The aim of this study is to identify, as hypotheses, all feasible sources and routes of infection for the BSE cases in Japan, and to study the probability of each hypothesis. The strategy of this epidemiological study is as follows. 1) BSE risk status in Japan is tentatively divided into 3 stages, i.e., before 1996 April when administrative guidance for feed ban of cattle MBM introduced. After that to 2001 September, the first case of BSE in Japan, then, after 2001 October with real feed ban in the law. 2) Make hypotheses depending on the invasive risk scenarios and propagation risk of BSE in Japan, and they are checked by evidences, case control study or statistics. 3) Grouping of BSE cattle was conducted time sequentially and spatially; that is Group-A (1995-96, born in Hokkaido, Kanto), Group-B (1999 in Kyushu), Group-C (1999-2001 in Hokkaido), Group-D (young cattle born after real feed ban) and Pre-A, Post-D groups. As a result, a milk replacer was considered one of the most probable cause of group-A contamination, and group-C outbreak might be caused by an indigenous BSE propagation of group-A in Hokkaido. If the hypothesis of Holland animal fat as causative material was accepted, however, there are several unexplainable points. Collection of scientific evidences on animal fat impurity and age dependent susceptibility to BSE will be needed to clarify the true causative material.
The Akt/protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) pathways are involved in cell survival. This study examined the temporal profiles and localization of Akt/PKB and ERK1/2 activation in rat testis after ischemia/reperfusion (I/R). Testicular tissue was collected from normal control rats and rats exposed to reperfusion for 6, 24, and 48 hr after ischemic injury; the tissues were analyzed via Western blotting and immunohistochemistry. Western blot analysis showed that the levels of phosphorylated Akt/PKB (pAkt/PKB) and ERK1/2 (pERK1/2) increased significantly during the first 6-24 hr of reperfusion after ischemia. However, both of these activated proteins were decreased slightly at 48 hr after reperfusion. Immunohistochemically, low levels of pAkt/PKB expression were observed in Sertoli cells from the normal control. After I/R, pAkt/PKB expression increased mainly in the adluminal portion of the Sertoli cells, as well as in spermatogenic cells. In addition, pERK1/2 expression was observed in Sertoli and Leydig cells in the normal control. After I/R, pERK1/2 expression increased in some surviving spermatogenic cells (mainly spermatocytes), as well as in the adluminal portion of Sertoli cells. These results suggest that both Akt/PKB and ERK1/2 are involved in the survival of testicular cells during the early phase of testicular I/R. These pathways may represent important targets for increasing cell survival in testicular injury, including testicular torsion.
We examined the age-related changes of calbindin D-28k (CB)-immunoreactive neurons and overall populations of neurons in the myenteric plexus of gerbil duodenum using whole mount preparations and immunohistochemistry. The circumference of duodenum increased age-dependently. CB-immunoreactive neurons were observed in all groups, and most of them had the Dogiel type II morphology. The fully developed cobweb-like structures were observed in the myenteric plexus of duodenum at postnatal month (PM) 3 to 24. Although the highest numbers of CB-immunoreactive neurons and overall population were observed in PM 1.5, it is related with significant increase of the size of circumference between PM 1.5 to PM 3. CB-immunoreactive neurons were slightly decreased with age between PM 3 to PM 24. We have also found that whole numbers of myenteric neurons were also significantly decreased in PM 24 group. These results suggest that loss of overall numbers of myenteric neurons and CB-immunoreactive neurons may be related with age-related neurodegeneration and functional loss of duodenum in the gerbil.
We performed comparative Nissl, Klüver-Barrera and Golgi staining studies of the mammalian facial nucleus to classify the morphologically distinct subdivisions and the neuronal types in the rat, rabbit, ferret, Japanese monkey (Macaca fuscata), pig, horse, Risso's dolphin (Grampus griseus), and bottlenose dolphin (Tursiops truncatus). The medial subnucleus was observed in all examined species; however, that of the Risso's and bottlenose dolphins was a poorly-developed structure comprised of scattered neurons. The medial subnuclei of terrestrial mammals were well-developed cytoarchitectonic structures, usually a rounded column comprised of densely clustered neurons. Intermediate and lateral subnuclei were found in all studied mammals, with differences in columnar shape and neuronal types from species to species. The dorsolateral subnucleus was detected in all mammals but the Japanese monkey, whose facial neurons converged into the intermediate subnucleus. The dorsolateral subnuclei of the two dolphin species studied were expanded subdivisions comprised of densely clustered cells. The ventromedial subnuclei of the ferret, pig, and horse were richly-developed columns comprised of large multipolar neurons. Pig and horse facial nuclei contained another ventral cluster, the ventrolateral subnucleus. The facial nuclei of the Japanese monkey and the bottlenose dolphin were similar in their ventral subnuclear organization. Our findings show species-specific subnuclear organization and distribution patterns of distinct types of neurons within morphological discrete subdivisions, reflecting functional differences.
