The effect and mechanism of selenium supplementation on the proliferation capacity of bovine endometrial epithelial cells exposed to lipopolysaccharide in vitro under high cortisol background.

Bovine endometritis severely inhibits uterine repair and causes considerable economic loss. Besides, parturition-induced high cortisol levels inhibit immune function, reduce cell proliferation, and further inhibit tissue repair. Selenium (Se) is an essential trace element for animals to maintain normal physiological function and has powerful antioxidant functions. This study investigated whether Se supplementation reduces endometrial damage and promotes tissue repair in cows with endometritis under stress and explored the underlying mechanism. Primary bovine endometrial epithelial cells were isolated and purified from healthy cows. The cells were treated with different combinations of lipopolysaccharide (LPS), cortisol, and various concentrations of Se. Data showed that LPS stimulation inhibited cell proliferation and increased cell apoptosis. High levels of cortisol further exacerbated these effects. Flow cytometry, scratch wound healing tests, and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays showed that Se supplementation promoted cell cycle progression, cell migration, and cell proliferation in the presence of LPS and cortisol. The quantitative PCR results showed that the expression of related growth factors was increased after Se supplementation. After administering various inhibitors, we further demonstrated that Se supplementation decreased the activity of glycogen synthetase kinase 3ß (GSK-3ß) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to reduce the degradation of ß-catenin except the Wnt signal to promote cell proliferation. In conclusion, Se supplementation attenuated the cell damage induced by LPS at high cortisol levels and increased cell proliferation to promote uterine repair by elevating the mRNA expression of TGFB3 and VEGFA and activating the PI3K/AKT/GSK-3ß/ß-catenin signaling pathway.

After parturition, endometritis is a common bovine disease, which hinders endometrial repair and reduces bovine economic value. Besides, parturition-induced high cortisol levels cause immunosuppression, aggravate infection, and further inhibit cell proliferation and tissue repair. As an essential trace element, adding selenium to feed helps to maintain the normal physiological function of animals. This study developed a cellular model using lipopolysaccharide (LPS) and cortisol to simulate cows with endometritis in stress conditions. The results showed that Se supplementation attenuated bovine endometrial epithelial cell damage and promoted their proliferation in the presence of LPS and high cortisol levels, which are positively correlated with the concentration of Se. Besides, this study proved another molecular mechanism for Se to regulate ß-catenin except for the Wnt signal by affecting the ß-catenin degradation pathway.

[The lipidosis in the liver of the dairy cow: Part 2 Genetic predisposition and prophylaxis]. / Die Leberverfettung der Milchkuh: Teil 2.

Hepatic lipidosis in dairy cows is the result of a disturbed balance between the uptake of non-esterified fatty acids (NEFA), their metabolism in the hepatocytes, and the limited efflux of TG as very-low-density lipoprotein (VLDL). Lipidosis and the associated risk for ketosis represents a consequence of selecting dairy cows primarily for milk production without considering the basic physiological mechanisms of this trait. The overall risk for lipidosis and ketosis possesses a genetic background and the recently released new breeding value of the German Holstein Friesian cows now sets the path for correction of this risk and in that confirms the assumed genetic threat. Ectopic fat deposition in the liver is the result of various steps including lipolysis, uptake of fat by the liver cell, its metabolism, and finally release as very-low-density lipoprotein (VLDL). These reactions may be modulated directly or indirectly and hence, serve as basis for prophylactic measures. The pertaining methods are described in order to support an improved understanding of the pathogenesis of lipidosis and ketosis. They consist of feeding a glucogenic diet, restricted feeding during the close-up time as well as supplementation with choline, niacin, carnitine, or the reduction of milking frequency. Prophylactic measures for the prevention of ketosis are also included in this discussion.

Dietary supplementation of acetate-conjugated tryptophan alters feed intake, milk yield and composition, blood profile, physiological variables, and heat shock protein gene expression in heat-stressed dairy cows.

