaminating viruses: BoHV-1, Equid alphaherpesvirus 1 (family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus), Bovine gammaherpesvirus 4 (family Herpesviridae, subfamily Gammaherpesvirinae, genus Rhadinovirus), OvHV-2), Orf virus (family Poxviridae, subfamily Chordopoxvirinae, genus Parapoxvirus), Pseudocowpox virus, (family Poxviridae, subfamily Chordopoxvirinae, genus Parapoxvirus), Bovine papular stomatitis virus (family Poxviridae, subfamily Chordopoxvirinae, genus Parapoxvirus), Lumpy skin disease virus (family Poxviridae, subfamily Chordopoxvirinae, genus Capripoxvirus), Bluetongue virus-4 and -8 (family Reoviridae, subfamily Sedoreovirinae, genus Orbivirus), Pestivirus (formerly: Bovine viral diarrhea virus; family Flaviviridae, genus Pestivirus), and samples containing virus of the genus Orthopoxvirus (family Poxviridae, subfamily Chordopoxvirinae) or of the family Papillomaviridae. PCR protocols were also analyzed for reproducibility and sensitivity. Sensitivity was calculated in numbers of genome copies that were reproducibly detectable. The efficiency of nucleic acid purification as well as the influence of inhibitory effects was controlled for all samples detecting beta-actin sequences by real time PCR as it has been described [42].