Clinicopathological Phenotype of Autosomal Recessive Cholesterol Deficiency in Holstein Cattle

Mock, T.; Mehinagic, K.; Menzi, F.; Studer, E.; Oevermann, A.; Stoffel, M.H.; Drögemüller, C.; Meylan, M.; Regenscheit, N.

Five Holstein calves (3 females, 2 males) and a 2.5-year-old heifer were referred to the Clinic for Ruminants of the Vetsuisse Faculty, University of Bern, Switzerland. The calves had a history of failure to thrive and intermittent diarrhea unresponsive to treatment since birth. The mean age of the calves was 86 days (min-max: 18-224 days), and the mean weight was 69 kg (min-max: 41-153 kg), as shown in Table 1 . The heifer (472 kg) had shown poor development and intermittent diarrhea in the first months of life, but had no longer shown diarrhea later on; it was presented to the Clinic with a primary complaint of lesions in the buccal cavity. A complete clinical examination of all animals was performed, and a CBC and blood chemistry profile was performed for calves numbered (no.) 1, 2, and 3. In addition, cholesterol and triglycerides were measured in all animals and were compared to a group of 5 healthy Holstein calves and a group of 5 inappetent Holstein calves with other diseases than diarrhea (Table 1) . Fecal samples were sent for routine viral, bacterial, and parasitological analyses to exclude the most common pathogens causing calf diarrhea (rotavirus, coronavirus, cryptosporidia, coccidia, E. coli, and Salmonella spp.). Dietetic causes for diarrhea were excluded by clinical history. The animals were tested for bovine viral diarrhea virus (BVDV) using an antigen-ELISA a on a skin biopsy (≤180 days in life) or an EDTA blood sample (>180 days in life). All animals were euthanized (Phenobarbitalum natricum, 100 mg/ kg i.v.) b 1-14 days after admission to the Clinic, based on the poor prognosis and suspicion of CD. A complete necropsy was performed on all animals. Fresh tissue samples were immediately fixed in 4% neutral buffered formalin, embedded in paraffin, cut at 4 lm, and stained with hematoxylin and eosin (HE). As lipids are removed from tissues during routine histological processing, for the visualization of lipids, optimal cutting temperature compound (O.C.T.) (Tissue-Tek) c -embedded frozen sections of small and large intestine and liver were stained with Sudan stain. Selected brain, spinal cord, and nerve sections of the heifer were stained with Luxol fast blue and Bielschowsky, respectively. Electron microscopical analysis was performed on small intestinal villi and liver of animals no. 5 and 6. Formalin-fixed tissue samples were postfixed with 1% OsO4 in 0.1 M cacodylate buffer for 1 hour at 4°C and embedded in Epon. d Semi-thin sections were stained with toluidine blue and used to narrow down further regions of interest. Sections were collected on collodion-coated 200 mesh copper grids. e Sections were double stained with 0.5% uranyl acetate f for 30 minutes at 40°C and 3% lead citrate g for 10 minutes at 20°C in an Ultrastain h and examined in a Philips CM12 transmission electron microscope i at an accelerating voltage of 80 kV. Micrographs were captured with a Mega View III camera using the iTEM software. j The presence of the APOB mutation was demonstrated by a PCR-based direct gene test described elsewhere. 3