The purpose of this study was to investigate the effects of dietary supplementation of rumen-protected tryptophan (RPT) at four levels on milk yield, milk composition, blood profile, physiological variables, and heat shock protein gene expression in dairy cows under conditions of moderate-severe heat stress (MSHS, THI = 80~89). Sixteen early-lactating dairy cows (body weight = 719 ± 66.4 kg, days in milk = 74.3 ± 7.1, milk yield = 33.55 ± 3.74 kg, means ± SEM) were randomly assigned in a factorial arrangement to one of the four treatments: control group (n = 4, no RPT supplementation), 15 g/d RPT (n = 4), 30 g/d RPT (n = 4), or 60 g/d RPT group per cow (n = 4) supplemented to the TMR. A higher dry matter intake (DMI) and milk yield were found in the 30 g RPT group compared with the other groups, and the 3.5% fat-corrected milk yield, energy-corrected milk yield, milk fat, protein, ß-casein, mono-unsaturated fatty acid, and poly-unsaturated fatty acid contents, and serum glucose content were observed in the 30 g RPT group (p < 0.05). The milk lactose concentration was significantly higher in the 30 g RPT group compared with the control and 60 g RPT groups (p < 0.05). The plasma cortisol level was lower, while the serotonin and melatonin concentrations were higher in the 30 g group compared with the other groups (p < 0.05). Heat shock protein (HSP) 70 expression was downregulated in the control and 15 g RPT groups, whereas the expression of HSP90 and HSPB1 remained unchanged among the groups. In particular, the 30 g RPT group was considered to have an improved DMI, milk yield, and lactose concentration, as well as anti-heat stress effects due to the simulation of serotonin and melatonin during MSHS.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Selenium influences mmu-miR-155 to inhibit inflammation in Staphylococcus aureus-induced mastitis in mice.

Mastitis, a major disease affecting dairy cows, is most commonly caused by Staphylococcus aureus (S. aureus). Selenium (Se) can activate pivotal proteins in immune responses and regulate the immune system, and microRNA-155 (miR-155) is a key transcriptional regulator for inflammation-related diseases. We constructed the model of mouse mastitis in vivo and primary mouse mammary epithelial cells (MMECs) in vitro, which were induced by S. aureus. Se content of the mammary was estimated using an atomic fluorescence spectrophotometer. Histopathological analysis was performed via hematoxylin and eosin (H&E) staining. The mmu-miR-155-5p mimic was transfected in MMECs, and viability was determined through the MTT assay. Transfected efficiency was evaluated by qPCR and fluorescence staining. Cytokines including TNF-α, IL-1ß, IL-10 and TLRs were detected with qPCR. In addition, western blotting was used to evaluate the expression of the NF-κB and MAPKs signaling pathways. The results demonstrated that a Se-supplemented diet improved the content of Se in mammary tissues. Histopathological studies indicated that the mammary glands were protected in the Se-supplemented group after S. aureus infection. Se-supplementation suppressed the production of MPO, mmu-miR-155, TNF-α, IL-1ß, and TLR2 and significantly inhibited the phosphorylation of NF-κB and MAPKs in vivo and in vitro. All the data indicated that mmu-miR-155 played a pro-inflammatory role in our study, and Se-supplementation could suppress the expression of mmu-miR-155 to inhibit inflammation in S. aureus-induced mastitis in mice.

Zinc Deficiency-Like Syndrome in Fleckvieh Calves: Clinical and Pathological Findings and Differentiation from Bovine Hereditary Zinc Deficiency.

BACKGROUND: Zinc deficiency-like (ZDL) syndrome is an inherited defect of Fleckvieh calves, with striking similarity to bovine hereditary zinc deficiency (BHZD). However, the causative mutation in a phospholipase D4 encoding gene (PLD4) shows no connection to zinc metabolism. OBJECTIVES: To describe clinical signs, laboratory variables, and pathological findings of ZDL syndrome and their utility to differentiate ZDL from BHZD and infectious diseases with similar phenotype. ANIMALS: Nine hospitalized calves with crusting dermatitis and confirmed mutation in PLD4 and medical records from 25 calves with crusting dermatitis or suspected zinc deficiency. METHODS: Prospective and retrospective case series. RESULTS: The 9 calves (age: 5-53 weeks) displayed a moderate to severe crusting dermatitis mainly on the head, ventrum, and joints. Respiratory and digestive tract inflammations were frequently observed. Zinc supplementation did not lead to remission of clinical signs in 4 calves. Laboratory variables revealed slight anemia in 8 calves, hypoalbuminemia in 6 calves, but reduced serum zinc concentrations in only 3 calves. Mucosal erosions/ulcerations were present in 7 calves and thymus atrophy or reduced thymic weights in 8 calves. Histologically, skin lesions were indistinguishable from BHZD. Retrospective analysis of medical records revealed the presence of this phenotype since 1988 and pedigree analysis revealed a common ancestor of several affected calves. CONCLUSIONS AND CLINICAL IMPORTANCE: ZDL syndrome should be suspected in Fleckvieh calves with crusting dermatitis together with diarrhea or respiratory tract inflammations without response to oral zinc supplementation. Definite diagnosis requires molecular genetic confirmation of the PLD4 mutation.

