Receptor cell degeneration and regeneration within the vomeronasal organ (VNO) of the rat was studied using both electron microscopy and histochemical methods. Electron microscopy was employed to examine the morphological changes along the surface of the sensory epithelium, and histochemical markers were used to monitor the changes in the epithelial cell layers. Transection of the vomeronasal nerves induced selective degeneration of the receptor cells, and within six days, a significant decrease in the number of receptor cells was observed. During the subsequent stage of receptor cell regeneration, cilia and bud-like structures characteristic of a developing sensory epithelium were seen. By day 15, thin microvilli covering the surface of the receptor cells reappeared in the sensory epithelium. The neural cell adhesion molecule (NCAM) and two vomeronasal system-specific lectins; 1) Bandeiraeasimplicifolia lectin (BSL-I) and 2) Vicia villosa agglutinin (VVA) were used as the histochemical markers. NCAM immunoreactivity on the surface of the epithelium was observed to be decreased significantly six days after nerve transection, and was restored during receptor cell regeneration (day 15). The reactivity of the two lectins, BSL-I and VVA, was decreased slightly during degeneration, but was still detectable at the time of maximum receptor cell degeneration (day 6). Lectin reactivity was restored to control levels by day 15. These findings suggest that (1) NCAM is a useful marker for vomeronasal receptor cells and that the vomeronasal system-specific lectins may bind to both receptor and supporting cells and (2) degeneration of vomeronasal receptor cells occurs during the first week (day 6) following nerve transection and the receptor cell population begins to recover within 15 days. The morphological changes observed during receptor cell regeneration suggest that the stages of VNO receptor cell regeneration are similar to those observed during development.
Effects of liquid paraffin on antibody responses and local adverse reactions after intramuscular injection of oil adjuvanted vaccines containing Newcastle disease (ND) and infectious bronchitis (IB) virus were investigated in chickens. Each vaccine was prepared with a liquid paraffin such as Carnation®, Crystol 52® and Lytol®. These vaccines induced sustained antibody responses against ND and IB. Among local adverse reactions, Lytol® induced granulomatous reactions and abscesses, but Carnation® and Crystol 52® did not. The residual weight of liquid paraffin at the injection site decreased in the order Carnation®, Crystol 52®, Lytol®. Crystol 52® was composed of relatively few short-chain hydrocarbons (i.e., <n-C16H 34) and long-chain hydrocarbons (i.e., >n-C20H 42). The vaccine with liquid paraffin mainly composed of n-C16H34~n-C20H42 was suggested to induce fewer adverse reactions.
The MET95 strain of a lentogenic Newcastle disease virus (NDV) isolated from a broiler in Japan, showed unique hemagglutination (HA) activity. The MET95 strain failed to show HA when examined by rapid glass plate method although they showed HA titer of 1:1,024 by micro-plate method. This unique HA was also observed after the MET95 strain was passaged ten times in chickens. The failure of HA by rapid glass plate method was not shown in any other NDVs examined.
To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected.
Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. chauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.
Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for production of cholesteryl esters in plasma. The LCAT activity is reduced in cows with fatty liver developed during the nonlactating stage and those with the fatty liver-related postparturient diseases such as ketosis. The purpose of the present study was to examine whether reduced LCAT activity during the nonlactating stage could be detected before the occurrence of postparturient diseases. Sera from 24 cows were collected at approximately 10-day intervals from -48 to +14 days from parturition. Of the 24 cows, 14 were apparently healthy, whereas 7 had ketosis and 3 had milk fever at around parturition. Of the 14 healthy cows, 7 had unaltered LCAT activity during the observation period, whereas 7 showed reduced activity from -20 to +14 days. Ketosis and milk fever occurred at from -3 to +10 days, but reductions of LCAT activity in diseased cows had already been observed from days -20 to 0. These results suggest that LCAT activity is virtually unaffected during the peripartum period at least in some healthy cows and also that the reduction in LCAT activity can be detected before the occurrence of ketosis and milk fever.
