The olfactory organ of African lungfish, Protopterus annectens, contains two distinct sensory epithelia: the lamellar olfactory epithelium and the recess epithelium. These epithelia correspond to the olfactory epithelium and the vomeronasal organ of tetrapods, respectively. In contrast to the lamellar olfactory epithelium, which has no associated gland, the recess epithelium is equipped with associated glands. Although the glandular cells and/or the supporting cells are generally presumed to secrete proteins involved in the function of olfactory sensory epithelia, the properties of these proteins in lungfish have not been evaluated to date. In this study, we investigated the associated glands in the olfactory organ of lungfish by transmission electron microscopy and found that the glandular cells contain numerous secretory granules and secrete them from the apical membrane. In addition, we analyzed the olfactory organ by lectin histochemistry using 16 biotinylated lectins. All lectins labeled the secretory granules in the glandular cells with different staining patterns from those of the supporting cells in the lamellar olfactory epithelium or in the recess epithelium. Furthermore, lectin blotting analysis showed that multiple bands were detected by the lectins which specifically labeled the glandular epithelium of the olfactory organ. These results indicate that the secretory products of the associated glands in the recess epithelium have different properties from those of the supporting cells in the olfactory sensory epithelia and contain multiple glycoproteins with different carbohydrate moieties.
The relationship between the invasion of indigenous bacteria into intestinal crypts and the proliferation of epithelial cells was histoplanimetrically investigated in the rat ascending colon. Indigenous bacteria preferentially adhered to the intestinal superficial epithelial cells in the mesenterium-attached mucosa (MAM) compared to those in the mesenterium-non-attached mucosa (MNM). Intestinal crypts with indigenous bacteria were also significantly more frequently found in MAM than in MNM. Total epithelial cells, columnar epithelial cells and goblet cells were significantly more abundant in the intestinal crypts with no-indigenous bacteria in MAM (MAM-C) than those in MNM (MNM-C), whereas the columnar epithelial cells were less abundant in MAM-C than in the intestinal crypts with indigenous bacteria in MAM (MAM-C-B). Columnar epithelial cells and goblet cells immuno-positive for proliferating cell nuclear antigen (PCNA) in MAM-C were more abundant than those in MNM-C, but less abundant than those in MAM-C-B. Toll-like receptor (TLR)-2, -4 and -9 were immuno-positive in the striated borders of the intestinal superficial epithelial cells, but their positive intensities were weaker in MAM than in MNM. From these findings, indigenous bacteria were confirmed to preferentially settle on the intestinal superficial epithelium of MAM in the rat ascending colon, and low TLRs-expression might contribute to the preferential settlement of indigenous bacteria in MAM. The increase of proliferating epithelial cells is probably induced by the invasion of indigenous bacteria into the intestinal crypts of MAM.
The aim of this study was to show that a 39-kDa protein or OmpH of Pasteurella multocida strain P-1059 is essential for cross protection. Strain PBA322, a thinly capsulated strain of P. multocida strain P-1059, was used as a live vaccine in chickens. Strain PBA322 is a thinly capsulated strain in comparison with the parental strain P-1059. Chickens were vaccinated by single injection and then challenge-exposed with strains P-1059 or X-73 at two weeks post vaccination. Moreover, immune responses were also evaluated for both humoral and cellular immune response by ELISA and lymphocyte proliferation assay, respectively. The results showed that the live vaccine induced efficient immunity to protect chickens from challenge-exposure to the parent strain, but that the heterologous protection was poor. We concluded that the 39-kDa protein is essential for cross protection.
The prevalence of hemotropic mycoplasmas in wild rodents is largely unknown. Here, we report the presence of hemoplasmas in blood samples collected from brown sewer rats (Rattus norvegicus) trapped during rodent control around an animal hospital in Morioka, Japan. We examined nine rats using real-time PCR and end-point PCR, and found one rat (11.1%) that was positive for a hemoplasma infection. The 16S rRNA gene and 16S to 23S rRNA intergenic spacer region of the hemoplasma detected in a wild-caught rat were amplified using PCR. The nucleotide sequences of the PCR products were further determined and compared to those of other hemoplasmas. Our examinations revealed the presence of a hemoplasma that has not previously been described in rodents. The pathogenic traits of this hemoplasma remain unexplored.