Spontaneously hypertensive rats (SHRs) are widely accepted in medical research because this model has been used for studies in neurodegenerative diseases such as vascular dementia and stroke. In the present study, we observed newly generated neuronal precursors using doublecortin (DCX, a marker of neural proliferation and differentiation) in the subgranular zone of the dentate gyrus in SHRs compared to Sprague-Dawley rats (SDRs) at various age stages. DCX immunoreactivity, immunoreactive cell numbers and its protein level in the dentate gyrus of the SHRs were higher than those in the SDRs at postnatal month 1 (PM 1). At PM 8, DCX immunoreactivity, immunoreactive cell numbers and protein levels in both groups were markedly decreased compared to those at PM 1; however, they were higher than those in the SDRs. They were decreased in the both groups with age: DCX immunoreactive cells in the SDRs were few at PM 12. Our results indicate that newly generated neuronal precursors are more abundant in SHRs than in SDRs during their life.
The expression of phospholipase D (PLD) isozymes was examined in the hearts of rats at different stages of development. Immunoprecipitation and Western blot analysis revealed weak PLD1 expression in the hearts of day 17 embryos. The level of PLD1 protein increased transiently 0 and 3 days postpartum, and declined gradually beginning 7 days after birth. Immunohistochemistry revealed weak PLD1 immunostaining in some cells at embryonic day 17. In contrast, some vascular endothelial cells and cardiomyocytes were immunostained typically at days 0, 3, and 7 after birth. After postnatal day 21, weak PLD1 expression was immunodetected in some vascular endothelial cells and cardiomyocytes. This suggests that the PLD1 protein in the heart is strongly associated with the early postnatal development of the heart in rats.
To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 μM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 μM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.
Chicken monoclonal antibodies are potentially useful for diagnostic research and have clinical applications, as chicken show higher potential for antibody production with mammalian-conserved biological molecules. However, the applications of chicken antibodies are limited because of their immunogenicity in mammals. To overcome this problem, we have constructed a chicken-mouse chimeric antibody containing the chicken variable region and the mouse constant region. This chimeric antibody retained similar binding affinities as the parental chicken antibody. The chimeric antibody was also producible as an ascitic antibody in BALB/c mice. Furthermore, when the chimeric antibody was administered to mice, it did not provoke the mouse anti-chicken antibody response. These results indicate that the chimeric antibody is suitable for application to preclinical mouse studies.
We developed a novel index to assess left ventricular (LV) relaxation as the ratio of transmitral early diastolic velocity to pulmonary diastolic velocity (E/D ratio). Mixed breed dogs (n=7) were anesthetized and their respiration was controlled. A 3.5-Fr micromanometer-tipped catheter was placed into the left ventricle. Dobutamine (5.0 or 10 μg/kg/min) or esmolol (100 or 500 μg/kg/min) was administered via the cephalic vein. The transmitral flow (TMF) and pulmonary venous flow (PVF) were recorded using transthoracic echocardiography from the apical long-axis view. The heart rate, systolic LV pressure, +dP/dt, and -dP/dt were significantly elevated by dobutamine, but significantly reduced by esmolol. Dobutamine significantly decreased τ, whereas esmolol significantly increased τ. The TMF-derived E and PVF-derived D wave velocities increased significantly with dobutamine, but decreased significantly with esmolol. A significant correlation was detected between the E and D wave velocities (r=0.92). Consequently, the E/D ratio was decreased significantly with dobutamine, and increased significantly with esmolol. Furthermore, the E/D ratio was significantly correlated with -dP/dt (r= -0.64) and τ (r=0.84). Our results suggest that the E/D ratio reflects LV relaxation, and may potentially provide further information on LV relaxation.
Topical 0.03% tacrolimus was used for treatment of a Korea Jindo dog diagnosed with pemphigus erythematosus. The dog was slowly improved following application of tacrolimus but did not achieve complete remission until end of this study. No adverse effects on clinical or laboratory parameters were noted during the topical tacrolimus therapy period.
Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (β-α-α-β) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 ± 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 ± 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases.