An enzyme-linked immunosorbent assay (ELISA) for canine blood apolipoprotein (apo) B-100 and A-I was developed. The working range for the assay was 1.8 to 28.7 ng/well for apoB-100 and it was 50 to 410 ng/well for apoA-I. The intra- and inter-assay coefficients of variation for the assay for apoB-100 were 5.4 and 6.9%, respectively, and for apoA-I they were 5.8 and 10.6%, respectively. The average concentrations of apoB-100 and A-I in 25 beagles (males, aged 3-4 years) were 0.084 +/- 0.028 (mean +/- SD) mg/ml and 6.29 +/- 1.55 mg/ml, respectively. The ratios of canine (C) apoB-100 to CapoA-I were 1.41 +/- 0.58%. The respective concentrations in one case of hyperlipidemia with systemic atherosclerosis were 0.454 mg/ml and 11.28 mg/ml (a ratio of 4.03%). These values were larger than those of the controls. These results suggest that the measurements of CapoB-100 and A-I concentrations by this newly developed ELISA are helpful for diagnosis of lipidosis.
Plasma lipoprotein cholesterol in 64 clinically healthy Shetland sheepdogs was evaluated to assess whether the breed is more susceptible to hypercholesterolemia. The incidence of hypercholesterolemia was clearly higher in Shetland sheepdogs and mean plasma cholesterol level was significantly higher in Shetland sheepdogs than in control dogs. Blood biochemical examinations did not evidence the abnormalities, which imply the causative disorders, and thyroid hormone levels were not significantly different from the controls. These results suggest that the cholesterolemia is a primary disorder. Cholesterol fractionation by agarose gel electrophoresis and ultracentrifugation revealed that accumulation of α2-migrating lipoproteins was the common characteristic of dogs showing cholesterol level over 250 mg/dl in the breed. Increase in preβ~ β-lipoproteins was also found in Shetland sheepdogs with marked hypercholesterolemia over 500 mg/dl. Therefore, Shetland sheepdogs may include more dogs with primary disorders in lipoprotein metabolism, which cause hypercholesterolemia, at least in Japan.
Rescula (0.120noprostone isopropyl) is the first docosanoid compound approved for treatment of glaucoma in humans. It is commercially available in Japan, and is undergoing clinical testing elsewhere. The aim of this study was to evaluate the effect of Rescula on intraocular pressure (IOP) in normotensive dogs. After establishing a baseline diurnal IOP curve, six dogs were unilaterally treated with Rescula while the contralateral eye was treated with a placebo. Applanation tonometry was performed in both eyes, and pupil size was evaluated, 30 min after treatment, and at 1-hr intervals for the next 9 hr. Rescula caused a significant (p=0.014) and long-lasting decrease in IOP, from 20.49 ± 2.02 mm Hg in control eyes to 15.49 ± 0.69 mm Hg in treated eyes. These results suggest that Rescula is potentially efficacious in treatment of canine glaucoma.
To determine the prevalence of capsular polysaccharide (CP) types of Staphylococcus aureus isolated from bovine mastitic milk in Korea, the protective effect of the conjugates, composed of microencapsulated S. aureus clinical isolate type 8 CP bound to Pseudomonas aeruginosa exotoxin A (ETA) was evaluated in mice. Of 107 S. aureus isolates, serotype 5 and 8 accounted for only 26 or 24.2%. When serotype 336 antiserum was employed, fifty of the remaining 81 isolates were typed as 336, 26 reacted with two serotypes, and 5 were nontypeable. Mice challenged with the same strain used for immunization had fewer S. aureus cells in their kidneys than mice challenged with the heterologous strain. But the magnitudes of difference on bacterial clearance were similar in both groups, indicating that the significance of this result remains to be determined. Mice immunized with the conjugate elicited an antibody response 3 days post injection, which persisted for 13 days of the observation period after second injection in some mice. The mice immunized with the CP8-ETA conjugates developed antibodies significantly higher than those immunized with CP-Freund's adjuvant or PBS. In invivo bacterial challenge experiment, the survival rate of mice immunized with CP8-ETA conjugate was significantly higher than that of mice immunized with PBS. It was suggested that CP8-ETA vaccine had a potential to protect mice against experimental S. aureus bacteremia.
