Horizontal transmission and host-virulence attenuation of totivirus in violet root rot fungus Helicobasidium mompa

Monokaryotic strains of Helicobasidium mompa were used as vectors of a mycovirus between various H. mompa isolates to examine the transmissibility of one of the mycoviruses, totivirus (HmTV1–17 virus) in the hypovirulent isolate V17 of H. mompa. The isolates that acquired HmTV1–17 virus were also examined for any alteration in the virulence. Twelve dikaryotic isolates of H. mompa, belonging to 11 mycelial compatibility groups (MCGs) and being mycelially incompatible with isolate V17, were used as recipients of HmTV1–17 virus. Two monokaryotic isolates that were mycelially incompatible with isolate V17 and all of the recipients were also used as vectors of HmTV1-17 virus between isolate V17 and the recipients. When isolate V17 and recipients were directly paired on plate media, HmTV1-17 virus was transmitted from isolate V17 into 2 of the 12 recipients (i.e., 2 of the 11 MCGs). Two monokaryotic strains were paired with isolate V17, and the monokaryotic strains that acquired HmTV1-17 virus were then used as new virus donors. When the monokaryotic strains containing HmTV1-17 virus were paired with the 12 recipients, HmTV1-17 virus was transmitted into 7 of the 12 recipients from the monokaryotic strains (i.e., 7 of 11 MCGs). Based on these results, we concluded that monokaryotic strains could act as vectors to transmit HmTV1-17 virus into H. mompa isolates. When four of the H. mompa isolates that acquired HmTV1-17 virus were used to inoculate apple rootstock Malus prunifolia, the virulence of all of the isolates was attenuated from that of their parental isolates. Moreover, because the DNA fingerprints of the fungal isolates that acquired HmTV1-17 virus were the same as those of their parental isolates, the infection with HmTV1-17 virus is considered the cause of virulence attenuation of H. mompa.     

Prevention of estrus in the queen with chlormadinone acetate administered orally.

The possibility of estrus prevention in the queen by the oral administration of chlormadinone acetate was examined. The animals used were 29 mature and 15 immature queens. For 16 mature females, 4-12.5 mg was given daily by mouth for 7 days every 3 months. Ten of the 16 queens given this treatment came into estrus within 4 months of the first treatment. For 28 females including the immature, 2-12.5 mg was given once a week throughout the experiment. This treatment prevented estrous activity for at least 1 year. In the queens in this study, the side effects were not observed excepting an increase in body weight during treatment. Our results showed that oral administration of this drug weekly is safe and reliable for long-range prevention of estrus in queens.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

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.     

Changes in the myocardial performance index during dobutamine administration in anesthetized cats

OBJECTIVE: To investigate the relationship between myocardial performance index (MPI; also known as the Tei index) and cardiac function in anesthetized cats administered dobutamine. ANIMALS: 6 adult cats. PROCEDURES: Cats were anesthetized by administration of propofol (6 mg/kg, IV), and anesthesia was maintained by administration of isoflurane. Heart rate and systolic arterial pressure (SAP) were monitored. Stroke volume, cardiac output, and aortic blood flow (ABF) were measured by use of transesophageal ultrasonography. Left ventricular fractional shortening (LVFS), mitral E-wave velocity-to-A-wave velocity (E:A) ratio, and ejection time were measured by use of transthoracic echocardiography. Dobutamine was administrated via a cephalic vein at rates of 2.5, 5.0, and 10 microg/kg/min. RESULTS: Heart rate, SAP, cardiac output, and ABF increased with dobutamine administration, whereas stroke volume significantly decreased. The LVFS significantly increased, and the E:A ratio significantly decreased. Total isovolumic time and the MPI significantly decreased. The MPI was negatively correlated (r=-0.63) with LVFS. Conversely, the MPI was positively correlated with the E:A ratio (r=0.47), stroke volume (r=0.66), and total isovolumic time (r=0.95). However, the MPI was not significantly correlated with heart rate, SAP, cardiac output, or ABF. CONCLUSION AND CLINICAL RELEVANCE: Analysis suggested that the MPI provides a sensitive clinical assessment of cardiac response to medication in cats, which may be similar to the usefulness of the MPI reported in humans.     

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.     