Genetic parameters of calcium, phosphorus, magnesium, and potassium serum concentrations during the first 8 days after calving in Holstein cows.

Calcium, Mg, P, and K are of great importance for the health and productivity of dairy cows after calving. So far genetic studies have focused on clinical hypocalcemia, leaving the genetic parameters of these macroelements unstudied. Our objective was to estimate the genetic parameters of Ca, Mg, P, and K serum concentrations and their changes during the first 8d after calving. The study was conducted in 9 herds located in northern Greece, with 1,021 Holstein cows enrolled from November 2010 until November 2012. No herd used any kind of preventive measures for hypocalcemia. Pedigree information for all cows was available. A total of 35 cows were diagnosed and treated for periparturient paresis and, therefore, excluded from the study. The remaining 986 cows were included in genetic analysis. The distribution of cows across parities was 459 (parity 1), 234 (parity 2), 158 (parity 3), and 135 (parity ≥4). A sample of blood was taken from each cow on d1, 2, 4, and 8 after calving and serum concentrations of Ca, P, Mg, and K were measured in each sample. A final data set of 15,390 biochemical records was created consisting of 3,903 Ca, 3,902 P, 3,903Mg, and 3,682K measurements. Moreover, changes of these concentrations between d1 and 4 as well as 1 and 8 after calving were calculated and treated as different traits. Random regression models were used to analyze the data. Results showed that daily heritabilities of Ca, P, and Mg concentrations traits were moderate to high (0.20-0.43), whereas those of K were low to moderate (0.12-0.23). Regarding concentration changes, only Mg change between d1 and 8 after calving had a significant heritability of 0.18. Genetic correlations between Ca, P, Mg, and K concentrations and their concentration changes from d1 to 4 and 1 to 8 after calving were not significantly different from zero. Most phenotypic correlations among Ca, P, Mg, and K concentrations were positive and low (0.09-0.16), whereas the correlation between P and Mg was negative and low (-0.16). Phenotypic correlations among macromineral concentrations on d1 and their changes from d1 to 4 and 1 to 8 after calving varied for each macromineral. This study revealed that genetic selection for normal Ca, P, Mg, and K concentrations in the first week of lactation is possible and could facilitate the management of their deficiencies during the early stages of lactation.

Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows.

During the periparturient phase, cows are typically in an inflammation-like condition, and it has been proposed that inflammation associated with the induction of stress of the endoplasmic reticulum (ER) in the liver contributes to the development of fatty liver syndrome and ketosis. In the present study, the hypothesis that supplementation of dairy cows with a plant product consisting of green tea (95%) and curcuma extract (5%) rich in polyphenols attenuates inflammation and ER stress in the liver during early lactation was investigated. Twenty-seven cows were assigned to two groups, either a control group (n=14) or a treatment group (n=13). Both groups of cows received a total mixed ration, and the ration of the treatment group was supplemented with 0.175 g of the plant product per kg dry matter from week 3 prepartum to week 9 postpartum. Dry matter intake and energy balance during week 2 to week 9 postpartum were not different between the two groups. However, cows supplemented with the plant product had a greater amount of energy-corrected milk during week 2 to week 9 postpartum and lower concentrations of triacylglycerols and cholesterol in the liver in week 1 and week 3 postpartum than cows of the control group (p<0.05). Cows supplemented with the plant product showed a trend towards a reduced mRNA concentration of haptoglobin (p<0.10), while relative mRNA concentrations of eight genes of the unfolded protein response considered in the liver were not different between the two groups of cows. Relative hepatic mRNA concentration of fibroblast growth factor, a stress hormone induced by various stress conditions, was reduced at week 1 and week 3 postpartum in cows supplemented with the plant product (p<0.05). Overall, the data of this study suggest that--although there were only minor effects on the occurrence of ER stress and inflammation--a supplementation of polyphenols might be useful to improve milk yield and prevent fatty liver syndrome in dairy cows.

