Biallelic IARS Mutations Cause Growth Retardation with Prenatal Onset, Intellectual Disability, Muscular Hypotonia, and Infantile Hepatopathy.

tRNA synthetase deficiencies are a growing group of genetic diseases associated with tissue-specific, mostly neurological, phenotypes. In cattle, cytosolic isoleucyl-tRNA synthetase (IARS) missense mutations cause hereditary weak calf syndrome. Exome sequencing in three unrelated individuals with severe prenatal-onset growth retardation, intellectual disability, and muscular hypotonia revealed biallelic mutations in IARS. Studies in yeast confirmed the pathogenicity of identified mutations. Two of the individuals had infantile hepatopathy with fibrosis and steatosis, leading in one to liver failure in the course of infections. Zinc deficiency was present in all affected individuals and supplementation with zinc showed a beneficial effect on growth in one.

Alterations of growth, blood biochemical components and hormone profiles by intensified nutrition in growth retarded Japanese Black cattle.

In order to determine the clinical conditions of Japanese Black (JB) cattle with growth retardation, we determined the changes of body growth, blood profiles of metabolism and hormones caused by intensified nutrition (sufficient total digestible nutrients and digestible crude protein for a target daily gain set at 1.2-1.3 kg/day) in three cattle. The daily gain (DG) was increased during the intensified period (Intense) compared with the preparation period (Pre), but the DG in the Intense period was 36-66% of the target DG. Serum albumin, total cholesterol, insulin and IGF-1 increased during the Intense period compared with the Pre period. Serum GH showed high levels in the Pre period, whereas it showed lower levels in the Intense period. These results suggested that the present growth retarded cattle had abnormalities in their metabolic systems and lacked nutrient absorption.

Relationship between immune function and serum vitamin A in Japanese black beef cattle.

This study evaluated the relationship between the serum vitamin A level and immune function in seventy non-diseased Japanese Black (JB) cattle during their fattening stages. The animals were divided into two groups, a Low Vitamin A (VA) group (N=9) with below 30 IU/dl of serum VA and a Control group (N=61). Blood samples were collected for biochemical analysis and examination of the leukocyte population and cytokine mRNA expression. The numbers of CD3(+)WC1(+) T cells and MHC class-ll(+)CD14(-) B cells were significantly lower in the Low VA group than in the Control group (P<0.05). The IFN-gamma/IL-4 rate was significantly lower in the Low VA group, while IL-4 was higher and IFN-gamma was lower in the Low group compared with the Control group. This study indicated that immune function imbalance was present in JB cattle with low serum VA levels during the fattening stage.

Effect of lidocaine hydrochloride on the function of bovine peripheral leukocytes.

To clarify the effect of lidocaine hydrochloride (Lid) on bovine peripheral granulocyte phagocytosis, adhesion molecule expression of leukocytes and peripheral blood mononuclear cell mRNA expression of cytokines were investigated. Lid was added to blood samples at a final concentration of 0 (only PBS; Cont), 0.2 mg/ml or 2.0 mg/ml. Phagocytosis of granulocytes was significantly decreased by addition of 2.0 mg/ml of Lid. CD18 expression of granulocytes and mononuclear cells were significantly reduced by addition of 2.0 mg/ml of Lid. IL-1beta and IL-8 mRNA expressions of mononuclear cells were also significantly reduced by addition of 2.0 mg/ml of Lid other hand. These results suggest that Lid might reduce the protective immunity of cows. On the other hand, reduction of CD18, IL-1beta and IL-8 mRNA expression also indicates that Lid has an anti-inflammatory effect in cows.

Peripheral blood leukocyte subpopulation of dairy cows with digital dermatitis and effect of hoof trimming with antibiotic treatment.

In the present study, 30 cows were used to evaluate the changes in the peripheral blood leukocyte subpopulation of dairy cows with digital dermatitis (DD) following hoof trimming and antibiotic treatment. The cows were divided into two groups; 18 cows (DD group) had DD on both hind feet, and 12 cows (control group) had four feet with no clinical abnormalities. The DD group was further divided into two groups based on the treatment; the antibiotic group (8 cows) was treated with only 2% lincomycin liquid spray once daily for 3 days, and the trimmed group (10 cows) received trimming of hooves as well as treatment with 2% lincomycin liquid spray. The plasma cortisol concentration was significantly higher in both DD groups before treatment than in the control group, and it decreased significantly after hoof trimming in the trimmed group. The number of CD3(+), CD4(+), WC1(+) and CD21(+) cells in both DD groups before treatment was significantly lower than that of the control group. The number of CD3(+), CD4(+), WC1(+) and CD21(+) cells in the trimmed group increased after treatment. These results indicated that cows with DD suffer from stress and reduced number of T and B cells. Treatment of DD with both hoof trimming and 2% lincomycin liquid spray was effective for reducing the stress and bringing the immune cell number back to the normal range.

