Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.     

Lactobacillus rhamnosus GG SpaC pilin subunit binds to the carbohydrate moieties of intestinal glycoconjugates

Lactobacillus rhamnosus GG (LGG) is a well‐established probiotic strain. The beneficial properties of this strain are partially dependent on its prolonged residence in the gastrointestinal tract, and are likely influenced by its adhesion to the intestinal mucosa. The pilin SpaC subunit, located within the Spa pili structure, is the most well studied LGG adhesion factor. However, the binding epitopes of SpaC remain largely unknown. The aim of this study was to evaluate the binding properties of SpaC to the carbohydrate moieties of intestinal glycoconjugates using a recombinant SpaC protein. In a competitive enzyme‐linked immunosorbent assay, SpaC binding was markedly reduced by addition of purified mucin and the mucin oligosaccharide fraction. Histochemical staining revealed that the binding of SpaC was drastically reduced by periodic acid treatment. Moreover, in the surface plasmon resonance‐based Biacore assay, SpaC bound strongly to the carbohydrate moieties containing β‐galactoside at the non‐reducing terminus of glycolipids. We here provide the first demonstration that SpaC binds to the oligosaccharide chains of mucins, and that the carbohydrate moieties containing β‐galactoside at the non‐reducing termini of glycoconjugates play a crucial role in this binding. Our results demonstrate the importance of carbohydrates of SpaC for mucus interactions.     

Effects of a high milk intake during the pre‐weaning period on nutrient metabolism and growth rate in Japanese Black cattle

This study aimed to determine the effects of feeding an increased volume of high‐fat milk during the early post‐natal life on metabolite concentrations in the blood, the expression of key genes regulating intermediary metabolism in the skeletal muscles, and the rate of growth of Japanese Black cattle. All calves were fed a high‐fat milk replacer (crude protein, 26%; crude fat, 25.5%; total dissolved nitrogen, 116%). Control calves (n = 4) were nursed with 500 g milk replacer until 3 months of age, whereas calves in the experimental group (n = 4) were nursed with 1800 g milk replacer until 3 months, and then the volume was gradually reduced until 5 months. Body weight was significantly higher in the experimental group than in the control group at 7 months. Plasma glucose concentrations were significantly lower in the experimental group. Expression of glucose‐transporter‐4 messenger RNA (mRNA) was lower, whereas that of glucose transporter 1, cluster of differentiation 36, and carnitine palmitoyltransferase‐1b mRNA was significantly higher in the Longissimus thoracis of the experimental group. Nutritional status during early post‐natal life appears to strongly influence the growth rate and glucose and lipid metabolism in Japanese Black cattle.     

Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of reproductive function in immature female Japanese quail (Coturnix japonica)

In a previous study, we found that 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust particles and also a degradation product of the insecticide fenitrothion, exhibits reproductive toxicity in the adult male Japanese quail. The present study investigated the toxicity of PNMC in the female Japanese quail and its ability to influence reproduction in immature females. The quail (21-day-old) were injected intramuscularly (im) with PNMC at doses 0.1, 1 or 10 mg/kg body weight daily for 3 days. There was no significant difference in body growth between the PNMC-administered and control birds. However, the weights of the oviducts were significantly lower in the PNMC-treated birds at all doses. Furthermore, the plasma concentrations of luteinizing hormone (LH) and estradiol-17 beta were significantly decreased with 1 and 10 mg/kg of PNMC. These findings suggest that PNMC might influence the hypothalamo-pituitary-gonadal axis with decreasing in secretion of GnRH, LH and ovarian steroid hormones and subsequently disturb growth of the reproductive organs of immature female quail. This study indicates that PNMC induces reproductive toxicity at the central level and disrupts reproductive function in the immature female quail.     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.     