For genotyping of feline major histocompatibility complex (FLA) class II DRB, the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method using group-specific primers was tried. Sixty-six DRB genes were classified into 8 groups according to differences in the first 5' amino acid sequences. The group-specific primers were designed as forward ones, which were specific for 5' base sequences of genes in each group. Three to 7 appropriate restricted enzymes were selected by computer analysis for RFLP typing of the genes divided into each group. In 6 out of 9 cats, the results of DRB typed by direct sequence method agreed with results of the PCR-RFLP method using group-specific primers. In the other 3 cats, the number of genes amplified by group-specific primers was 1 or 2 more than those detected by direct sequence method. The direct sequence method in 9 cats identified 5 new FLA-DRB genes. The PCR-RFLP method using group-specific primers could divide 66 genes into 37 genes and 10 subgroups from the RFLP pattern. One to 6 genes in each cat, and a total of 203 genes and subgroups were detected in 68 domestic cats. The genes detected might be biased to the subgroup G1-1a (28.8%), DRB*0501 (10.3%), G1-2a (9.4%) and G6b (7.4%). The PCR-RFLP method using group-specific primers may be useful in typing FLA class II DRB.
Malignant histiocytosis was diagnosed in 4 cows. In all cases the tumor tissues were composed of cytologically atypical histiocytes with evidence of erythrophagocytosis. The tumor in case 1 appeared highly anaplastic with marked nuclear pleomorphism, and had areas of spindle cell differentiation, but had no relation to malignant fibrous histiocytoma. The neoplastic tissue in case 2, characterized by cohesive growth of tumor cells, was distinguishable from anaplastic carcinoma cells by cytokeratin immunostaining. There were many hemosiderin-laden neoplastic cells suggestive of high phagocytic activity in a lymph node of case 3. The neoplastic cells in case 4, frequently multinucleated, were less atypical than in the other cases. All cases expressed histiocyte-associated markers (lysozyme and HAM56), and were negative for cytokeratin, S100, and T- and B-cell lineage-specific markers (CD3 and CD79a). The most frequent HAM56 immunoreactivity was detected in case 4, and the giant, multinucleated forms, reminiscent of epithelioid cell differentiation, seemed not to indicate cytological pleomorphism as a result of neoplastic transformation.
Oval cells which appear in the liver after hepatic injuries are suspected to be progenitor cells for both hepatocytes and bile duct cells. Oval cell isolated from the livers of the hamsters treated with diethylnitrosamine and 2-acetylaminofluorene and infected with Clonorchis sinensis (CS), cultured for 2 weeks and evaluated for differentiation and plasticity by electron microscopy and immunohistochemistry. In the CS-uninfected group, glycogen granules and peroxisomes were noted in the cells that were cultured for 2 weeks. Starting at 1 week postculture, immunoreactivity of the cells to cytokeratin 19 markedly decreased but that to albumin and alpha-fetoprotein gradually increased. This means that oval cells isolated from hamsters that were not infected with CS differentiated toward hepatocyte lineage. However, in the CS-infected group, cultured cells contained numerous rough endoplasmic reticulum and showed immunoreactivity that was generally in reverse to that of CS-uninfected group, meaning that cells isolated following CS infection were primed by CS and differentiated toward bile duct cell lineage. The results of this study suggested that oval cells are indeed bipolar progenitor cells for hepatocytes and bile duct cells and can differentiate toward either lineage depending upon the priming factor.
The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.
Three suspected cases of cat scratch disease were diagnosed by indirect immunofluorescence antibody assay and/or polymerase chain reaction. Patient 1 was a 10-year-old female who presented swelling of the right axial lymph nodes with pain and fever. She kept a kitten, and many scratches were observed on her both legs and dorsum manus. Antibody titers against Bartonella (B.) henselae were 1:32 for IgM 3 weeks after the onset of the symptoms and 1:64 for IgG 8 weeks after the onset. The DNA for 16S rRNA type I of B. henselae was detected from the blood sample obtained 3 weeks after the onset of symptoms by polymerase chain reaction for the first time in Japan. Patient 2 was a 22-year-old female veterinary student with a cat scratch at the bottom of her neck by a male kitten. She developed a papule at the scratch, slight fever, and neck pain. Although both Bartonella-specific IgG and IgM antibodies were negative before the scratch, the IgG antibody titer rose to 1:512 14 weeks after the onset. B. henselae was isolated from the kitten and its DNA found to be for 16S rRNA type I by PCR. Patient 3 was a 23-year-old female veterinary student with a cat scratch on her left forearm. A small reddish papule developed on the scratch, and she experienced swelling of the left axial lymph node and pain. Both the IgG and IgM antibodies against B. henselae were negative before the cat scratch, and the IgG titer rose significantly to 1:128 and 1:1,024 in 2 and 5 weeks, respectively, after the onset of the symptoms.