Changes in peripheral blood mononuclear cells after calving in lactating cows with serious decrease of body weight before and after calving.

The aim of this study was to clarify the hormonal and immunological changes in periparturient dairy cows with excessive weight loss following calving. We analyzed leukocyte populations and the peripheral blood mononuclear cell (PBMC) mRNA of IFN-gamma, IL-4, growth hormone (GH)-receptor (R) and insulin -R and measured the GH and insulin concentrations. Ten dairy cows fed in one herd were used in this study and were divided into two groups. Five cows that experienced loss of body weight following calving of over 10% comprised the Weight Loss Group, and the remaining five cows, which experienced loss of body weight of 5% or less following calving, served as the Control Group. The Weight Loss Group had high serum cholesterol and low blood urea nitrogen levels compared with the Control Group throughout the period of observation. In regard to the leukocyte populations, there were significantly fewer CD4(+)CD45R-T cells in the Weight Loss Group 1 to 2 months after calving compared with the Control Group. The percentage of IFN-gamma/IL-4 among PBMCs in the Weight Loss Group tended to be lower than in the Control Group throughout the observation period. In the Weight Loss Group, the levels of GH-R during month 2 were lower than in the Control Group. We concluded that excessive loss of body weight in dairy cows following calving is associated with depressed cellular immune function.

Effect of hoof trimming before the dry period on productive performance in perinatal dairy cows.

To investigate the effect of hoof trimming before the dry period, the hooves of 10 cows (trimmed group) were trimmed at 79.6 +/- 8.6 days before parturition and the hooves of 52 cows were left untrimmed (control group). Blood biochemistry and hormone concentrations were investigated for 6 cows from each group. The daily milk yield after parturition in the trimmed group tended to be higher than that of the control group. Between 0 and 30 days after parturition, the levels of non-esterified fatty acids were significantly lower stet, and the plasma glucose and glucose disposal rates were significantly higher in the trimmed group. The plasma cortisol concentration was stable before and after parturition in the trimmed group. Hoof trimming before the dry period appears to reduce stress and maintain the nutritional conditions of perinatal dairy cows.

Cervical ganglioneuroblastoma in a new born Japanese Black calf.

This case report describes a congenital ganglioneuroblastoma in a 38-day-old male Japanese Black calf. The cervical multinodular mass was present at birth and grew rapidly. The cut surface was pale gray-to-yellow and had a gelatinous appearance. Hemorrhagic cysts of various sizes were observed in the nodule. Histologically, the mass contained clusters of neuroblastic cells, ganglionic cells, and Schwann-like cells. Immunohistochemically, the ganglionic cells showed strong positivity for neuron-specific enolase, neurofilament, synaptophysin, and chromogranin A, whereas the Schwann-like cells strongly expressed S-100, glial fibrillary acidic protein, and vimentin. Ultrastructurally, neurosecretory granules resembling catecholamine were observed in the neuroblastic and Schwann-like cells. Based on the pathology, the diagnosis was congenital cervical nodular ganglioneuroblastoma.

Uterine and ovarian blood flow in a Holstein Friesian cow with aplasia of one uterine horn.

The ovarian dynamics and uterine and ovarian blood flows of a 6-year-old (2 parturitions) Holstein Friesian cow with right uterine horn aplasia were observed during two estrous cycles. In one estrous cycle, a corpus luteum (CL) formed in the right ovary, but regression of the CL and subsequent ovulation were not observed. In the other estrous cycle, a CL formed in the left ovary and delayed regression of the CL and subsequent ovulation were observed. The blood velocity of the right uterine artery was lower than that of the left uterine artery throughout both estrous cycles when a CL formed in either the right and left ovary. The blood velocities of the right and left ovarian arteries were unaffected by right uterine horn aplasia and changed depending on the presence of a CL. These results indicated that the blood flow of the right uterine artery was very weak and that aplasia of one uterine horn affects the estrous cycle, especially CL regression.

Evaluation by weight change rate of dairy herd condition.

The purpose of this study was to clarify whether weight change rate (WCR) is useful in evaluating herd health. Five herds (A, B, C, D, and E) were examined monthly for approximately 1 year to measure both WCR and BCS. WCR was calculated using the following equation: WCR (%) = (body weight of current month-body weight of the preceding month)/body weight of current month x 100. In addition, blood profiles were performed in each herd before and after calving. The incidence of periparturient diseases was higher in Herds A, B, and C compared with Herds D and E. Reproductive performance in Herds A and B was rather poor compared with Herds C, D, and E. There were significantly lower WCRs in Herd A compared with Herd C during the period of day -30 to day 30 after calving. According to the blood profiles, high levels of non-essential fatty acids (NEFA) were observed during the period of day -30 to -1 and high beta-hidroxybutyricacid (BHB) and low levels of glucose were observed at day 30 after calving in Herd A. A marked poor condition in Herd A during the peripaturient period was clearly expressed by low WCR in this experiment. WCR can express body condition of herds and is suggested to be a useful parameter in the determination of herd performance in field experiments.