Bovine Neonatal Pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the MHC haplotype.

Bovine Neonatal Pancytopenia (BNP), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. A commercial BVD vaccine is the likely source of alloantigens eliciting BNP associated alloantibodies. We hypothesized that the rare occurrence of BNP in calves born to vaccinated dams could be associated with genetic differences within dams and calves. We found that the development of BNP within calves was a heritable trait for dams, not for calves and had a high heritability of 19%. To elucidate which genes play a role in the development of BNP we sequenced candidate genes and characterized BNP alloantibodies. Alloantigens present in the vaccine have to be presented to the dam's immune system via MHC class II, however sequencing of DRB3 showed no differences in MHC class II haplotype between BNP and non-BNP dams. MHC class I, a highly polymorphic alloantigen, is an important target of BNP alloantibodies. Using a novel sequence based MHC class I typing method, we found no association of BNP with MHC class I haplotype distribution in dams or calves. Alloantibodies were detected in both vaccinated BNP and non-BNP dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in BNP dams. We concluded that the development of BNP in calves is a heritable trait of the dam rather than the calf and genetic differences between BNP and non-BNP dams are likely due to genes controlling the quantitative alloantibody response following vaccination.

Investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16S rRNA gene.

The objective of this study was the use of metagenomic pyrosequencing of the 16S rRNA gene for the investigation of postpartum dairy cows' uterine bacterial diversity. The effect of subcutaneous supplementation of a trace mineral supplement containing Zn, Mn, Se, and Cu (Multimin North America, Inc., Fort Collins, CO) at 230 days of gestation and 260 days of gestation on dairy cows' uterine microbiota was also evaluated. Uterine lavage samples were collected at 35 DIM and were visually scored for the presence of purulent or mucopurulent secretion. The same samples were also used for the acquisition of bacterial DNA. The 16S rRNA genes were individually amplified from each sample. Pyrosequencing of the samples was carried at the Cornell University Life Sciences Core Laboratories Center using Roche 454 GS-FLX System Titanium Chemistry. The Ribosomal Database Project online tools were used for the analysis of the obtained sequences library. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., Peptostreptococcus spp., Sneathia spp., Prevotella spp. and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that had a higher uterine lavage sample score. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that were not pregnant by 200 DIM. Anaerococcus spp., Peptostreptococcus spp., Parabacteroides spp., and Propionibacterium spp. prevalence was significantly (P<0.05) lower in samples derived from cows that were trace mineral supplemented.

Bovine CD14 receptor produced in plants reduces severity of intramammary bacterial infection.

CD14 is a high-affinity receptor protein for the complex of bacterial LPS (LPS) and LPS binding protein in animals. Binding of the soluble form of CD14 (sCD14) to LPS, found in the outer membrane of Escherichia coli and other Gram-negative bacteria, enhances host innate immune responses, reduces the severity of mastitis, and facilitates clearance and neutralization of LPS, thus protecting against an excessive immune response to LPS and development of endotoxic shock. A truncated form of sCD14, carrying a histidine residue affinity tag for purification, was incorporated into Potato virus X for transient expression in Nicotiana benthamiana plants. Western blots probed with CD14-specific antibodies demonstrated that crude plant extracts and affinity-purified samples contained immunoreactive sCD14. Biological activity of plant-derived recombinant bovine sCD14 (PrbosCD14) was demonstrated in vitro by LPS-induced apoptosis and interleukin (IL) -8 production in bovine endothelial cells, and in vivo by enhancement of LPS-induced neutrophil recruitment. Finally, in PrbosCD14-infused glands subsequently infected with E. coli, lower numbers of viable bacteria were recovered and there was an absence of clinical symptoms, demonstrating prophylactic efficacy of PrbosCD14. This is the first report of a functionally active animal receptor protein made in plants and the prophylactic use of a plant-derived protein to reduce the severity of bacterial infections in animals.