We analyzed the distribution of 11 periodontitis-related bacterial species in dental plaque collected from 176 Japanese dogs divided into young (less than 2 years of age), middle-aged (2–7 years of age) and elderly (more than 8 years of age) groups using a polymerase chain reaction method. Clinical examination revealed that no dogs in the young group were affected by periodontitis, whereas the rates for gingivitis and periodontitis were high in the middle-aged and elderly groups. In addition, the total numbers of bacterial species in the middle-aged and elderly groups were significantly greater than in the young group. Our findings suggest that age is an important factor associated with the distribution of periodontitis-related bacteria and periodontal conditions in dogs.
Diet therapy is an important treatment component available for obese cats. In this study, the impact of four commercially available prescription diet regimens (1 for general use and 3 aimed at treating obesity and diabetes mellitus (DM)) on short-term postprandial serum glucose, insulin, triglyceride and nonesterified fatty acid (NEFA) concentrations was investigated with five obese cats. The diet regimens used were as follows: C/D dry (general use: moderate protein, moderate fat, high carbohydrate and low fiber), M/D dry (DM: high protein, high fat, low carbohydrate and high fiber), W/D dry (DM: high protein, low fat, high carbohydrate and high fiber) and Diabetic dry (DM: high protein, low fat, low carbohydrate and high fiber). A significant reduction (10–13%) in postprandial glucose (area under the curve; AUC) was observed with the M/D and Diabetic diets, which both contained lower concentrations of carbohydrates than the C/D diet. An accompanying significant reduction (30–36%) in postprandial insulin AUC was also observed with the three DM diets, which all had higher amounts of fiber, as compared with the C/D diet. Lastly, a significant increase (32–65%) in postprandial NEFA AUC was observed with the M/D and Diabetic diets as compared with the C/D diet. Therefore, dietary amounts of carbohydrates and fiber, as opposed to protein content or dietary fat, appear to have a very significant impact on postprandial glycemia and subsequent insulin requirement levels in obese cats. In addition, dietary amounts of carbohydrates may also impact lipid metabolism in obese cats.
In humans, increased levels of GFAP in the CSF and blood have been reported with various neural diseases. However, there has been no study describing the usefulness of GFAP in the blood for disease of the spinal cord in dogs. The aim of this study was to describe the utility of GFAP in serum for a diagnosis of progressive myelomalacia. Fifty-six dogs with acute thoracolumbar IVDD diagnosed by computed tomography with myelography or MRI were included. Serum specimens were collected at initial presentation from all cases and at follow-up examinations from some cases. Serum samples were assayed for GFAP concentrations using a commercially available GFAP ELISA Kit. Progressive myelomalacia was the final diagnosis in 8/51 cases (15.6%). Eight dogs had clinical signs suggestive of progressive myelomalacia, of which 6 were positive and 2 were negative by GFAP. Seven dogs had a detectable level of serum GFAP, of which 6 had the onset of progressive myelomalacia. The sensitivity and specificity of the GFAP to progressive myelomalacia were 75% and 97.7%, respectively. The results suggest the utility of GFAP in serum in the diagnosis of progressive myelomalacia.
Isolates of the yeast Malassezia pachydermatis obtained from skin samples of healthy dogs and of dogs with atopic dermatitis in Japan, Taiwan and Korea were molecularly characterized using intergenic pacer 1 (IGS1) region analysis. The percentage of IGS1 subtype isolates detected in healthy skin was as follows: 1A (6%), 1B (27%), 1C (11%), 2A (6%), 2B (6%), 3A (11%), 3B (6%), 3C (3%) and 3D (24%). In contrast, the most prevalent isolates detected in skin lesions of atopic dermatitis were subtype 3D in Japan and Taiwan and subtype 3C in Korea. All subtype isolates grew well on acidic medium (pH 6). However, subtype 3C and 3D isolates grew better than the other subtype isolates on medium at pH 8.
