Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog

A capillary reversed passive latex agglutination test (capillary RPLA) was developed which allows quantification of serum C-reactive protein (CRP) within approximately 15 min. The logarithmic regression line (calibration curve) obtained after measuring each CRP concentration three times in twofold dilutions of a standard canine serum containing 222 g/ml of CRP was y=6.394+0.030x (r=0.995). Capillary RPLA permitted quantification of CRP in the range 6.9–222 g/ml. The coefficients of variation ranged from 10.28% to 12.40%. The recovery rates (percentage recovery) of CRP by capillary RPLA were within the range 87% to 106%. On measuring the CRP concentrations in sera from 78 dogs by capillary RPLA, single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), close correlations were demonstrated between SRID and capillary RPLA (y=7.250+1.109x, r=0.978), between SRID and ELISA (y=3.042+1.059x, r=0.967), and between capillary RPLA and ELISA (y=1.778+0.929x, r=0.962). Capillary RPLA may be considered useful as a routine biochemical technique for measurement of serum CRP concentration in the dog.Abbreviations CRP C-reactive protein - ELISA enzyme-linked immunosorbent assay - RPLA reversed passive latex agglutination test - SRID single radial immunodiffusion     

Monitoring of genetic diversity in the Japanese Black cattle population by the use of pedigree information

The gene pool of the Japanese Black cattle has been completely closed to foreign breeds during the last 100 years. Genetic diversity of the Japanese Black cattle from 1960 to 2000 was monitored with three estimates of effective number of ancestors. Founder genome equivalent (N_ge) accounts for all the causes of reduction of diversity. Effective number of founders (N_ef) and non‐founders (N_enf) explain reduced diversity because of unequal genetic contributions of founders and random genetic drift in non‐founders, respectively. Further examination using gene dropping simulation was conducted to obtain information on survival of founder alleles. Unique founder alleles were dropped down along the actual pedigree with Monte Carlo procedure following Mendelian segregation rules, and generated genotypes of all the current live animals (612 959 heads). Pedigree records consisted of 2 075 188 animals was used for these analysis. The estimates of three effective numbers (N_ef, N_ge, and N_enf) decreased from 418.6 to 50.3, 86.6 to 7.3, and 109.2 to 8.5, respectively, during the period 1960–2000. The increasing differences between two kinds of genetic diversity indices derived from N_ge and N_ef showed that large part of the reduced diversity from 1980 was attributed to genetic drift caused by the intensive use of particular limited number of sires. In gene dropping analysis, probabilities of extinction of founder alleles were derived from their distributions of frequency in the current animals. Several founders showed low probabilities of allele extinction, irrespective of their relatively low genetic contributions. This suggests that these founders have lineages through which their alleles are surely transmitted to the current breed. The use of these founders as a strategy for recovering the genetic diversity was discussed.     

Prognostic significance of surgical margin, Ki-67 and cyclin D1 protein expression in grade II canine cutaneous mast cell tumor

The prognosis for canine cutaneous mast cell tumor (CCMT) is thought to be correlated with histopathological grading. However, the wide variety of histopathologic types of grade II is one of the most troublesome and difficult points for prognosis. The objective of this study is to determine the prognostic value of surgical margin, ki-67 and cyclin D1 protein expression in grade II tumor. Surgically resected specimens of solitary grade II CCMT from 48 dogs with follow-up periods over 360 days (median was 1080 days) were used in this study. The expression of cyclin D1 and ki-67 proteins was determined by morphometrically using slides stained immunocytochemically, and the correlations among the results, survival rate, and recurrence and/or metastasis rate of each dog were analyzed statistically. The recurrence and/or metastasis and mortality rate in the incomplete surgical excision group within 30 months postoperatively were higher than that of the complete surgical excision group. In the incomplete surgical excision group, dogs with low positive staining of ki-67 had a significantly better survival, but the recurrence and metastasis rate and ki-67 positivity failed to show a significant correlation. Only a small number of cases showed cyclin D1-positive tumor cells, but most of them had a poor outcome with a high recurrence rate. In grade II CCMT, incomplete excision induced a relatively high metastasis rate and poor prognosis. Ki-67 positivity is a marker for the estimation of overall survival in incomplete surgical excision cases. Cyclin D1 positivity was low and may not have a prognostic role.     

Changes in the expression of toll-like receptor mRNAs during follicular growth and in response to lipopolysaccharide in the ovarian follicles of laying hens

The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F(5)-F(1), respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1beta in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F(3). Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F(5)-F(1), with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1beta in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1beta by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.     