The sperm count required were investigated to obtain a conception rate of 80% by unilateral intrauterine insemination (UIUI) of fresh semen in cats. The conception rates obtained by insemination before and after ovulation were also examined. Thirty-six female cats aged 1-7 years were used in the experiments, and the number of experimental cases was 44. Seven male cats aged 2-12 years from which semen could be collected by the artificial vagina method were used. In artificial insemination, 100 iu × 2 or 250 iu of hCG was administered on days 2-4 of estrus, and sperm were introduced into the uterine horn with a greater number of ovulations (or mature follicles) 15, 20 and 30 hr after hCG administration by laparotomy. The inseminated sperm counts were 2 × 106 (Exp. 1), 4 × 106 (Exp. 2), and 8 × 106 (Exp. 3). As a result, ovulation was induced in 42 of 44 cases (induction rate: 95.5%) regardless of the dosage of hCG. Conception was obtained by UIUI in two of 16 animals (conception rate: 12.5%) in the Exp. 1, five of 16 animals (31.3%) in Exp. 2, and eight of 10 animals (80.0%) in Exp. 3. Regarding the relationship between the ovulation state at insemination and conception, the conception rate obtained by insemination before ovulation was clearly higher than that obtained by insemination after ovulation (p<0.05). Regarding the number of kits compared to the number of ovulations on the inseminated side, the percentages of cases in which the number of kits exceeded the number of ovulations on the inseminated side were similar in all groups inseminated with a different number of sperm. It is therefore necessary to investigate conception rates obtained by bilateral insemination to increase the fertility rate. Based on the above findings, it was shown that the sperm count required for fertilization by UIUI is 8 × 106.
Frozen feline semen was prepared using two types of extenders, egg yolk Tris-fructose citric acid (EYT-FC) and egg yolk sodium citrate solution (EYC), and the semen qualities after thawing and the conception rates obtained by unilateral intrauterine horn insemination (UIUI) were investigated. Cats used in the experiment were six males and 11 females aged 2-12 years (the number of experimental cases was 17). For preparation of frozen semen, semen collected by the artificial vagina method was adjusted to 1 × 108 sperm/m l and 7% glycerol, put in 250 μl straws, and then frozen using a cell freezer. The mean sperm motility after thawing was 30.0 ± 9.7 (SE) % in the semen prepared with EYT-FC and 30.0 ± 3.3% in the semen prepared with EYC. Four of seven animals were fertilized by UIUI using two straws in both extenders, and the conception rate was 57.1%. The mean ratios of number of kits to the number of ovulations in the inseminated side were 61.1 ± 24.5% and 30.5 ± 3.4% for EYT-FC and EYC, respectively, showing that the ratio tended to be higher in the semen prepared with EYT-FC. The above findings, comparing the two extenders for preparation of frozen feline semen, showed that EYT-FC is slightly superior to EYC. To increase conception and fertility rates, it may be important to increase the sperm count for insemination and to inseminate both uterine horns.
Plasma levels of the chemokine RANTES were examined in monkeys infected with either a pathogenic simian and human immunodeficiency chimeric virus (SHIV) or a non-pathogenic SHIV to determine whether RANTES levels were related to the pathogenicity of the virus, the plasma viral load, or the kinetics of CD4+ T-cells. In the results no significant correlation was found between the RANTES kinetics and changes in the CD4+ T-cell numbers nor the plasma viral loads in any of the monkeys, although a transient decrease of the RANTES level was observed in the pathogenic virus-infected monkeys. At least, the plasma RANTES level can not be used as an index of the pathogenicity of the virus at the early stage of infection.