In the present work, a retrospective study on the localization of vegetal foreign bodies (VFBs) inside the tracheobronchial tree of 41 dogs is reported. In total, 47 grass awns were found, and only three dogs presented more than one foreign body. Most VFBs were found inside right bronchial ramifications (76.6%, no. 36), while 23.4% (no. 11) were found inside the ventrocaudal branch of the left cranial lobar bronchus. The impossibility of predicting the location and the concomitant presence of more than one foreign body in a single patient are conditions that impose the need for careful evaluation of the whole bronchial tree in all cases.
The cross-reactivity of the anti-human D-dimer antibody 1C9-6F10 to the canine D-dimer was evaluated by western blotting using purified canine fibrinogen, fibrin degradation products (XDPs) and plasma from a dog with suspected disseminated intravascular coagulation (DIC). The antibody cross-reacted with canine XDPs and plasma from the dog with suspected DIC. A latex agglutination assay using 1C9-6F10 measured the canine XDPs in dogs. In conclusion, the antibody 1C9-6F10 cross-reacted to the canine D-fragment with high specificity, but lower affinity compared with the human D-dimer. The latex agglutination assay using the 1C9-6F10 antibody was used to measure canine plasma D-dimer concentrations.
Myeloid cell leukemia sequence 1 (MCL1) is a potent antiapoptotic protein that plays a critical role in cell survival and drug resistance in various cancers. However, to the best of our knowledge, the role of MCL1 in mast cell tumors (MCTs) has not been investigated in dogs. Here, we detected increased MCL1 expression in MCT cell lines, regardless of the presence of a c-kit mutation. MCL1 expression increased when the cells were exposed to specific inhibitors of mitogen-activated protein kinase or Janus kinase-signaling pathways, thus protecting the cells from apoptosis, but not when KIT or phosphatidylinositol-3 kinase signaling cascades were inhibited. These results indicate that MCL1 expression may contribute to MCT survival and confer drug resistance.
Two dogs that exhibited sudden astasia, anorexia and fever higher than 40°C were suspected of having Lyme disease in July 2011. Clinical symptoms gradually improved with antibiotic treatment in both cases. Polymerase chain reaction and sequence analysis revealed Borrelia garinii DNA fragments in the peripheral blood in the acute disease phase. Serological tests, including enzyme linked immunosorbent assay and Western blot analysis, showed an increased IgG antibody titer against Borrelia pathogens in one of the dogs. These findings suggested that diagnosis of the two dogs was Lyme disease related to B. garinii infection.
An 8-year-old male Shiba dog presented with chronic vomiting and diarrhea. Upper gastrointestinal endoscopy revealed severe enteritis and infection of the duodenal mucosa with Echinostoma hortense. We performed therapy for parasites and enteritis. The therapy was successful for deworming and temporarily improved the symptoms, but the dog died soon thereafter. To the authors’ knowledge, this is the first case report of an antemortem diagnosis of E. hortense infection in a dog.
DDD.Cg-Ay female mice developed massive obesity as compared with B6.Cg-Ay female mice. We previously identified quantitative trait loci (QTLs) for obesity on chromosomes 1, 6, 9 and 17 in F2 female mice, including F2Ay (F2 mice with the Ay allele) and F2 non- Ay mice (F2 mice without the Ay allele), produced by crossing C57BL/6J and DDD.Cg-Ay strains. We here addressed the question whether the obesity QTLs share genetic bases with putative QTLs for plasma glucose, insulin and leptin concentrations. We performed QTL analyses for the first principal component (PC1) extracted from these metabolic measurements to identify the genes that contributed to the comprehensive evaluation of metabolic traits. By single QTL scans, we identified two significant QTLs for insulin concentration on chromosomes 6 and 12, three for leptin concentration on chromosomes 1, 6 and 17, and five for PC1 on chromosomes 1, 6, 12 (two loci) and 17. Although insulin and leptin concentrations and PC1 were not normally distributed in combined F2 mice, results of single QTL scans by parametric and non-parametric methods were very similar. Therefore, QTL scan by the parametric method was performed with the agouti locus genotype as a covariate. A significant QTL × covariate interaction was found for PC1 on chromosome 9. All obesity QTLs had significant metabolic effects. Thus, obesity- and diabetes-related traits in DDD.Cg-Ay mice were largely controlled by QTLs on chromosomes 1, 6, 9, 12 and 17.