Identification of allelic variants of the bovine immune regulatory molecule CEACAM1 implies a pathogen-driven evolution.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), the primordial member of the carcinoembryonic antigen (CEA) family, functions as a MHC-independent natural killer (NK) cell inhibitory receptor, regulates T and B cell proliferation, and induces dendritic cell (DC) maturation. Despite these fundamental functions, CEACAM1 and most of the CEA family members differ significantly in primates and rodents. A number of diverse murine and human pathogens use CEACAM1 as a cellular receptor, indicating that the observed species-specific differences are the result of divergent molecular pathogen/host coevolution. To gain deeper insight into its evolution and function, we cloned CEACAM1 cDNA from cattle as a representative of a third mammalian order. Bovine CEACAM1 differs considerably from rodent and primate CEACAM1 due to deletion of the B domain exon which was most likely caused by insertion of LINE/SINE sequences and reveals alternative splicing within the transmembrane exon. However, the characteristic long and short isoforms exist which contain or lack the typical immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tails, respectively. Bovine peripheral blood lymphocytes (PBL) express only ITIM-containing CEACAM1 isoforms, and upregulate their expression upon stimulation, suggesting an inhibitory function in these cells. As found in rodents, two clearly distinct CEACAM1 alleles exist in cattle. In the a allele, a unique deletion of three amino acids is found in the N domain, which is important for pathogen binding in mice and humans. This is consistent with the notion that CEACAM1 serves or has served as a pathogen receptor in cattle.

[Recent findings on the genetics of gastro-intestinal nematode resistance in ruminants]. / Recenti acquisizioni sulla genetica della resistenza ai nematodi gastro-intestinali dei ruminanti.

The control of helminthiases in ruminants raised in open pasture has been mainly undertaken by using prophylactic measures in the environment, but these are often inadequate due to incorrect application. With the appearance of anthelmintics, the strategy for controlling these parasitoses, passed to pharmacological treatments which became effective in reducing their impact. However, the frequent and incorrect utilisation of these molecules resulted in resistance to anthelmintics and the presence of chemical residues in animal products for human consumption. Anthelmintic resistance is widespread throughout the world, heterogeneous and probably underestimated. This has encouraged the introduction of homeopathic agents and products derived from plants whose effectiveness has not been scientifically assessed. It is well known that it is possible to detect differences in resistance to the most important parasites between breeds. In Europe, it has been reported that some ovine autochthonous breeds, Scottish Blackface and Lacaune, showed higher resistance. The implementation of breeding strategies aimed at obtaining animals with naturally low susceptibility to nematode infestations could therefore play an increasingly important role. Standard animal breeding techniques have been largely successful in improving the performance of domestic animals in the last century. Standard quantitative selection requires field data on: i) individual phenotype performance; ii) expected covariance among animals due to blood relationship between them. The whole process of predicting the breeding value of animals in order to select subsequently the genetically superior parents of the next generation is entirely based on sophisticated computations (BLUP-animal model). In sheep, the main objective is always selecting for milk yield and sometimes, in addition, milk composition. However, due to the evolution of the EU agricultural policy and consumer demand in terms of healthy and organic food, more attention is now being given to traits related to health (resistance to EST, mastitis or parasitic diseases). Some studies conducted in New Zealand and Australia showed that nematode resistance is genetically controlled with high heritabilities and quite low genetic correlations with production traits. In this sense, some studies showed that it is possible to decrease the number of parasites in the framework of a traditional breeding programme. However, in most situations, this trait is not extensively recorded due to the high cost of individual recording. Therefore, it would be useful to implement breeding strategies based on the knowledge of the genes involved in this trait expression. Traditionally, two approaches are available to locate a gene: i) genome scan; ii) candidate gene approach. The candidate gene approach attempts to link general resistance to some particular genes. To date, genetic resistance against parasites is considered to be linked with the MHC and IgE genes. Furthermore, several gene detection studies based on the genome scan approach for this trait are currently being carried out on both crossed experimental populations (fat x lean Blackface lines and Sarda x Lacaune) and pure breeds (Churra). The preliminary results seem promising as to the use of marker assisted or genotype assisted selection for this trait, which is difficult and expensive to measure on a population scale.

Growth of Holstein calves from birth to 90 days: the influence of dietary zinc and BLAD status.