Androgen induces production of male effect pheromone in female goats

Previously we showed that the primer pheromone responsible for the "male effect" was produced in specific skin regions of castrated male goats by androgen treatments. In the present study, we examined whether androgen can also induce production of the male effect pheromone in female goats. Capsules containing dihydrotestosterone (DHT) or testosterone (T) were subcutaneously implanted into six ovariectomized (OVX) goats for 28 days. Small skin samples were collected from the head and rump regions, and the pheromone activity of their ether extracts was examined using a bioassay that monitors the electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator as multiple-unit activity. Behaviors of OVX goats towards ovary-intact estrous goats were also examined before and at the end of DHT or T treatment. Before androgen treatment, neither the head nor rump skin samples in OVX goats showed pheromone activity. DHT treatment induced pheromone activity in the head skin sample of six OVX goats and in the rump skin sample of two OVX goats. Similar results were obtained by T treatment. In addition, OVX goats treated with T showed masculine-type sexual behaviors such as courtship and mounting behaviors towards the estrous goats. These results demonstrate that androgen is capable of inducing primer pheromone activity in the female and suggest that the synthesis pathway of the male effect pheromone exists in both sexes in the goat.     

Effects of long-term administration of recombinant bovine tumor necrosis factor-alpha on glucose metabolism and growth hormone secretion in steers

OBJECTIVE: To investigate the effects of long-term administration of recombinant bovine tumor necrosis factor-alpha (rbTNF) on plasma glucose and growth hormone concentrations, and to determine whether treatment with rbTNF causes insulin resistance in steers. ANIMALS: 5 steers treated with rbTNF and 5 steers treated with saline (0.9% NaCl) solution (control). PROCEDURES: In experiment 1, rbTNF (5.0 microg/kg of body weight) or saline solution (5 ml) was administered SC daily for 12 days. Blood samples were obtained before treatment, and plasma was harvested for determination of glucose, insulin, and growth hormone (GH) concentrations. In experiment 2, insulin, glucose, or growth hormone-releasing hormone (GHRH) was administered IV on days 7, 9, and 11, respectively, after initiation of rbTNF or saline treatment in experiment 1. Plasma glucose and insulin concentrations were measured before and at various times for 4 hours after insulin or glucose administration. Plasma GH concentrations were measured at various times for 3 hours after GHRH administration. RESULTS: In experiment 1, administration of rbTNF resulted in hyperinsulinemia without hypoglycemia and decreased plasma GH concentrations. In experiment 2, plasma glucose concentrations were higher in steers treated with rbTNF and insulin than in controls. Plasma GH concentrations were lower in steers treated with rbTNF and GHRH than in controls. CONCLUSIONS AND CLINICAL RELEVANCE: Prolonged treatment with rbTNF induced insulin resistance and inhibited GHRH-stimulated release of GH in steers. Results indicate that rbTNF is a proximal mediator of insulin resistance and inhibits release of GH during periods of endotoxemia or infection.     

Examination of the pathogenesis of diabetic nephropathy in OLETF rats

We conducted protein loading to examine the progression and pathogenesis of diabetic nephropathy. For this experiment, male OLETF, LETO, F344 and BN rats were used. This experiment was performed on rats between 5 and 30 weeks of age. Examination parameters included body weight, food intake, oral glucose tolerance test (OGTT), urinary protein level (UP), urinary albumin level (UA), glomerular filtration rate (GFR), kidney weights, light microscopy (LM) and electron microscopy (EM). In the protein-loaded OLETF group, the UP level was markedly increased 20 weeks or more after birth. In OLETF control group, GFR were higher than those in other strains. Glomerular hypertrophy and kidney weights were markedly increased in protein-loaded groups in OLETF rats. Thirty weeks after birth, EM showed that the number of polyethyleneimine (PEI) of the glomerular basement membrane (GBM) in protein-loaded OLETF group was significantly decreased compared to that in control group. These changes in OLETF rats were more marked in the protein-loaded group than those in the control group. LM showed that the number of exudative lesions with fibrin-cap in the protein-loaded OLETF group was significantly increased than those in control group. In OLETF rats, protein loading caused deterioration of nephropathy at 30 weeks of age. Therefore, it was demonstrated that not only blood sugar control but also protein intake factors play important roles in the deterioration of nephropathy in OLETF rats.