Sendai virus (SeV) is one of the most prevalent viral pathogens infecting laboratory mice and rats. To date, mature SeV virions have been used as antigens for serological diagnosis. To develop antigens that are more specific and easier to prepare for diagnosis, we examined the antigenic sites in the nucleocapsid protein (NP) of SeV with antisera from experimentally SeV-infected mice and a peptide array membrane containing overlapping 10-mer peptides covering the entire NP. We found antigenic linear sequences in two regions, amino acids 120–160 and 420–500, of the SeV-NP. From these antigenic sequences, we applied two synthesized peptides, IVKTRDMEYERTTEWL and FVTLHGAERLEEETNDE, which correspond to positions 119–134 and 458–474 of the SeV-NP, respectively, as antigens in an enzyme-linked immunosorbent assay (ELISA). Evaluation of the ELISAs using these peptides revealed that they were specific to anti-SeV antisera. Furthermore, the ELISAs using these peptides were able to distinguish between SeV-positive and SeV-negative mouse sera to the same extent as a commercial ELISA kit. These results indicate that these peptides are useful for the serological diagnosis of SeV infection.
Cattle are major hosts of Cryptosporidium. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. Fecal samples were collected from calves in Ishikari District, Hokkaido, Japan and examined by PCR and sequence analyses. Among the 107 fecal samples collected in May and June 2012, 25 (23%) were positive for Cryptosporidium, including 8 samples (7%) having C. parvum, 10 (9%) having C. bovis and 7 (7%) having C. ryanae. This is first time C. ryanae has been detected in Hokkaido. Furthermore, it is the first detection of C. ryanae from pre-weaned calves in Japan. Microscopic observation with the flotation method is powerful and traditional tool for screening for Cryptosporidium species, but it sometimes leads to low detection of Cryptosporidium with low oocyst shedding intensity. If calves with or without diarrhea are examined using the molecular diagnostic tools, C. bovis and C. ryanae might be detected in other areas of Japan including Hokkaido. Here, the zoonotic species, C. parvum, was also observed. Therefore, calves can be potential sources of cryptosporidial infections for humans and other animals. The detection of C. parvum was statistically correlated with diarrhea in calves.
Babesia microti is a rodent tick-borne blood parasite and the major causative agent of emerging human babesiosis. Here, we identified a candidate of common antigenic protein BmP41 of B. microti by serological screening of cDNA library of human-pathogenic Gray strain with antisera against rodent Munich strain. Immunofluorescent antibody test using mouse anti-recombinant BmP41 (rBmP41) serum revealed that native BmP41 was expressed in each of the developmental stages of B. microti merozoites. An enzyme-linked immunosorbent assay (ELISA) using rBmP41 detected specific antibodies in sera from hamsters infected with B. microti Gray strain and mice infected with B. microti Munich strain. Taken together, BmP41 could be a promising universal serological marker for diagnosis of human babesiosis.
A total of 250 blood samples were collected from clinically healthy cattle in five provinces of Philippines. DNA was extracted from the samples and analyzed by nested PCR assays for an epidemiological survey of Babesia bovis and Babesia bigemina infections. Out of the 250 samples, 27 (10.8%) and 16 (6.4%) were positive for B. bovis infection and B. bigemina infection, respectively. Mixed infections were detected in a total of 4 samples (1.6%). Our data provide baseline information regarding the epidemiology of B. bovis and B. bigemina infections in cattle in Philippines, which can be utilized in developing proper strategies for disease control and management.
