Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.
Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3Bov.Yamagata, is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3Bov.Yamagata. These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.
Hyperlipemia in horses is a disorder of lipid metabolism peculiar to ponies. This study reports changes of blood biochemical values from the acute to the postconvalescent phases in 3 Shetland ponies with hyperlipemia in Japan. Diseased ponies (all 7 to 9 years old, in late pregnancy, and obese) were fed in the same farm. The periods of their hospitalizations ranged from 30 to 45 days. Twelve well-conditioned ponies (3 to 13 years old) around parturition were used to establish baseline values for blood test results. Main clinical findings in the affected ponies were depression, dysphagia, anorexia, ventral edema and milky-appearing plasma. Hypertriglyceridemia (40- to 70-fold rise of controls) was found in the acute phase of the disease in the affected ponies, and was derived from increased very-low density lipoproteins. Aspartate transaminase and γ-glutamyl transpeptidase activities, blood urea nitrogen, and creatinin concentrations were increased in acute ponies compared to controls, suggesting impairment of liver and kidney functions. However, these values gradually recovered until the end of postconvalescent phase. Hyperinsulinemia was observed in the acute phase of the hyperlipemia of all affected ponies. And an exaggerated insulin response to intravenous glucose was observed in the 2 ponies given intravenous glucose tolerance tests. These findings suggest decreased insulin sensitivity in hyperlipemic ponies.
An eardrop solution of β-thujaplicin was examined for therapeutic effects on canine Malassezia-related otitis externa. Half to one ml of β-thujaplicin solution of 100 μg/ml including DMSO 2% was injected everyday into both external ear canals of 31 cases for test-of-cure agreement. Fifteen score phases were established from the symptoms and cerumen smear biopsy findings, and score changes were recorded at least once a week. The means of the second and the third inspection day scores decreased significantly more than the previous value of each. In addition, the numbers of yeast-like organisms clearly decreased. These results suggest that β-thujaplicin eardrops are effective for Malassezia-related otitis externa in dogs.
A 14-year-old male miniature schnauzer was referred to us because it was circling to the right. A mass in the diencephalon was noted on brain magnetic resonance images. The dura was thickened with marked linear enhancement after contrast administration. Based on diagnostic image analysis, this lesion strongly suggested meningioma. The patient's symptoms were well controlled by a combination therapy of prednisolone and lomustine (CCNU), and survived for thirteen months after diagnosis. This case was diagnosed as a meningioma based on histopathological findings. This report describes the clinical findings, imaging characteristics, and pathologic features of a diencephalic and mesencephalic meningioma and long-term survival after lomustine and prednisolone therapy.
Blood samples were taken from eight multiparous cows at a dairy farm on eight occasions between the prepartum period and peak lactation to study the serum concentrations of amino acids and biochemical constituents. The cows were classified as having either severe hepatic lipidosis (HL) or non-hepatic lipidosis (non-HL) according to their clinical condition after calving and changes in serum biochemical parameters. The serum concentrations of β-hydroxybutyric acid were higher in the HL group than in the non-HL group (ANOVA: p<0.01). The serum concentrations of methionine (Met), phenylalanine, and arginine were significantly different between the two groups (ANOVA: p<0.05). In particular, the Met levels were significantly low for 14 days after calving in the HL group (p<0.05), although Met levels in the HL group tended to be lower than the values in the non-HL group until 30 days after calving, starting 14 days before calving. The results suggest that an insufficiency of Met during the periparturient period is related to the development of hepatic lipidosis.
A 9-year-old female Pug was presented to us with chronic diarrhea. Hematologic findings indicated severe hypoproteinemia and hypoalbuminemia, and endoscopy revealed severe edema of the duodenal mucosa. Based on these results and on additional histopathological findings, we made a diagnosis of protein-losing enteropathy caused by lymphocytic-plasmacytic enteritis with lymphangiectasia. The dog was initially treated with prednisolone and cyclosporine. This treatment regimen was not effective. However, when methotrexate was substituted for cyclosporine, progress was obtained and the diagnosis was confirmed.