The 18S rRNA gene and the piroplasm major immunodominant protein gene (p33/34) of Theileria from various subspecies of sika deer in 8 different locations of Japan were analyzed. The similarity between 633 bp partial sequences of the 18S rRNA gene among various subspecies of sika deer was found to be between 99.7% and 100%. While the percent identities of the 412 bp partial p33/34 gene sequence and deduced amino acid sequences between Theileria of sika deer from Yamaguchi Prefecture and those found in deer from other Prefectures, were comparatively low, 68.7% to 70.1% and 64.1% to 70.0% respectively. These findings suggest that there are at least two genetically distinct strains of Theileria of sika deer in Japan.
Five rabbits suffering from diarrhea were diagnosed with proliferative enteropathy (PE). Histopathology revealed a thickened mucosa consisting of hyperplastic intestinal epithelium and infiltration of inflammatory cells mainly consisted of macrophages. In the affected epithelial cytoplasm, numerous curved bacillus-like organisms were observed in the Warthin-Starry silver stain and electron microscopy observation. In polymerase chain reactions, Lawsonia intracellularis-specific DNA fragment were amplified from affected ileal tissue extracted DNA in each case and present 5 cases were confirmed to be L. intracellularis infection. Serum collected from the affected rabbit was immunohistochemically reactive with L. intracellularis in tissue sections from pigs with porcine proliferative enteropathy, as well as with tissue sections from the five affected rabbits. Thus, serum obtained from the affected rabbit may be applicable to immunohistochemical detection for L. intracellularis infection in other species.
Effects of electro-acupuncture (EA) stimulation on the rhythm of the autonomic nervous system in dogs were studied. Six healthy beagles were used in this study. Each dog was separately kept in a cage, and repeatedly exposed to light for 12 hr and dark for 12 hr alternately. Fixed subject dogs were stimulated by use of 5-V, 250-μsec, 2-Hz biphasic square pulses for 15 min at the Xuan Shu (GV-5) and Bai Hui (GV-20) points on the spine. After EA stimulation, electrocardiogram was recorded for 24 hr. From the electrocardiogram data, the heart rate (HR), coefficient of variation in the R-R intervals (CVRR; index of autonomic nervous activity), power of high frequency component (HF; index of vagal nervous activity), and ratio of powers of the low and high frequency components (LF/HF; index of sympathetic nervous activity) were obtained. Cosinor analysis demonstrated that these indices exhibited a significant rhythmicity (P<0.05), irrespective of EA stimulation. In LF/HF, EA stimulation advanced the acrophase (from 22:55 to 21:33, P=0.012), and elevated the midline-estimating statistic of rhythm (from 0.653 to 0.725, P=0.006). However, there was no significant difference in HR, CVRR, or HF. In conclusion, EA stimulation markedly influenced the rhythm of sympathetic nervous system in dogs.
Antibiotic therapy administered for treatment of gram-negative bacterial infections can exert a negative effect in the form of endotoxin shock. Therefore, a sterilization method that releases little endotoxin is required. This in vitro study aimed to compare the amounts of endotoxin released from Escherichia coli exposed to antibiotics (aminobenzylpenicillin, kanamycin, oxytetracycline, sulfadimethoxine and enrofloxacin) or ozone. The bacterial growth and amount of endotoxin released were measured at specific time points. Compared with antibiotic treatment, ozone sterilization induced release of smaller amounts of endotoxin from the bacteria. Moreover, no major differences were observed when ozone was used against the purified endotoxin. We conclude that compared with antibiotic treatment, ozone sterilization may release smaller amounts of endotoxin.
The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.
Susceptibility of DT40 cells to pathogenic field strains of infectious bursal disease virus (IBDV) including very virulent and classical virulent strains were studied. After the first and second passage of the virus in DT40 cells, IBDV-specific antigen was readily detected in DT40 cells inoculated with the pathogenic field strain infected bursal homogenates. Nucleotide sequence analysis in the VP2 hypervariable domain, which is critical for the virulence of IBDV, revealed no common amino acid substitutions among the pathogenic IBDVs in accordance with the propagation in DT40 cells. These results indicate that DT40 cells are a useful tool for rapid isolation of pathogenic field strains and successive in vitro analysis of IBDV.
Swine influenza virus (SIV) was isolated from a farm in Miyazaki Prefecture in Japan in July 2006. An isolate was genetically subtyped as H1N2 and was designated A/swine/Miyazaki/1/2006 (H1N2). The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. All segments were shown to be closely related to those of Japanese SIV H1N2 isolates, which have been circulating since the 1980s. The results indicate the persistence of the SIV H1N2 subtype in the Japanese pig population for more than two decades and emphasize the importance of continuous surveillance for SIV.