Peripheral mononuclear cell response in Japanese black calves after oral administration of IFN-alpha.

To investigate the effects of oral administration of an interferon (IFN)-alpha drug on the immune reaction of healthy Japanese Black (JB) calves, peripheral leukocyte populations and their ability to produce cytokine mRNA were analyzed after oral administration of IFN-alpha. Fourteen calves fed in one herd were divided into two groups; seven calves were orally administered 0.1 g/day of IFN-alpha from the day of birth to day 5 on each day (group 1, N=7), and the other seven calves were used as the control (group 2, N=7). Blood samples were collected from the jugular veins of all calves before administration and in weeks 1, 2, 4, 8, and 16 after birth. The number of MHC class II+CD14+ monocytes in the leukocytes population of group 1 increased gradually after birth, and significantly higher numbers were detected in week 4 compared with group 2. MHC class II-CD14+ monocytes in group 1 peaked in week 1, and a significant increase was detected compared with group 2. The level of IL-12 in the cytokine mRNA of group 1 increased gradually between weeks 1 and 2, and a significantly higher level of IL-12 was found compared with group 2. These results suggest that oral administration of IFN-alpha induces activation of the monocyte functions in JB calves.

Comparison of two different nutritive conditions against the changes in peripheral blood mononuclear cells of periparturient dairy cows.

To clarify the relationship between cellular immune status and nutritive condition in periparturient dairy cows, feeding content, blood profiles, and immune condition were observed in cows from two dairy herds with different types of feed content. Immunological analyses such as leukocyte population and peripheral blood mononuclear cell (PBMC) mRNA of IFN-gamma, TNF-alpha, IL-4, and IL-10, quantified by real-time RT-PCR were performed. With regard to feed content during dry periods, there were six cows in the herd with insufficient non-structural carbohydrate (NFC) intake (group I) and six cows in the herd with sufficient NFC intake (group II). Significantly lower levels of blood glucose were observed in group I between weeks -12 and 16 compared with group II. Serum cholesterol level was significantly lower in group I between weeks 2 and 10 than in group II. The numbers of CD3+ and CD4+ T cells in group I were significantly lower than those in group II in weeks 6 and 14. The numbers of CD21+ B cells were significantly lower in group I than in group II in weeks -16, -12, 2, and 10. On the other hand, the CD4+/CD8+ ratio in group II was significantly higher than group I between weeks 2 and 14. The IFNgamma/IL-4 mRNA rate in group I was significantly lower than group II in week 6. We concluded that cellular immune depression occurrs after calving in dairy cows with low nutritional status in the periparturient period.

Comparison of the insulin reaction of peripheral blood T cells between healthy Holstein dairy cows and JB during the periparturient period.

To compare the changes in the insulin reaction of Holstein dairy cows and Japanese Black cows (JB) during the periparturient period, the insulin resistance test in vivo and lymphocytes proliferation with insulin in vitro were performed. Ten healthy Holstein dairy cows (Holstein group) and 10 healthy JB cows (JB group) used in this study were observed on days 60, 40, and 20 before calving and days 7 and 20 after calving. In insulin resistance reaction in vivo and in vitro, a low insulin-stimulated glucose disposal rate and lymphocyte proliferation with insulin were observed in the Holstein group compared with the JB group during the experimental period. An analysis of the lymphocytes cultured with insulin showed that the percentage of CD4+CD45R- T cells in the Holstein group was significantly lower than that of the JB group before day 20. These findings indicate that T cells reaction to insulin in healthy periparturient Holstein cows is lower than that in Japanese Black.

Carrier rate of Factor XI deficiency in stunted Japanese black cattle.