The population and distribution of feral raccoons (Procyon lotor) are expanding in Japan after escape or release from animal-owners. Wakayama Prefecture is one of the most typically devastated areas by this exotic carnivore, particularly in the last five years after a latent distribution for more than ten years. Official control measures of feral raccoons commenced in the summer of 2002 by several municipalities, and 531 animals collected in 12 municipalities between May 2003 and April 2005 were submitted for parasitological examination of gastrointestinal helminths. Detected parasites included six nematodes (Physaloptera sp. [prevalence; 5.1%], Contracaecumspiculigerum [0.9%], Strongyloides procyonis [25.5%], Ancylostoma kusimaense [0.8%], Arthrostoma miyazakiense [0.4%], and Molineus legerae [1.1%]), seven trematodes (Isthmiophora hortensis [4.9%], echinostomatid sp. with 34-39 collar spines [1.7%], Metagonimus takahashii [12.4%], M. yokogawai [0.8%], Plagiorchis muris [0.2%], Macroorchis spinulosus [1.9%], and Consinium ten [0.2%]), one cestode (Mesocestoides sp. [0.2%]), and six acanthocephalan spp. (Centrorhynchus bazalenticus [0.2%], Centrorhynchusteres [5.5%], Sphaerirostris lanceoides [2.4%], Plagiorhynchus ogatai [0.6%], Porrorchis oti [1.5%], and Southwelina hispida [1.9%]). Most of the collected parasites are food-borne, indigenous helminth species. Physaloptera sp. has never been recorded in indigenous wild carnivores in Japan, and resembles closely P. rara, prevalent in raccoons of North America, in morphology. The position of a pair of phasmids in the posteroventral region of the adult male, however, could differentiate it from P. rara. Since Strongyloides procyonis is known to cause creeping eruption as well as intestinal infection in a healthy human volunteer, we should be concerned about the rapid increase in the population and distribution of feral raccoons in Japan from the viewpoint of public health as well.
Life cycle of Eimeria krijgsmanni-like coccidium isolated from the feces of naturally infected mice purchased from commercial sources was examined. The parasite was purified by single oocyst isolation and maintained by passage in the mice before experiments. The sporulated oocysts were ovoid or ellipsoid, measuring 19.3 × 14.8 μm on average. One or two small polar granules were present. Micropyle and oocyst residuum were absent. Sporocysts were ellipsoid, measuring 11.6 × 7.2 μm on average with a small Stieda body and sporocyst residuum. Six groups of respective 5 mice (4-week-old) were inoculated with doses varying from 2.0 × 101 to 106 oocysts. All the mice examined began to shed oocysts from 7 day postinoculation (PI) and their maximum number of oocysts per gram of feces were 106 on day 8 PI. Patency was 6 or 7 days. This parasite had severe virulence to the mice that is, the mice given 106 oocysts showed anorexia, diarrhoea and rough hair from 1 day and all of them died on day 3 PI. The mice given 103 or more oocysts showed the clinical signs described above from day 5 and 4 of them received 105 died on day 9 or 10 PI. The parasites occurred within the epithelial cells of cecum, colon and rectum of infected mice. Sporozoites, 13.9 × 3.0 μm, with two large refractil bodies on side of the nucleus located subcentrally were observed on day 1 and 2 PI. Merozoites were first observed at 24 hr PI, and sexual stages were found from 4 day PI. No parasites were detected in the small intestine and mecenteric lymph nodes.
Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes.
Thirty-six flukes were collected from the livers of wild deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan, and were served for morphometry. The length and/or the width of the body, suckers, testes, ovary, vitelline glands, cirrus pouch and eggs in the uterus of the flukes were measured. The distance between anterior end of the body and position of the maximal body-width or upper end of the testes were also determined. A remarked morphological characteristic was that the right and left testes did not lie tandem but lined bilaterally. Also the position of the maximal body-width did not always locate in the posterior part of the body of the fluke. The property was in accordance with those for Dicrocoelium chinensis.
To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondiiSAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan.
