Kestose, a fructooligosaccharide (FOS) with one fructose monomer linked to sucrose, is a key component of the prebiotic activity of FOS. This study aimed to evaluate the prebiotic potential of Kestose in terms of the impact on population change in the intestinal microbiota and fecal short-chain fatty acid (SCFA) concentration in dogs. Kestose 2 g per dog was administered daily with conventional diet to 6 healthy, adult beagle dogs for 8 weeks followed by 4 weeks of follow-up period without Kestose supplementation. Fresh fecal samples were obtained before and every 4 weeks until the end of the follow-up period. Genomic DNA extracted from the fecal samples was subjected to 16S rRNA gene analysis using next generation sequencer and to quantitative polymerase chain reaction (qPCR). Fecal acetate, propionate, butyrate, lactate and ethanol concentrations were measured by high-performance liquid chromatography. 16S rRNA gene analysis and qPCR showed increasing trend of genus Bifidobacterium after Kestose supplementation while genera Bacteroides and Sutterella decreased. Clostridium perfringens decreased below the detection limit within first 4 weeks after starting Kestose supplementation. Fecal butyrate concentration was significantly increased at week 8 and returned to the base level after 4 weeks of the washing period. To the best of our knowledge, this is the first study to reveal effect of Kestose on the populational changes in fecal microbiota and fecal butyrate concentration in dogs.
Streptococcus suis strains isolated from porcine endocarditis and tonsils in the Tokai area of Japan during 2004–2007 and 2014–2016 (n=114) were tested for antimicrobial susceptibility and distribution of selected resistance genes. No strains showed resistance to penicillin, ampicillin, cefotaxime, meropenem, vancomycin, and levofloxacin. High resistance to tetracycline (80.7%), clindamycin (65.8%), erythromycin (56.1%), and clarithromycin (56.1%) was observed. In chloramphenicol and sulfamethoxazole-trimethoprim, there was a trend towards increased resistance between the first (2004–2007) and second (2014–2016) periods. tet(O) and erm(B) genes were the most frequently detected, and tet(M) and mef(A/E) genes were only detected in strains isolated during 2014–2016. These results indicate that chloramphenicol and sulfamethoxazole-trimethoprim resistance, and tet(M) and mef(A/E) genes emerged in S. suis of this area after 2014.
Instrument cost is a major problem for the transduction of DNA fragments and proteins into cells. Water-in-oil droplet electroporation (droplet-EP) was recently invented as a low-cost and effective method for the transfection of plasmids into cultured human cells. We here applied droplet-EP to livestock animal cells. Although it is difficult to transfect plasmids into bovine fibroblasts using conventional lipofection methods, droplet-EP enabled us to introduce an enhanced green fluorescent protein (EGFP)-expressing plasmid into bovine earlobe fibroblasts. The optimal transfection condition was 3.0 kV, which allowed 19.1% of the cells to be transfected. For swine earlobe fibroblasts, the maximum transfection efficacy was 14.0% at 4.0 kV. After transfection with droplet-EP, 69.1% of bovine and 76.5% of swine cells were viable. Furthermore, droplet-EP successfully transduced Escherichia coli recombinant EGFP into frozen-thawed bovine sperm at 1.5 kV. Flow cytometry analysis revealed that 71.5% of spermatozoa exhibited green fluorescence after transfection. Overall, droplet-EP is suitable for the transfection of plasmids and proteins into cultured livestock animal cells.
The complement systems play an important role in innate and adaptive immunity. In this study, the complement C3 gene, designated CagC3, was cloned and sequenced from Gibel carp (Carassius auratus gibelio). The expression pattern of CagC3 in different tissues of healthy Gibel carp and after challenge with Cyprinid herpesvirus 2 (CyHV-2) were evaluated using quantitative real-time PCR. The full-length CagC3 cDNA was 5131 bp with an ORF of 4950 bp, encoding a predicted protein of 1649 amino acids. The deduced amino acid sequence showed that CagC3 has conserved domains and residues known to be critical for C3 function. Phylogenetic analysis demonstrated that CagC3 clustered with homologs from common carp and grass carp (Ctenopharyngodon idella). CagC3 is expressed in all examined tissues of healthy Gibel carp, with the highest expression in liver. In vivo, after CyHV-2 challenge, CagC3 transcription was significantly upregulated in liver, spleen and kidney with the peaks at 24 hr, 2 d, and 2 d, respectively. In vitro, CagC3 expression in the Gibel carp brain cell line showed the same pattern as that in vivo after stimulation with CyHV-2 or poly(I:C). However, CagC3 expression was downregulated at 24 hr after induction with lipopolysaccharide (LPS), and then reached the peak at 2 d. These results suggest that CagC3 is involved in the innate immune response of Gibel carp to viral infection.
Karyolysis is the complete dissolution of nuclear components of a dying cell. However, the generation mechanism has not been clarified. We studied a necrotic DNA fragmentation factor DNase γ (also known as DNase1L3) and previously found that karyolysis was inhibited in DNase γ deficient (DNase γ−/−) mice. To confirm this, we transiently expressed DNase γ in the liver of DNase γ−/− mice and caused hepatocyte necrosis by acetaminophen overdose. As expected, karyolysis was induced in the necrotic hepatocytes. We also found that the depletion of Kupffer cells from wild type mice reduced the expression and activity of DNase γ in the liver. Thus, we concluded that DNase γ produced from Kupffer cells caused karyolysis of necrotic hepatocytes.
The immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed in the clinical cases with Mycoplasma (M.) bovis infection. Seventy-eight Holstein calves in one farm were used. These calves were divided into three groups; the calves with M. bovis infection of poor outcome after treatment (Non-Recovery Group), the calves with M. bovis infection recovered (Recovery Group) and clinically healthy calves (Control Group). Blood samples were collected at days of the first medical treatment and the final treatment or euthanasia. IL-17A levels in the Non-Recovery Group were higher than those in the Recovery Group on both days. Our result suggested that the IL-17A of PBMC is an important factor to affect outcome of the calves with M. bovis infection.
A 3-year-old male Rottweiler presented with the chief complaint of recurrent vomiting, diarrhea, hypothermia, and lethargy. Hypovolemic shock was noted with abnormal electrolytes (Na/K ratio, 27.9) and anemia (hematocrit, 17.3%). Since the hematocrit was 49.2% four days earlier when the primary veterinarian examined the dog, acute anemia was diagnosed. Melena was observed on the next day. The general condition and hydration improved with treatment, and an adrenocorticotropic hormone stimulation test identified hypoadrenocorticism. However, the hematocrit decreased further to 9%, necessitating blood transfusion. The cause of severe acute anemia was thought to be gastrointestinal hemorrhage. It should be noted that hypoadrenocorticism can lead to potentially fatal anemia with gastrointestinal tract bleeding, and blood transfusion may be required.
Microparticle (MP)-associated tissue factor (TF) activity in plasma might play a role in human disseminated intravascular coagulation (DIC). The aim of this study was to compare MP-TF activity between non-DIC and DIC groups. Ten clinically healthy beagles and 26 diseased dogs were enrolled. The proportion of dogs with increased MP-TF activity was significantly higher in the DIC group than the non-DIC group (P=0.014). MP-TF activity in the DIC group was significantly higher than the non-DIC group (P=0.021). MP-TF activity positively correlated with plasma D-dimer concentration (r=0.42, P=0.034). Moreover, MP-TF activity was decreased by the time of recovery in some dogs with DIC. Larger prospective studies are warranted to assess its value as a diagnostic and prognostic biomarker in DIC.
We performed a clonality analysis using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) gene rearrangement, specifically with regard to its utility as a method to diagnose bovine B-cell lymphoma. PCR for IgH gene rearrangement indicated monoclonal proliferation of B-cells in 24 of 35 cattle with B-cell lymphoma. In contrast, PCR for IgH gene rearrangement in lymph nodes and tumor tissues from 65 cattle diagnosed with tumors other than B-cell lymphoma and non-tumors revealed polyclonal population of B-cells. Sensitivity, specificity, positive predictive value, and negative predictive value for PCR for IgH gene rearrangement for bovine B-cell lymphoma were 68.6%, 100%, 100%, and 85.5%, respectively. Clonality analysis using PCR for IgH gene rearrangement may be useful for adjunctive diagnosis of bovine B-cell lymphoma.
Brachycephalic airway syndrome (BAS) is a common disease in certain “flat-faced” dog breeds. This syndrome includes stenotic nares, elongated and thickened soft palate, laryngeal collapse, and tracheal hypoplasia. Pharyngeal collapse is also commonly observed, but it is unclear if laryngopharynx motions are merely sequelae or actually contribute to BAS respiratory symptoms. Laryngopharynx motion was imaged using dynamic four-dimensional computed tomography (4D-CT) during spontaneous respiration in four dogs with different BAS types. Dynamic 4D-CT showed laryngopharynx motion in the following order during inspiration: pharyngeal collapse, contraction, and laryngospasm. We concluded that dynamic 4D-CT is a highly-detailed diagnostic approach for detecting laryngopharynx motion. Pharyngeal contraction during inspiration appears to contribute toward the worsening of clinical respiratory signs of BAS.
Recently, a mixture of medetomidine, midazolam and butorphanol (MMB) has been used as an injectable general anesthetic agent for laboratory animals. The purpose of this study was to establish data to encourage practical usage of MMB, and to clarify the effects of MMB on the respiratory function in rats. To compare the anesthetic efficacy between the injection routes, the anesthetic effects of MMB by subcutaneous (s.c.) or intraperitoneal (i.p.) injection were evaluated in rats. To assess the respiratory function, the blood gas parameters and electrolytes were assessed in serial venous blood samples collected from before s.c. injection of MMB to 270 min after the injection. Recovery from anesthesia and the respiratory changes after atipamezole injection at 30 min after MMB injection was also examined. Subcutaneous injection of MMB was associated with more rapid induction and a longer duration of anesthesia as compared to i.p. injection. The blood gas analysis findings showed MMB had effects on respiratory function, that is, elevations of the partial pressures of carbon dioxide and bicarbonate and reduction of the blood pH. Atipamezole injection resulted in recovery from the MMB-induced anesthetic effect as well as respiratory depression. In conclusion, MMB provides more effective anesthesia administered by s.c. injection compared to i.p. injection and induces respiratory change. These changes were counteracted by atipamezole. Therefore, we recommend MMB administered by s.c. injection for anesthesia, followed by injection of atipamezole after the operative procedure to allow recovery.
Patients with hypoglycemic coma show abnormal signals in the white matter on magnetic resonance imaging. However, the precise pathological changes in the white matter caused by hypoglycemic coma remain unclear in humans and experimental animals. This study aimed to reveal the distribution and time course of histopathological and immunohistochemical changes occurring in the white matter during the early stages of hypoglycemic coma in rats. Insulin-induced hypoglycemic coma of 15–30-min duration was induced in rats, followed by recovery using a glucose solution. Rat brains were collected after 6 and 24 hr and after 3, 5, 7, and 14 days. The brains were submitted for histological and immunohistochemical analysis for neurofilament 200 kDa (NF), myelin basic protein, olig-2, Iba-1, and glial fibrillary acidic protein (GFAP). Vacuolation was observed in the fiber bundles of the globus pallidus on days 1–14. Most of the vacuoles were located in GFAP-positive astrocytic processes or the extracellular space and appeared to be edematous. