The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.
Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.
One hundred dogs that were positive for canine distemper virus antigen and inclusion bodies in the tonsils were examined for the distribution of inclusion bodies in various tissues. Inclusion bodies were found in the lungs (70 dogs), brains (20 dogs), urinary bladders (73 dogs), stomachs (78 dogs), spleens (77 dogs), and lymph nodes (81 dogs) of the dogs. Based on these results, the tonsils may be the most suitable tissue for detection of inclusion bodies in canine distemper.
This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.
The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1β, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-α and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-α and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1β as well as TNF-α, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1β and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1β/IL-18 production through caspase-1 induction.
This study evaluated the suitability of using a chitosan formulation as an adjuvant to enhance both the mucosal and systemic immune responses against recombinant transferrin-binding protein B (rTbp B) of Actinobacillus pleuropneumoniae via direct tracheal administration. The chitosan formulation was found to enhance mucosal immune response, as measured by the secretory IgA level in lung lavage fluid and lung homogenate extracts, and systemic immune response, as measured by the serum IgG level.
In the present study, we tested the hypothesis that vasopressin administration prior to crystalloid resuscitation can be used to improve hemodynamic and oxygen delivery functions. Hemorrhagic shock was experimentally induced by maintaining mean arterial pressure at 60 mmHg for 30 min in sixteen healthy dogs weighing from 8 to 10.6 kg. Vasopressin was administered and then volume resuscitation was performed for the 6 dogs of V-C group, while vasopressin was administered at the end of volume resuscitation in the 5 dogs of C-V group. The control group (n=5) was administered 0.4 IU/kg of vasopressin after induction of shock without fluid resuscitation. In all groups, hemodynamic parameters were measured pre- and post-hemorrhage and for 60 min after fluid resuscitation. The dogs in V-C group had substantially increased systolic arterial pressure (SAP) for 60 min and improved pulmonary capillary wedge pressure (PCWP), cardiac output (CO), oxygen delivery, and oxygen consumption indexes compared with C-V and control groups. Diastolic pressure and systemic vascular resistance was significantly lower in the V-C group than those in the C-V and control groups (P<0.05). In the V-C group, there was effective and rapid restoration of the SAP, CO, PCWP, and oxygen delivery parameters after treatment. This study indicates that vasopressin administration before crystalloid resuscitation is a more efficient way of improving hemodynamic and oxygen delivery functions in hemorrhagic shock in dogs.
We evaluated the relationship between depression score and acid-base status in 84 purebred and crossbred Japanese Black calves. The bicarbonate (p<0.001) and base excess concentrations (p<0.001) were significantly and negatively correlated with the depression scores of the calves. The proposed diagnostic cutoff point for a depression score that indicates severe metabolic acidosis (BE < -10 mM) is 6.5 based on analysis of the ROC curve. The sensitivity and specificity were 88.4% and 81.2%, respectively. The depression scoring system is a useful tool for evaluation of the acid-base status of purebred and crossbred Japanese Black calves. In addition, a depression score of 6.5 suggests severe metabolic acidosis and that intravenous infusion of sodium bicarbonate solution is necessary.
The effectiveness of combination therapy using clindamycin, metronidazole and doxycycline against canine babesiosis, and the usefulness of platelet count and the plasma C-reactive protein (CRP) concentration as an estimation factor for treatment, were evaluated in four dogs experimentally infected with Babesia gibsoni. The combination therapy successfully eliminated B. gibsoni in peripheral blood in 3 of 4 dogs, however the remaining dog showed obvious uncontrolled relapse after a temporary recovery. In addition, it was shown that CRP levels decreased in an inverse relationship to the recovery of packed cell volume and therefore CRP levels could be used as an optional clinical marker to estimate the response to treatment.
The nucleotide sequences of 18S rDNA and internal transcribed spacer (ITS) regions were used for studying the relationships of Trypanosoma evansi isolate from a buffalo. The sequences were analyzed and compared to 18S rDNA and the ITS regions of the other Trypanosoma spp. Maximum likelihood phylogenetic trees were constructed using Leishmania major as the outgroup. The tree of 18S rDNA indicated that T. evansi (buffalo B18) isolate was closely related to those of Taiwan and T. brucei stock. The ITS tree showed the genetic diversity among 32 clones of T. evansi (B18) within a single host. This data will be useful for epidemiological and dynamic studies for designing the rational control programs of the disease.