The expression of osteopontin (OPN) in the hearts of rats with experimental autoimmune myocarditis (EAM) was evaluated. Western blot analysis showed that OPN was significantly increased in the hearts with EAM compared with those of complete Freund's adjuvant (CFA) immunized control. Immunohistochemically, OPN was weakly expressed in the cardiomyocytes in the heart with normal and CFA immunized controls. In EAM lesions, OPN was intensely immunostained in some inflammatory cells, mainly ED1 positive macrophages. These findings suggest that OPN is significantly increased in EAM lesions and that OPN mediates the inflammatory process in the course of rat EAM model.
Fourteen diseased pigs from four farms in which there had been an outbreak of salmonellosis were investigated. Granulomatous inflammation with depletion of lymphocytes was observed in the swollen lymph nodes in these pigs. Antigens to porcine circovirus type 2 (PCV2) were immunolabeled in the lesions along with detection of viral DNA as PCV2 by polymerase chain reaction (PCR). In addition, antigens to porcine reproductive respiratory syndrome virus (PRRSV) were immunodetected in the lungs and Salmonella Choleraesuis was isolated from the affected pigs. The nine salmonellosis affected pigs, five (55.6%) with salmonellosis and PMWS concurrently infected with PRRSV were much higher than those infected with salmonellosis and PMWS (22.2%) or with salmonellosis and PPPRV (22.2%).
Pharmacokinetics (PK) of probenecid including plasma probenecid concentrations, in vitro plasma protein binding properties, and in vivo PK parameters were determined in dogs. Probenecid concentrations were best determined by HPLC, which showed good linearity and good recovery with simple plasma preparation. The quantification limit of probenecid was approximately 50 ng/ml at S/N ratio = 3, by simple procedure with HCl and methanol treatment. Probenecid showed two types of binding characteristics, i.e., high-affinity with low-capacity and low-affinity with high-capacity binding. This result indicated 80-88% of probenecid was bound to plasma protein(s) at observed concentrations (< 80 μg/ml) in vivo at an intravenous dose of 20 mg/kg. Plasma probenecid concentration-time profile following i.v. administration in dogs showed biphasic decline and well fitted a two-compartment open model. The total body clearance was 0.34 ± 0.04 ml/min/kg, volume of distribution at steady-state was 0.46 ± 0.07 l/kg, elimination half-life was 18 ± 6 hr, and mean residence time (MRT) was 23 ± 6 hr. Since probenecid has been known as a potent inhibitor of renal tubular excretion of acidic drugs and highly binds to plasma proteins, our observation in relation to plasma protein binding and PK parameters will serve as the basic information concerning drug-drug interactions in dogs and in other mammalian species.
Cq3 was identified in C57BL/6J (B6) × KK-Ay F2 mice as a quantitative trait locus (QTL) that controls plasma cholesterol and phospholipid levels, and normolipidemic B6 allele was associated with increased lipids. Cq3 was statistically significant in F2-a/a, but not in F2-Ay/a; probably because the Cq3 effect was obscured by introduction of the Ay allele, which in itself has a strong hyperlipidemic effect. Because the peak LOD score for Cq3 was identified near D3Mit102 (49.7 cM) on chromosome 3, linkage analyses with microsatellite markers located at 49.7 cM were performed in KK × RR F2, B6 × RR F2, and KK × CF1 F2. However, even a suggestive QTL was not identified in any of the three F2. By testing all pairs of marker loci, I found a significant interaction between Cq3 and the Apoa2 locus, and F2 mice with the Apoa2KK/Apoa2KK; D3Mit102B6/D3Mit102B6 genotype had significantly higher cholesterol levels than did F2 mice with other genotypes. The results showed that the `round-robin' strategy was not always applicable to the search for QTL genes; probably because specific gene-to-gene interaction limited the validity of the strategy to the utmost extent.