The main objective of this study was to describe Holstein neonatal growth and development as influenced by dietary zinc supplementation and the CD18 genotype, both of which may affect immune competence. Holstein calves (n = 421), after being fed colostrum, were brought to a calf facility, randomly assigned to one of four zinc supplementation groups (control at 40 mg Zn/kg DM or the control diet supplemented with an additional 60 mg Zn/kg DM provided as either zinc sulfate, zinc lysine, or zinc methionine), weighed, and measured for morphometric growth parameters. Measurements were repeated at 30, 60, and 90 d. Calves were also genotyped for the presence of the mutant D128G CD18 allele, which, if present in two copies, causes bovine leukocyte adhesion deficiency. Zinc supplementation above 40 mg Zn/kg DM, regardless of the chemical form, did not accelerate growth (P > 0.25). Further, overall calf growth performance was not suppressed or improved (P > 0.4) in calves heterozygous at the CD18 locus relative to calves homozygous for the normal CD18 allele, although genotype negatively affected some morphometric measurements (P < 0.05). Using these data, quadratic models of early growth were generated as a preliminary step to develop growth criteria that will allow producers, veterinarians, and animal scientists to identify poor growth performance early in neonatal life. Such criteria provide the basis for tools to improve economic performance.

Clinical features of renal tubular dysplasia, a new hereditary disease in Japanese Black cattle.

A new hereditary disease characterised by renal failure, poor growth and long hooves in Japanese Black cattle (wagyu) has been recognised in a region of central Japan since 1990. The number of calves affected has increased gradually, with the incidence reaching 17 of 485 (3.51 per cent) in 1995. Almost all the calves were slightly undersized at birth, and repeatedly had diarrhoea during the neonatal period. They began to show signs of growth retardation with proportional body and elongation of the hooves from about two to five months of age, but they had an almost normal or only slightly decreased appetite. The concentrations of urea nitrogen, creatinine and inorganic phosphorus in serum were high, and the affected calves excreted diluted urine frequently. Among 25 cases, the urine of 21 contained occult blood, 24 contained protein and two contained glucose. In 29 calves observed for 30 to 130 days, the course of the disease varied; in 21 of them it remained unchanged, six became gradually worse and two became severely debilitated and died. The disease was diagnosed as renal tubular dysplasia by histopathological examination.

A novel stop codon mutation (X417L) of the ferrochelatase gene in bovine protoporphyria, a natural animal model of the human disease.

Protoporphyria (PP) is caused by a deficiency of ferrochelatase (FC) activity, which catalyzes the final step in the heme biosynthesis pathway. Bovine are the only species other than man with naturally occurring PP. For expression of the PP phenotype, two copies of the mutated gene are necessary in bovine, whereas one copy is sufficient in humans. We report the first potential disease-causing mutation in the bovine FC gene. The coding region of FC was sequenced from the liver tissue of protoporphyric and normal bovine. A transversion was identified at nucleotide position 1250 which changed the stop codon to leucine (TGA-->TTA) in the protoporphyric FC sequence. As a consequence, the mutant protein is predicted to have an additional 27 amino acids. To screen other bovine for the G-->T transversion, cDNAs from liver tissue of clinically and biochemically normal, and from heterozygous and homozygous affected animals were used for allele-specific polymerase chain reaction. Three normal animals had only the G allele, five affected animals had only the T allele, and three heterozygous animals had both the G and T alleles. These results support our hypothesis that this mutation causes PP in bovine.

Zinc metabolism in animals: pathology, immunology and genetics.

Deficiency of dietary Zn rapidly reduces both appetite and growth, the latter effect being apparently caused by a failure of cell replication. Diagnosis of presymptomatic Zn deficiency depends largely on estimation of plasma Zn concentration but is complicated by reductions of it during a range of stressful conditions. In the latter cases, the decrease in plasma Zn concentration does not appear to be associated with inadequate Zn intake. Only two genetic defects of Zn metabolism are known in animals. One is associated with lethally inadequate concentrations of Zn in the milk of mice, the other with the A46 trait in Friesian cattle. A46 is a recessively inherited defect of Zn absorption which is lethal in the absence of major Zn supplementation of the diet. The characteristics of the disease are very similar to those of acrodermatitis enteropathica in man.

Intestinal absorption of 65 Zinc in A46 (Adema disease) after treatment with oxychinolines.

In two calves with the genetic defect A46 (Adema disease) intestinal absorption of 65Zn was increased during a period of oral treatment with oxychinolines. Oral supplementation with zinc oxide resulted in extremely high plasma zinc concentrations and a moderate increase in 65Zn absorption.