Histopathologically, fibrosis in Fasciola-infected cattle livers was characterized by inflammatory cell infiltration, such as eosinophils and macrophages, pseudo-lobule, pseudo-bile ducts and fibrotic bridges separating pseudo-lobules; the fibrotic lesions were developed in the Glisson’s sheath. Pseudo-bile ducts consisting of epithelial cells reacted clearly to cytokeratin (CK) 19, indicating cholangiocyte origin. Immunophenotypes of macrophages and myofibroblasts were investigated in the fibrotic livers. Macrophages positive for CD68 (reflecting phagocytosis) and CD163 (representing proinflammatory cytokine production) were increased, and those for CD204 (implying lipid metabolism) and Iba-1 (a calcium-binding protein playing role in chemotaxis) decreased in fibrotic livers compared to control livers. Spindle-shaped myofibroblasts positive for vimentin, desmin and α-smooth muscle actin (α-SMA) increased in the peribiliary connective tissues, although the desmin-positive cells were fewer. In addition to the usefulness of these antibodies for macrophage detection in cattle livers, this study shows that macrophages with different immunophenotypes participate in Fasciola-infected cattle livers, in relation to development of myofibroblasts expressing mainly vimentin and α-SMA.
To determine hemodynamic effects of hydroxyethyl starch (HES) infusion during anesthesia in horses, incremental doses of 6% HES were administered to 6 healthy Thoroughbred horses. Anesthesia was induced with xylazine, guaifenesin and thiopental and maintained with sevoflurane at 2.8% of end-tidal concentration in all horses. The horses were positioned in right lateral recumbency and administered 3 intravenous dose of 6% HES (5 ml/kg) over 15 min with 15-min intervals in addition to constant infusion of lactated Ringer’s solution at 10 ml/kg/hr. Hemodynamic parameters were measured before and every 15 min until 90 min after the administration of 6% HES. There was no significant change in heart rate and arterial blood pressures throughout the experiment. The HES administration produced significant increases in mean right atrial pressure, stroke volume, cardiac output (CO) and decrease in systemic vascular resistance (SVR) in a dose-dependent manner. There was no significant change in electrolytes (Na+, K+, Cl-) throughout the experiment, however, packed cell volume, hemoglobin concentration, and total protein and albumin concentrations decreased in a dose-dependent manner following the HES administration. In conclusion, the HES administration provides a dose-dependent increase in CO, but has no impact upon arterial blood pressures due to a simultaneous decrease in SVR.
Bone marrow cell infusion (BMI) has recently been suggested as an effective therapy for refractory liver disease; however, the efficiency of BMI using canine bone marrow cells (cBMCs) has not been reported. We evaluated the accumulation potential of cBMCs in a mouse model of acute liver failure. Acute hepatitis was induced by carbon tetrachloride (CCl4) treatment in NOD/SCID/γcnull(NOG) mice and wild-type (WT) C57BL mice, and the characteristics of liver dysfunction and the degree of hepatic injury and regeneration were compared between the two mouse models. Next, female CCl4-treated NOG mice were xenotransplanted with male PKH26-labeled cBMCs, and the potential of cBMCs to accumulate in injured liver tissue compartments was examined. Fluorescence microscopy was performed to histologically detect the infused cBMCs, and DNA polymerase chain reaction was performed for detection of the male Y chromosome (SRY gene) in the recipient female NOG mice. The number of PKH26-positive cBMCs transplanted in the liver tissue gradually increased in the NOG mice. The infused cBMCs were located in the necrotic area of the liver at an early stage after transplantation, and most had accumulated a week after transplantation. However, the therapeutic efficacy of the xenotransplantation remained unclear, because no significant differences were observed concerning the extent liver injury and regeneration between the cBMC-transplanted and saline control mice. These results suggest that cBMCs will specifically accumulate in injured liver tissue and that BMC transplantation may have the potential to repair liver deficiency.