We developed a neurosurgical operating table for restraining adult cattle in the sternal recumbent position during long-time inhalation anesthesia, and examined intracranial pressure (ICP), blood pressure and blood gases during isoflurane anesthesia. We confirmed that the maintenance of inhalation anesthesia, the restraint of cattle in the sternal recumbent position and bringing the cattle out of anesthesia could all be carried out safely using the operating table we produced. For the purposes of the present experiment, the cattle were divided into 2 groups: the SR group, which underwent sternal recumbency for 8 hr under isoflurane anesthesia using the neurosurgical operating table, and the RR group, which underwent right lateral recumbency for 3 hr under isoflurane anesthesia on a standard operating table. The mean ICP was found to be significantly lower in the SR group than in the RR group during anesthesia, and PaO2 was significantly higher in the SR group. In the SR group, no complications such as regurgitation or ruminal tympany occurred for 8 hr after the induction of anesthesia, and recovery from anesthesia was uneventful. In contrast, all RR cattle showed ruminal tympany and regurgitated ruminal fluid at 3 hr after the induction of anesthesia. Thus, the neurosurgical operating table developed in the present study may be useful for long-time anesthesia and neurosurgery of adult cattle.
111In-DTPA octreotide (DTPAOC) has been used for detecting somatostatin receptor positive tumor for years. In-111 DOTA-Tyr3-octreotide (DOTATOC) is newly developed for diagnostic and therapeutic purposes. In this study, we compared the biodistribution and tumor uptake ratio after injection of In-111 DTPAOC and In-111 DOTATOC in rats. Twelve rats bearing pancreatic tumors were divided into two groups: six rats were sacrificed at 4 hr after injection of 3.7 MBq of In-111 DTPAOC and another 6 rats were sacrificed at the same time after injection of 3.7 MBq of In-111 DOTATOC. Samples of various organs were obtained and counted to calculate the tissue concentration. In addition, 12 rats bearing pancreatic tumors were scanned at 4, 24, and 48 hr after injection of 37 MBq of In-111 DTPAOC or In-111 DOTATOC. The tumor uptake ratios (T/N ratio) were calculated. The biodistribution data showed that the activity in the tumor as well as in the kidney was significantly higher in the In-111 DOTATOC group than in the In-111 DTPAOC group, although both radiopharmaceuticals had the expected high affinity to the tumor. The T/N ratios in the In-111 DOTATOC group were also significantly higher than those in the In-111 DTPAOC group at 24 hr after injection. We conclude that In-111 DOTATOC showed lower clearance than In-111 DTPAOC in the rats bearing pancreatic tumors, although both of these radiopharmaceuticals showed expected high tumor uptake.
Electroencephalography (EEG) is an effective method for the evaluation of sedation or anesthesia. The purpose of this study was to examine the sedative effect of acupuncture by electroencephalographic spectral edge frequency (SEF) in Miniature Schnauzer dogs (4.2-6.1 kg, 1-2 years old). The acupuncture points "GV20 and Yintang" were applied for 20 min. Sedation level was assessed before, during, and after acupuncture by spectral edge frequency 95 values and the Ramsay sedation score. The spectral edge frequency 95 values were significantly reduced during acupuncture on GV20 or Yintang point and returned to the baseline values after acupuncture releasing. The Ramsay sedation score (RSS) also showed the acceptable sedation level during acupuncture. It was concluded that an acupuncture application at GV20 or Yintang point used in the present study would be a valuable method to induce the sedation in dogs.
To investigate the possibilities of two NA inhibitors [oseltamivir carboxylate (OC) and zanamivir (ZA)] as the clinical agents for equine nifluenza A virus (EIV) infection, we examined the efficacies of these inhibitors against twelve EIVs in vitro. OC and ZA inhibited NA activities of all EIVs with 50% inhibitory concentrations with ranging from 0.017 to 0.130 and from 0.010 to 0.074 μM, respectively. OC and ZA inhibited plaque-forming of all EIVs in MDCK cells with 50% effective concentrations with ranging from 0.015 to 0.097 and from 0.016 to 0.089 μM, respectively, except for one strain (13.328 μM and 6.729 μM). These results suggest that these inhibitors are effective against most EIVs and might be useful for treatment of EI in horses.