Species of the genus Syphacia are considered to have generally co-evolved with their rodent hosts. This study determined partial sequences of the CO1 gene from several species in the genus Syphacia and discuss the relationships between pinworms and their hosts. Syphaciamontana, which parasitizes Microtinae, was closely related to S. frederici and S. obvelata, which parasitize Murinae. Although both S. obvelata and S. ohtaorum parasitize rodents in the genus Mus, these two species were not found to be closely related to each other. Syphaciafrederici, S. emileromani and S. agraria are all pinworms of the Apodemus species, but genetic affiliation between these three species was not indicated. These facts suggest that the co-evolutionary relationship between species of the genus Syphacia and their host rodents may not so strict and host switching has probably occurred during the course of evolution.
A female adult bottlenose dolphin suddenly died at 17 days after the capture. Macroscopically, severe pulmonary congestive edema was found. Histopathology revealed many lungworms in the bronchioli and the worms were identified as Stenurus ovatus. Variously sized vessels proliferated around the lesioned bronchioli. Based on these findings, chronic bronchopneumonia due to the lungworm was diagnosed and vascular proliferation was similar to angiomatosis recently reported in Atlantic bottlenose dolphin.
The aims of this study were to measure the activity of the autonomic nervous system using heart rate variability (HRV) during learning tasks and to clarify the relationship between learning to overcome a difficult situation and the autonomic nervous system in monkeys. Two young male monkeys (Macaca mulatta) were given simple discrimination learning tasks (DL) and delayed matching to samples tasks (DMTS); Holter-type electrocardiography was done, and HRV was measured. We defined the frequency bands of HRV in rhesus macaques; the low frequency (LF) was 0.01-0.15 Hz, and the high frequency (HF) was 0.15-0.50 Hz. Based on these frequency bands, the LF/HF ratios during learning tasks were analyzed, and a significant increase in the ratio was found during the tasks. The variances in the HF differed between the DL and DMTS tasks; during DMTS tasks, HF variances had a tendency to increase. Our results indicate that increased sympathetic activity accompanied learning and suggest that the parasympathetic nervous system plays a key role during learning, particularly when difficult tasks are being learned.
In dogs, a variety of diseases of the retina and choroid have been reported, either separately or concomitantly; however, the canine choroid is difficult to evaluate by veterinary techniques currently available. Indocyanine green (ICG) angiography is widely used in human ophthalmology, but has not been investigated for use in canine ophthalmology. The aim of this study was to apply a new approach to ICG angiography and compare the resulting angiograms with fluorescein (FLUO) angiograms of the ocular fundus in dogs. With a fundus camera equipped with an infrared-sensitive charged coupled device (CCD), we performed angiography on eight healthy beagles under inhalation anesthesia. ICG angiography enabled clear visualization of the choroidal vasculature, whereas FLUO angiography showed only the retinal vessels. At 8.4 ± 3.6 sec after administration of ICG dye into the cephalic vein, the choroidal arteries could be seen extending radially from the optic disc, then the choroidal veins became apparent at 10.2 ± 4.1 sec, coursing alongside the choroidal arteries. Gradual fading of the choroidal vessels began 13.2 ± 2.2 min after the dye was administered, and overall diffuse fluorescence of the fundus appeared. Diffuse fluorescence of the fundus continued after the choroidal vessels and optic disc faded at about 58.3 ± 5.3 min from administration of the dye. In conclusion, ICG angiography provides clear resolution and is reliable and simple, thus offering promise as a diagnostic aid for clinical evaluation of the choroid in dogs.
The interactions of tumor cells with the extracellular matrix (ECM) are a crucial step in invasion and metastasis. Integrins are adhesive molecules forming heterodimers with α and β subunits that play a definitive role in these interactions. In this study, mastocytoma (mast cell tumor: MCT) cell-ECM interaction was investigated using 3 canine MCT cell lines: CM-MC (originating from cutaneous MCT), VI-MC (originating from intestinal MCT), and CoMS (originating from oral MCT). Flow cytometric analysis showed that all cells highly expressed the integrin β1 and α1 through α5 subunits and that they moderately expressed the α6 subunit. In adhesion studies, CoMS weakly but spontaneously adhered to fibronectin (FN), which was enhanced by phorbol ester (TPA), while CM-MC and VI-MC required cell activation by TPA to adhere to FN. Anti-β1 and α5 integrin antibodies strongly inhibited cell adhesion to FN in CM-MC and CoMS and moderately inhibited cell adhesion in VI-MC. Only VI-MC adhered to laminin (LN) under activation by TPA. Anti-β1 integrin antibodies strongly inhibited cell adhesion to LN, but all anti-α integrin antibodies failed to inhibit cell adhesion to LN. No cells adhered to collagen types I and IV. Canine MCT cells from different origins expressed similar integrin patterns; however, there were some differences in adhesive behavior in response to various ECM proteins and activating stimuli.
The purpose of this study was to evaluate the antagonism of acupuncture-induced sedation by electroencephalographic spectral edge frequency (SEF) 95 in 10 healthy intact male Miniature Schnauzer dogs (4.2-6.1kg; 2-3 years old) without neurological disorder. The GV20 and Yintang acupoints were administered for 20 min. While the dogs were conscious, SEF 95 baseline values were recorded at 2-min intervals for 4 min. Then acupuncture was administered at the GV20 and Yintang acupoints. During the acupuncture procedure, the SEF 95 values were recorded at 2-min intervals for 10 min. Subsequently, antagonist drugs, naloxone and atipamezole, were administered through the cephalic vein. The SEF 95 values were then measured again at 2-min intervals for 10 min. Those values were found to be significantly increased after administration of atipamezole in dogs sedated by acupuncture at the GV20 and Yintang acupoints (p<0.05). However, the SEF 95 values in the naloxone groups did not show any significant changes before and after administration of the antagonist. It was concluded that sedation induced by acupuncture at the GV20 and Yintang acupoints might be partially associated with the α2-adrenergic system.
Ichthyosis (fish scale disease) is a rare hereditary disease and characterized by excessive cutaneous scale formation. A male HanWoo calf born by natural service was found with fissures and thickened, scaly, cutaneous plates covering over 90% of its body. Histopathological feature was excess compact orthokeratotic hyperkeratosis involving surface of the epidermis and follicular epithelia. The calf had small malformed ears, ectropion, eclabium and an abnormal nose. Gross and histopathologic findings in calf were consistent with those of harlequin ichthyosis, and it was the first observed harlequin ichthyosis in HanWoo cattle.
Increased hepatic metabolism of estradiol may cause weakened estrous behavior in lactating dairy cows, but this hypothesis must be examined further, especially through diachronic study of the hepatic estradiol-17β glucuronidation activity of uridine diphosphate (UDP)-glucuronosyltransferases. Therefore, in order to develop a new tool for this purpose, we attempted to conduct biopsy of the livers of dairy cows with the aid of ultrasonography and to measure the UDP-glucuronosyltransferase activities of microsomes of the specimens using in vitro glucuronidation followed by HPLC analysis. We were able to measure the activities of the microsomes prepared from the liver biopsy, and the results seemed reliable. Therefore, this method may become a new tool in clinical studies to detect estradiol-17β glucuronidation activity.
The peripheral blood plasma testosterone (T) levels and superoxide dismutase (SOD) activity were measured in 5 azoospermic (AZ-) beagles. The mean values in the AZ-dogs were significantly lower than in 7 control beagles (P<0.001). Subcutaneous injections of 1 μg/kg GnRH analogue three times weekly in the AZ-dogs induced significant increases in mean T level and SOD activity (P<0.05) and improvement in spermatogenesis. Thus, spermatogenic function in the dog appears to be maintained by T and normal SOD activity in the testis.
Feces were collected from two female and one male Siberian tigers, Panthera tigris altaica. Steroid hormones were extracted from lyophilized feces and quantified by enzyme immunoassay. The fecal contents of estradiol-17β (E2) and testosterone in the females and male, respectively, changed markedly throughout the year. The fecal E2 contents of females Nos. 179 and 238 increased at 26.4 ± 8.0 and 28.0 ± 14.2 day intervals, respectively. However, the fecal contents of progesterone (P4) in the female kept alone did not change. In contrast, the other female, which was kept with a male, had increased fecal P4 contents after copulation. The fecal progesterone levels of the pregnant female remained high during her 106-day pregnancy.
The aim of this study was to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples and to determine whether a relationship exists between BoTV and diarrhea in Japan. Ninety-nine diarrheic and 114 normal fecal samples from calves in Hokkaido Prefecture and 38 diarrheic fecal samples from calves in 10 other prefectures were examined by reverse transcription (RT)-PCR with primers designed in the spike (S) gene for the presence of BoTV. The specimens were also examined for the presence of other enteric pathogens, bovine rotavirus, coronavirus and Cryptosporidium spp. BoTV RNA was detected in 15 (15.2%) of the 99 diarrheic samples from Hokkaido and in 9 (23.7%) of the 38 diarrheic samples from the other prefectures. The incidence of BoTV in control specimens was 7.0%. In 11 of the 15 BoTV-positive specimens from Hokkaido, BoTV was the only pathogen detected among those examined, and 11 BoTV-positive specimens were obtained from calves less than 2 weeks of age. Rotavirus was confirmed to be associated with calf diarrhea, but coronavirus and Cryptosporidium spp. were not. Nucleotide sequences of 17 different BoTV RT-PCR products were determined. Phylogenetic analysis based on the sequences revealed that Japanese BoTVs could be classified into at least two groups. This study showed that BoTV is a common virus in fecal specimens of calves with diarrhea in Japan and may be an important pathogen of cattle, principally in young calves less than 2 weeks of age.
The 475 strains of bovine viral diarrhea virus (BVDV) isolated from cattle in 12 prefectures of Japan in the last 7 years were phylogenetically classified as BVDV-1 or BVDV-2 on the basis of the nucleotide sequence of the 5'-untranslated region. BVDV-1 strains were further subtyped as 1a (101 strains), 1b (163), 1c (128), 1j (3), and So CP/75-like (1), and all of the 79 BVDV-2 strains belonged to subtype 2a. These 2a BVDVs contain two isolates that had high nucleotide identities with those of highly pathogenic BVDV-2 strains reported in North America (Pellerin et al., 1994). However, acute infection with severe mortality like North American outbreak was not observed and most of the present BVDV-2 strains were isolated from persistently infected (PI) cattle showing mild or no clinical sign. Moreover, it was revealed that 61.5% of the 39 PI cattle with cytopathogenic BVDVs did not show typical mucosal disease and 54.6% of the 405 PI animals only with non-cytopathogenic BVDVs were apparently healthy. The present results indicate that the prevention of the infection with an appropriate vaccine and active surveillance covering healthy cattle are required for the control of BVD.
In this study we report the first outbreak of camelpox in two provinces in Syria. Clinical symptoms started with fever, salivation and general exanthema. The main features were facial and legs oedema, pustules on the mucosa of the lips and a high rate of abortion. Lesions may also occur on the whole body including scrotum and udder. Specimens were investigated by electron microscopy, virus isolation in cell culture and embryonated eggs and by immunohistochemistry. The causative agent was identified as camelpox virus by polymerase chain reaction and sequencing of the hemagglutinin gene.
The pathogenicities of RacL11 and Kentucky D strains of equine herpesvirus 1 in the hamster infection model are different from those of Ab4p and the Japanese isolates. Virus genome restriction fragment length polymorphism analysis and sequence comparison of an intergenic region, glycoproteins and tegument genes showed higher conservation but with some strain-specific differences. These results indicate that point nucleotide differences in RacL11 and Kentucky D might be responsible for their pathogenicity in rodent models.