Blood examinations and genotyping of Factor XI (F11) were performed in growth retardation Japanese Black cattle and their dams. Genotyping of F11 revealed that the recessive homozygous and heterozygous genotype frequencies were 5.2% and 50.0% in the Claudin-16 (CL-16) deficiency group (n=58), 0% and 14.2% in the renal dysplasia group (n=7), 0% and 26.1% in the non-CL-16 deficiency nephritis group (n=23), 8.9% and 46.7% in the hypogenesis syndrome group (n=45), 6.2% and 25.0% in the neonatal weak calf syndrome group (n=32), 9.1% and 38.6% in the respective dams group (n=44), 0% and 23.1% in the normal cattle group (n=13), and 5.9% and 38.2% in total (n=222), respectively. These results showed that the carrier rate of F11 deficiency was high in Japanese Black cattle, and that the CL-16 deficiency, hypogenesis syndrome, neonatal weak calf syndrome, and dams groups had a large amount of recessive homozygous genotype than the other groups. No abnormal bleeding was observed clinically in the present study, and 4 of the recessive homozygous dams showed normal growth and parturition.

Abnormal development of nephrons in claudin-16-defective Japanese black cattle.

The kidneys of 37 Japanese Black calves aged 2 to 65 months diagnosed with Claudin 16 (CL-16) defect by the DNA-based test were examined pathologically. The animals exhibited clinical symptoms such as growth impairment, renal failure, overgrowth of hooves, and anemia at a young age. There was no correlation between the time of onset and age. Kidney weights relative to body weight were similar to those in normal animals, but both kidney net weights and size were reduced due to atrophy in animals that showed severe renal dysfunction. Histopathological examination of the kidneys showed reduction in the number of glomeruli, compensatory hypertrophy of glomeruli and tubules, and glomerular and tubular atrophy accompanied by interstitial fibrosis and lymphocytic infiltration. Glomeruli were clearly less in number in the kidneys of CL-16-defective animals than those of normal animals even in the cases with mild lesions. A small number of immature glomeruli and tubules were also detected, suggesting that there were fewer nephrons developed at birth in CL-16-defective animals. It was suggested that a defect of the CL-16 gene is involved in the "abnormal development of nephrons". Immunohistopathological examination of the kidneys showed that the epithelium of thick ascending limb of Henle was stained with anti-CL-16 antibody in the control animals, but not in the affected animals, suggesting a defect of CL-16 in the epithelium of renal tubules in the affected animals.

Hormonal differences in peripheral blood and gene profiling in the liver and lymphocytes in Japanese black cattle with growth retardation.

Japanese Black cattle occasionally demonstrate growth retardation despite sufficient nutrient intake. To clarify hormonal and transcriptional characteristics, we investigated differences in blood components, including hormones, and differences in exhaustive gene expressions in the liver and peripheral lymphocytes of six cattle with growth retardation (GR cattle) and eight control cattle of the same age and pedigree with normal growth. Hematocrit values and concentrations of hemoglobin, serum albumin, total cholesterol, insulin-like growth factor 1 (IGF-1), thyroxine and insulin in GR cattle were significantly lower than those in controls. GR cattle also excreted higher levels of GH. We used three GR and three control cattle for a microarray analysis in the liver and found that 279 gene expressions were significantly different. However, gene expressions related to the GH-IGF-1 axis, such as the GH receptor and IGF-1, were not significantly different from those of controls. Immune-related gene expressions were significantly lower. To clarify these gene expression levels, peripheral lymphocytes were used for real-time RT-PCR. The expression rates of genes that were significantly lower in the liver, such as chemokine ligand 8, interferon gamma receptor 1 and immunoglobulin light chain VJ region were also significantly lower in three GR cattle than those in the three control cattle. These results suggest that the cause of growth retardation in the present study was due to other factors, not abnormal gene expressions of factors related to the GH-IGF-1 axis in the liver, and that GR cattle were susceptible to infectious disease.

Mapping and exome sequencing identifies a mutation in the IARS gene as the cause of hereditary perinatal weak calf syndrome.

We identified an IARS (isoleucyl-tRNA synthetase) c.235G>C (p.Val79Leu) substitution as the causative mutation for neonatal weakness with intrauterine growth retardation (perinatal weak calf syndrome). In Japanese Black cattle, the syndrome was frequently found in calves sired by Bull A. Hence, we employed homozygosity mapping and linkage analysis. In order to identify the perinatal weak calf syndrome locus in a 4.04-Mb region of BTA 8, we analysed a paternal half-sibling family with a BovineSNP50 BeadChip and microsatellites. In this critical region, we performed exome sequencing to identify a causative mutation. Three variants were detected as possible candidates for causative mutations that were predicted to disrupt the protein function, including a G>C (p.Val79Leu) mutation in IARS c.235. The IARS c.235G>C mutation was not a homozygous risk allele in the 36 healthy offspring of Bull A. Moreover, the IARS Val79 residue and its flanking regions were evolutionarily and highly conserved. The IARS mutant (Leu79) had decreased aminoacylation activity. Additionally, the homozygous mutation was not found in any of 1526 healthy cattle. Therefore, we concluded that the IARS c.235G>C mutation was the cause of hereditary perinatal weak calf syndrome.