Frequency of and risk factors for epistaxis associated with exercise-induced pulmonary hemorrhage in horses: 251,609 race starts (1992-1997)

OBJECTIVE: To determine the frequency of epistaxis during or after racing among racehorses and identify factors associated with development of epistaxis. DESIGN: Retrospective study. SAMPLE POPULATION: 247,564 Thoroughbred and 4,045 Anglo-Arab race starts. PROCEDURE: Race start information (breed, age, sex, racing distance, and race type) was obtained for Thoroughbred and Anglo-Arab horses racing in Japan Racing Association-sanctioned races between 1992 and 1997. All horses that raced were examined by a veterinarian within 30 minutes of the conclusion of the race; any horse that had blood at the nostrils was examined with an endoscope. If blood was observed in the trachea, epistaxis related to exercise-induced pulmonary hemorrhage (EIPH) was diagnosed. RESULTS: Epistaxis related to EIPH was identified following 369 race starts (0.15%). Frequency of EIPH-related epistaxis was significantly associated with race type, age, distance, and sex. Epistaxis was more common following steeplechase races than following flat races, in older horses than in horses that were 2 years old, following races < or =1,600 m long than following races between 1,601 and 2,000 m long, and in females than in sexually intact males. For horses that had an episode of epistaxis, the recurrence rate was 4.64%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that frequency of EIPH-related epistaxis in racehorses is associated with the horse's age and sex, the type of race, and the distance raced. The higher frequency in shorter races suggests that higher intensity exercise of shorter duration may increase the probability of EIPH.     

Immunohistochemical detection of apolipoprotein B-100 and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs

Accumulation of lipids and hyalinosis in the splenic arteries of aged dogs are frequently detected by routine histopathologic examinations. The purpose of this study was to pinpoint the localization of canine apolipoprotein B-100 (CApoB-100) and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs (n = 80) through the use of immunohistochemical techniques. CApoB-100 deposits were found in the subendothelial space, extracellular matrix, and atheromatous lesions in the tunica media of the arteries in dogs > or = 6 years of age. Foamy cytoplasm of the infiltrated macrophages was also CApoB-100 immunopositive. In dogs > or = 10 years of age, almost all central arteries were CApoB-100 immunopositive. Hyaline deposits within the wall were characterized by immunopositivity against canine IgA, IgM, IgG, and albumin. Lipid accumulation in splenic arteries may be an age-related lesion and a precursor of the atheromatous plaques associated with splenic hemorrhage and infarcts later in life. In addition, deposition of immunoglobulins, probably mediated by immune complexes, may play an important role in the development of canine vascular diseases similar to human disease.     

Changes in serum thyroid hormone levels in newborn calves as a diagnostic index of endemic goiter

Maximum serum thyroxine (T4) and triiodothyronine (T3) levels of healthy calves were seen at 1 day after birth, and thereafter rapidly decreased until 5 days after birth. They stabilized until 2 weeks after birth, then gradually decreased until 4 weeks after birth. Serum T4 levels of calves with endemic goiter tended to be lower than those of healthy ones, but showed similar levels to those of adult cows. T3 levels of calves with goiter were similar to those of healthy ones, but showed higher variation. T4/T3 ratio of calves with goiter were significantly lower than those of healthy ones and adult cows. While individual levels of serum T4 and T3 at just after birth could not be considered as a diagnostic index, the T4/T3 ratio could be adopted as a diagnostic index of endemic goiter.     

Mitotic disruption by a novel pyrimidine herbicide, NS-245852, in oat (Avena sativa L.) roots

The effects of a novel pyrimidine herbicide, NS-245852 [2-chloro-6-fluorophenyl-4-(trifluoromethyl)thieno[2,3-d]pyrimidine-2-yl-ketone], on mitosis in oat ( Avena sativa L. cv. Zenshin) root tips were investigated by using light and immunofluorescence microscopy. The root growth was strongly inhibited at 10⁻⁷ mol L⁻¹ of NS-245852, and swollen root tips were induced at 5 × 10⁻⁸ mol L⁻¹. As observed by the use of light microscopy, the herbicide produced disrupted mitosis and large polynucleate cells in the meristematic root tissue. These symptoms were similar to those of mitotic disrupter herbicides. The immunofluorescence microscopy studies of the root tip cells treated for 30 min revealed that spindle fibers and the preprophase band were reduced, although kinetochore fibers and the phragmoplast were not affected. Kinetochore fibers remained as small fluorescence spots, and the phragmoplast disappeared after a 3 h treatment. No microtubule arrays were observed by a longer treatment (longer than 3 h). Among the microtubule arrays, spindle fibers and the preprophase band were found to be the most sensitive to the herbicide, whereas kinetochore fibers were the most resistant. The phragmoplast was intermediate. Thus, the primary action of NS-245852 is the inhibition of polymerization of tubulin into microtubules.