Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.     

Studies of foetuses from cows clinically affected with bovine leucosis

Five foetuses varying stages of gestation were recovered from cows showing clinical signs of infection with bovine leukaemia virus (BLV). No pathological changes were found in the foetuses although BLV was isolated from 2 of these foetuses. No antibody to BLV could be detected by virus-neutralisation or immunodiffusion (ID) techniques in these two foetuses, although neutralising antibody alone was detected in one of the other 3 foetuses which were all negative for virus.     

Analysis of 46-kDa protein in the perivitelline membrane of Japanese quail (Coturnix japonica)

The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. The PL is constructed with two major glycoproteins, ZPC and ZP1, which are synthesized in the ovarian granulosa cells and the liver, respectively. Although the properties of the major components in the PL have been examined, knowledge about the nature of its minor constituents is lacking. In this study we focused on PL protein, which migrates at 46‐kDa in the gel of SDS‐PAGE. N‐terminal sequence analysis demonstrated that the 46‐kDa protein is the C‐terminal fragment of ZP1. Analysis of lysylendopeptidase digests or cyanogens bromide‐degraded fragments of ZP1 confirmed this postulate. Western blot analysis using antiserum against 46‐kDa protein indicated the absence of 46‐kDa protein in the serum. Moreover, small immunoreactive bands, thought to be cleaved fragments of ZP1, were detected in the PL lysate by western blot analysis using antiserum against the N‐terminal peptide of ZP1. These results indicated that the N‐terminal proteolytic processing of ZP1 might take place after the arrival of ZP1 at the ovary, and the resulting product, 46‐kDa protein, is incorporated into the PL.     

Effect of a single polymorphism in the Japanese quail NK‐lysin gene on antimicrobial activity

NK‐lysins are cationic peptides that play important roles in host protection, and are an important constituent of innate immunity. We identified nine single‐nucleotide polymorphisms (SNPs) in the NK‐lysin open reading frame (ORF) from 32 Japanese quails in six strains: A, B, ND, K, P, and Y. The G to A substitution at nucleotide position 272 in the ORF resulted in a Gly (G) to Asp (D) amino acid substitution (Cj31G and Cj31D alleles). The Cj31D allele was detected in P (frequency 0.76) and Y (frequency 0.03) strains. We compared the antimicrobial activities of four synthetic peptides from the helix 2‐loop‐helix 3 region of avian NK‐lysins against Escherichia coli: Cj31G and Cj31D from quail and Gg29N and Gg29D from chicken. The antimicrobial activities of the four peptides decreased in the following order: Gg29N > Cj31G > Gg29D > Cj31D (P < 0.05). Although there were no differences in the predicted secondary structure of the Cj31G and Cj31D, the net charge of the Cj31G was higher than that of Cj31D. These data indicated that the antimicrobial activity of CjNKL is influenced by net charge, similar to that which has been observed in chicken. © 2015 Japanese Society of Animal Science     

Effect of water‐soluble carbohydrate content in orchardgrass pasture on grazing time and rumen fermentation in dairy cows

Two experiments were conducted to clarify the effect of water‐soluble carbohydrate (WSC) content in orchardgrass pasture on the diurnal distribution of grazing time. Six ruminally cannulated, non‐lactating dairy cows were grazed on either of two pastures with different orchardgrass cultivars containing low WSC (LWSC; cultivar: ‘Hokkai 28’) or high WSC (HWSC; cultivar: ‘Harunemidori’). The cows were grazed in morning and evening sessions in experiment 1, whereas the cows were grazed throughout the day in experiment 2. In experiment 1, grazing time of the cows on HWSC was longer than that of the cows on LWSC (P < 0.01). This difference was larger in the morning session than in the evening session (pasture × grazing session: P < 0.05). Effects on herbage intake were similar to those on grazing time. In experiment 2, daily total grazing time was longer for the cows on HWSC than for those on LWSC (P < 0.05). The cows on HWSC spent a longer time grazing than those on LWSC in the morning between 03.00 and 09.00 hours (P < 0.01). The results indicated that prolonged grazing time in the period between dawn and early morning could increase daily herbage intake in cows grazed on pastures of orchardgrass cultivars with high‐WSC content.     

Effects of ageing and gonadal steroid hormones on prolagtin concentration in the anterior pituitary of the chicken

1. Effects of ageing and gonadal steroid hormones on pituitary prolactin (PRL) concentration in the chicken were investigated.

2. No difference was found in the qualitative electrophoretic patterns of the anterior pituitary homogenates at different ages and between the sexes.

3. Pituitary PRL concentrations in males were generally higher than in females.

4. Pituitary PRL concentrations in castrated immature cockerels were higher than in intact birds, while PRL concentration was depressed by the injection of testosterone propionate or oestradiol benzoate.

5. Prolactin concentration in intact immature cockerels was decreased by daily injection of testosterone or oestradiol for 3 weeks but not by injection of the hormones for 1 week.     

Shape changes in caprine erythrocytes exposed to chlorpromazine and lysolecithin

Stomatocytic and echinocytic transformations of caprine erythrocytes were studied in vitro using chlorpromazine as a stomatocyyic agent and lysolecithin as an echinocytic agent. Morphologic changes in erythrocytes generally varied with the cell shape and the concentration of the substances used. Discoytic, triangular, and pear-shaped red cells common to normal goats, exhibited classical stomatocytic and echinocytic changes with formation of spherostomatocytes, sphero-echinocytes, and spherocytes. In comparison, fusiform and spindle-shaped red cells found in certain Angora goats seemed less prone to shape changes and required greater concentrations of the inducing agents to effect such changes. Chlorpromazine at higher concentrations also inflicted localized membrane damage in form of tiny pits and lysolecithin likewise induced formation of fragile smooth or beaded filaments.     

Effect of feeding non-fibrous carbohydrate before grazing on intake and nitrogen utilization in dairy cows throughout the grazing season

Ten lactating cows were used to determine the effect of feeding non-fibrous carbohydrate (NFC) supplement before grazing on feed intake and nitrogen (N) utilization throughout a grazing season. The experiment was conducted from June to September. Cows grazed twice a day (2.5 h × 2) under a set stocking system and were fed NFC supplement (1 kg/4 kg of milk yield) 2 h before grazing (PRE) or immediately after grazing (POST). Cows were also fed a grass and corn silage mixture ad libitum. Herbage dry matter intake (DMI) was greater for PRE than for POST throughout the experiment and decreased from June to September. Conversely, silage DMI was less for PRE than for POST throughout the experiment and increased over the grazing season. Consequently, total DMI for PRE did not differ from that for POST. Milk urea-N concentration and urinary urea-N excretion in June did not differ between the treatments, whereas that after July was higher for PRE than for POST. Proportion of urinary N excretion to absorbed N intake in June was lower for PRE than for POST, but that after July was higher for PRE than for POST. Feeding NFC supplement before grazing would improve N utilization when cows eat large amounts of herbage high in N.     

Serum level of apoptosis inhibitor of macrophage in dogs with histiocytic sarcoma and its association with the disease

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour-derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra-tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.