Calcium and phosphorus requirements for growing dogs can be calculated by different methods. The current standard feeding recommendations are based on experimental data derived from young giant breed puppies. In order to determine the absolute requirement, an extrapolation via metabolisable energy requirement is recommended. Another approach is to calculate the requirement factorially, taking into account the endogenous losses and the amount of calcium and phosphorus retained due to tissue accretion during growth as well as the expected availability of these nutrients. The working hypothesis was that both methods are valid and lead to comparable results in young puppies of a high mature body weight (BW). Yet, deviations for other age and mature BW groups were expected. Thus, the aim of the present study was to compare the results of both methods using exemplary puppies of different age and mature BW groups. The hypotheses could be verified for calcium. The extrapolated requirements overestimate the factorial requirements by up to 59.7% for puppies <60kg mature BW and/or >6 months of age. In case of phosphorus requirement, the deviations between both methods are overall very high in all stages. Taking into account the potentially harmful effects of calcium and phosphorus excess, the feeding recommendations based on the extrapolation should be reconsidered.

The correct assumption of metabolisable energy (ME) requirement is essential for the nutrition consultation and diet formulation. In young dogs, too high energy supply can accelerate growth and thus lead to developmental orthopaedic diseases. The aim of the present study was to collect the data on ME intake and body weight (BW) development in privately owned growing dogs in order to compare these data with the current recommendations. Our hypothesis was that the actual ME intake of healthy young dogs would be lower than the actual recommendation. The data of 493 privately owned puppies (median age at first consultation 21 weeks, the median expected mature BW 30 kg) on ME intake, actual and expected mature BW were collected and compared with recommendations of the Society of Nutrition Physiology (GfE, Meyer and Zentek and NRC). In 243 dogs, there was a follow‐up. The actual BW did not deviate systematically from the calculated expected BW (R2 = .929). The ME intake significantly decreased with age (p < .05) and significantly increased with expected mature BW (p < .05). There was no significant interaction between these two parameters (p > .05). Sex had no effect on the ME intake (p > .05). The ME intake of young dogs with a history of skeletal problems or of food allergy did not differ systematically from healthy dogs of similar age and expected mature BW. The ME intake was considerably below NRC recommendations, especially in younger puppies (>8–17 weeks: 78%, >17–26 weeks: 83% of NRC recommendation). A predictive linear equation for ME intake was developed: ME intake (MJ) = (1.063 − 0.565 × [actual BW/expected mature BW]) × actual BW0.75.

High-yielding dairy cows collapsed and died displaying signs of a disturbed central nervous system (muscle tremor, convulsion) and a considerably reduced body condition score. An intense diagnostic screening did not allow to confirm a definite diagnosis. Therefore, further analyses including an evaluation of feeds and feeding were initiated. The herd was fed a total mixed ration (TMR) based on corn and grass silage of moderate nutritive value supplemented with various amounts of chocolate chips.

Background Pattern recognition receptors (PRRs) serve as a hub of immune responses to microbes in the gut and play a critical role in maintaining intestinal homeostasis. Toll-like receptor 4 (TLR4) represents an important PRR that can recognize the gram-negative bacterial cell wall component lipopolysaccharide (LPS). Upon binding to LPS, TLR4 forms a multimeric complex with the indispensable co-receptor myeloid differentiation protein 2 (MD2) facilitating selection of TLR4 as cargo for endocytosis and TLR4-mediated activation of downstream signalling. Despite the critical role of TLR4 in innate immunity, no patients with TLR4 or MD2 deficiency have yet been reported. Methods To investigate potential genetic causes for two related patients presenting with very early onset Inflammatory Bowel Disease (VEO-IBD) and/or pneumonia, we have performed whole exome sequencing (WES). To assess the functional consequences of the identified LY96 (encoding for MD2) variant, we generated a CRISPR/Cas9-mediated knockout (KO) of MD2 or knockin (KI) of the patient mutation in induced pluripotent stem cells (iPSCs) and studied TLR4-mediated signalling, cytokine responses, and bacterial handling in iPSC-derived macrophages. Results Genetic analysis identified a 3 bp homozygous in-frame deletion in the LY96 gene (NM_015364.5, c.347_349delCAA; p.Thr116del) in our index patients following an autosomal recessive inheritance pattern. Immunoblotting revealed an altered protein expression of overexpressed Flag-tagged mutant MD2 protein due to impaired N-linked glycosylation. Correspondingly, iPSC-derived MD2-deficient macrophages showed an impaired LPS-induced TLR4 endocytosis. As a functional consequence, we could detect reduced NF-κB and MAPK signalling (phosphorylation of NF-κB p65 subunit, ERK1/2, and p38 MAPK) and dysregulated inflammasome activation (IL-1b production and secretion) upon challenge with LPS. Gentamycin protection assays and live cell imaging suggested that MD2-deficient macrophages exhibit an impaired phagocytosis of microbial pathogens. In addition, macrophages with mutant MD2 showed decreased cytokine expression (e.g., IFNB, IL6, IL10, and TNF) in response to LPS or gram-negative bacteria (E. coli and Salmonella typhimurium) but not gram-positive bacteria (Listeria monocytogenes). Conclusion Human MD2 deficiency is an inborn error of immunity that may predispose to VEO-IBD associated with altered TLR4-mediated signalling, cytokine responses, and bacterial handling. The first description of patients with MD2 deficiency provides critical insights on human TLR4/MD2 biology and warrants caution on therapeutics strategies targeting TLR4 signalling.