Influences of indigenous phototrophs on methane emissions from a straw-amended paddy soil

A pot experiment was conducted to investigate the influences of indigenous phototrophs on methane (CH₄) emissions from a paddy soil where rice straw was incorporated or was surface-applied. During the cultivation, half of the pots were covered with aluminum foil, except for the minimum space for rice plants, to prevent ambient light reaching the floodwater or the soil surface. Growth of oxygen-producing phototrophs was hardly observed in the unilluminated plots, whereas intensive growth of algae, duckweed and hydrophytes was found in the illuminated ones. Plant growth was not affected by the different treatments. Seasonal changes in CH₄ emission determined by a closed chamber method indicated that illumination had no or only minor effects on CH₄ emissions when rice straw was incorporated or was not applied, but significantly reduced CH₄ emissions when rice straw was surface-applied. Methanogenesis occurring in the soil-floodwater interface was further investigated in two lab-scale model experiments measuring methanogenic activity. As a result, more activated methanogenesis was found in the surface-applied rice straw and the soil around the straw compared with the soil incubated without rice straw. The magnitude of the methanogenic activity in the rice straw incubated under illuminated conditions was significantly lower than that incubated in the dark. Consequently, this study demonstrates that methanogenesis in paddy soil occurs even in the soil-floodwater interface if plant residues like rice straw exist, and such methanogenesis is likely to be suppressed by growth of indigenous phototrophs under illumination.     

Regression tree analysis of the relationship between the concentrations of antimicrobial components and the microbiota of normal milk from dairy cows

The purpose of this study was to determine the concentrations of antimicrobial components (immunoglobulin A (IgA), lactoferrin (LF), lingual antimicrobial peptide (LAP), and S100A7) in normal milk and their relation to host factors (Age, somatic cell count (SCC), days in milk, richness, and alpha diversity of the milk microbiota) in dairy cows using multivariate regression tree analyses, and to clarify how the milk microbiota is related to the obtained results. Thirty normal milk samples were collected from a commercial dairy farm in June 2020. The thresholds that predicted the concentration of each antimicrobial component in milk were obtained by regression tree analysis, and the beta-diversity of the milk microbiota composition between groups divided according to each threshold was compared by an analysis of similarities test. The IgA and LF concentrations were mainly predicted by the SCC (177,500 and 70,000 cells/ml, respectively), and the LAP and S100A7 concentrations were predicted by Age (29.667 and 40.3 months, respectively). No relationship was observed between the concentration of IgA, LAP, or S100A7 and the milk microbiota composition between the groups divided by the threshold for prediction, but the milk microbiota composition was significantly different between the groups divided by the threshold for predicting the LF concentration. Our results indicated that the LF concentration in normal milk may be associated with the milk microbiota composition.     

Present state of imposex in neptune whelk Neptunea arthritica inhabiting shallow waters around Hokkaido, Japan

ABSTRACT:   The state of imposex in Neptunea arthritica from seven sites along the coast of Hokkaido, Japan was examined in 2002 based on the criteria: (i) relative penis size index (RPSI); (ii) imposex frequency; (iii) stage frequency distribution of imposex in adult and immature whelks; and (iv) sex ratio. RPSI differed from site to site, although values from all sites were low (0.186–5.294). In particular, the RPSI values for four sites were very low (<1.0). In sites where immature whelks were also collected, the frequency of imposex was considerably lower in immature (7.7%–55.6%) than in adult whelks (50%–95.2%) except at one site. The imposex stage frequency distribution also differed among sites, and the trend in the adult whelks corresponded with their RPSI value. Female whelks showing severe imposex (Stage 3 and 3+) were restricted to large individuals.     

Inbreeding and effective population size of Japanese Black cattle

The objective of this research was to estimate the amount of inbreeding and effective population size of the Japanese Black breed using pedigree records from bulls and heifers registered between 1985 and 1997. Inbreeding was quantified by three F-statistics: actual inbreeding, inbreeding expected under random mating, and inbreeding due to population subdivision. During the period of 1985 to 1997, the inbreeding expected under random mating increased from 2.3% to 5.0%, whereas the increase of actual inbreeding was more gradual (from 4.7% to 5.4%). The inbreeding due to population subdivision decreased almost linearly and reached 0.5% in 1997, indicating that genetic subdivision of the Japanese Black cattle population has essentially disappeared. The effective size of the breed was estimated from the increasing rate of inbreeding expected under random mating. In the earlier half of this period (1986 to 1990), the breed maintained an effective size of approximately 30. However, after 1991 the effective size sharply decreased and the harmonic mean between 1993 and 1997 was only 17.2. The main cause of this reduction of the effective size was considered to be the intensive use of a few prominent sires. To increase the effective size, an upper limit in the use of AI semen per sire should be imposed.