Ethics statement
The study was conducted according to national and international guidelines for animal welfare. Permission was obtained from the cattle owner agreed for the samples to be used in the study. The data were obtained during diagnostic procedures which would have been carried out regardless. This is a very special situation in veterinary medicine. Since the data were from client-owned cattle which underwent veterinary exams, according to the legal definitions in Germany, no “animal experiment” took place.
Histopathological examination
Samples of various locations of the skin, oral mucosa, tongue, brain, lung, heart, muscle, liver, spleen, kidney and intestine were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 3 μm, and stained with haematoxylin/eosin according to routine methods. Selected sections were stained with PAS-reaction
Ultrastructural examinantion
For transmission electron microscopy skin and oral mucosa tissue were fixed in 2.5% glutaraldehyde/cacodylate buffer for 24 h, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of alcohol and embedded in epon (Serva, Heidelberg, Germany). Sixty nm thick ultra-thin sections were contrasted with 2% aqueous uranyl acetate and lead citrate and examined with a Zeiss EM 10C electron microscope (Zeiss, Oberkochen, Germany).
Animals and genotyping
Blood samples were taken from the affected animal and from a total of 162 animals belonging to the herd into which the affected calf was born. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. In addition, archived DNA samples of 81 Charolais bulls and 50 animals from different cattle breeds were used for genotyping the ITGB4 deletion.
The genotyping of the affected animal was carried out using the BovineHD BeadChip (illumina, San Diego, USA), including 777,961 evenly distributed SNPs and standard protocols as recommended by the manufacturer.
Whole genome sequencing and variant calling
A fragment library with 300 bp insert size was prepared and one lane of illumina HiSeq2000 paired-end reads (2 × 100 bp) were collected. The reads were mapped to the cow reference genome UMD3.1/bosTau6 and aligned using Burrows-Wheeler Aligner (BWA) version 0.5.9-r16 [22] with default settings. The SAM file generated by BWA was then converted to BAM and the reads were sorted by chromosome using samtools [23]. The PCR duplicates were marked using Picard tools [24]. The Genome Analysis Tool Kit (GATK version 2.4.9) [25], was used to carry out local realignment and to produce a cleaned BAM file. Variant calls were then made with the unified genotyper module of GATK. The variant data for each sample were obtained in variant call format (version 4.0) as raw calls for all samples and sites flagged using the variant filtration module of GATK. Variant filtration was performed, following the best practice documentation of GATK version 4. The snpEFF software [26] together with the UMD3.1/bosTau Ensembl annotation was used to predict the functional effects of the variants detected. The pindel package using split-read approaches to identify large deletions and medium-size insertions in pair-end reads was used to detect structural variants in cleaned BAM files [27]. Hence, in order to avoid missing large inserts, deletions and false positives of all the variants detected in the region of EB genes (Additional file 3) were also manually inspected using the Integrative Genomics Viewer (IGV) [28].
The variants of a total of 50 genomes from various cattle breeds (14× Holstein, 6× Simmental, 5× Angler, 4× Brown Swiss, 3× Hinterwalder, 3× Vorderwalder, 2× Galloway, 2× Eringer, 2× Romagnola, 2× Scotish Highland Cattle, 2× Tyrolean Grey Cattle, 1× Hereford, 1× Limousin, 1× Pezzata Rossa Italiana, 2× crossbred), which had been sequenced in our laboratory in the course of other ongoing studies, were used as controls during filtering.
Genetic testing
Primers for the amplification of the deletion were designed using the Primer3 software [29] after masking repetitive sequences with RepeatMasker [30]. The positions of the three primers used for genotyping (fwd GTGAGGGCTTCGTATGGGTA; rev 1 TGAACGAGGTGTACCGACAA; rev 2 AGTCGCTCTACACGGACACC) are displayed in Figure 4. Sanger sequencing was used to confirm the illumina sequencing results. For these experiments, PCR products using AmpliTaqGold360Mastermix (Life Technologies, Darmstadt, Germany) were amplified. The PCR products were loaded on 2% agarose gels for visual inspection of band size. The PCR products were directly sequenced on an ABI3730 capillary sequencer (Life Technologies) after treatment with exonuclease I and shrimp alkaline phosphatase. The sequence data were analysed using Sequencher 5.1 software (GeneCodes, Ann Arbor, USA).
Availability and requirements
The genome data were made available freely at the European Nucleotide Archive [ENA:PRJEB7528] [31]. Further supporting data are included as additional files.