Stem cell transplantation is one of the most promising yet enigmatic treatments for spinal cord injury (SCI), a common problem in dogs. As pre-differentiated mesenchymal stem cells (MSCs) can be expanded and differentiated into neurospheres in vitro, before being transplanted back, they may prove to be more beneficial for treating SCI. Therefore, we compared the endogenous differentiation potential, including the neuronal cell differentiation, of neurospheres from canine bone marrow MSCs (cBMMSCs) with that of the adipose tissue-derived MSCs (cADMSCs). Nestin-positive neurospheres were generated from MSCs derived from the bone marrow and adipose tissue. Neuronal cells were differentiated from the neurospheres derived from both these tissues. Gene expression analysis revealed that Nestin, βIII-tubulin, NCAM, OCT4 and SOX2 were expressed in MSCs and the corresponding neurospheres. Notably, cBMMSC-derived neuronal cells expressed higher levels of βIII-tubulin. The mRNA expressions of NANOG, Nestin, OCT4 and SOX2 were upregulated in neurospheres derived from both. Immunofluorescence analysis detected the expression of neuronal markers, namely, βIII-tubulin, GFAP, S100, NF200 and MAP2, in differentiated neuron-like cells. Our findings highlight that both cBMMSCs and cADMSCs could be differentiated into neurospheres and neuron-like cells, and therefore, these cells are suitable candidates for cell transplantation. Further, cADMSCs form a more suitable cell source, as larger number of cells could be harvested from cADMSC-derived neurospheres. Future studies employing in vivo transplantation models to investigate the effectiveness of MSCs for treating SCI are warranted.
To investigate influence of general anesthesia on immunological anti-tumor activity, the natural killer (NK) cytotoxic activity of peripheral lymphocytes (PBLs) was measured in 7 dogs anesthetized for 3 hr with isoflurane following propofol-induction (anesthesia group) and 6 dogs without anesthesia (control group). Blood samples were collected before (baseline) and 24, 120 and 192 hr after the anesthesia. The PBLs were isolated via centrifugation with Ficoll-Hypaque solution (density, 1.073), and adherent cells were removed. The NK cytotoxic activity of the isolated PBLs against canine thyroid cancer cells was detected by the colorimetric rose Bengal assay. Significant decrease in the NK cytotoxic activity was observed at 24 hr after the anesthesia, compared with the baseline values and the control group. The NK cytotoxic activities were recovered to the baseline values until 120 hr after the anesthesia. The general anesthesia with isoflurane following propofol-induction decreased the NK cytotoxic activities of PBLs in dogs. This finding has a clinical relevance to the risk of tumor recurrence or metastasis induced by the suppression of immunological anti-tumor activity after general anesthesia in dogs. The results further emphasized the importance of the need to evaluate immune suppression following general anesthesia in animals.
Craniomandibular traits of the water deer from the Korean peninsula were examined to assess size change in growth between age groups and sexes. Univariate and multivariate analyses were conducted based on 34 cranial and 11 mandibular measurements from both sexes. Statistical comparisons of skull measurements revealed a significantly different growth pattern between the sexes. For the male, the size change of the cranium and mandible was straight through age groups, constantly. On the other hand, the size of the cranium and mandible of the female was changed relatively steeper than that of the male in age groups 2 to 3, and the growth curves from age group 3 to 4 were more gradual than age groups 2 to 3. Principal component analysis showed that these 2 sexes have a similar trend. In the allometry analysis, there were differences in growth in 5 traits in both sexes. In conclusion, our study suggests that the male and the female Korean water deer had a similar trend for their growth, although there was a small difference of skull growth for age groups.
This epidemiological survey was conducted to determine the prevalence of Hepatozoon, Babesia and Theileria infection in the Iriomote cat (IC) and the Tsushima leopard cat (TLC). Blood samples from 43 ICs and 14 TLCs were collected between November 2002 and January 2012. Polymerase chain reaction and DNA sequencing analyses detected a Hepatozoon felis infection prevalence of 72.0% (31/43 cats) and 100% (14/14 cats) in ICs and TLCs, respectively. The degree of Hepatozoon parasitemia observed on blood smears ranged from 0.1 to 4.7%. However, no cases had obvious clinical signs of hepatozoonosis. Neither Babesia- nor Theileria-infected wildcats were detected in this study.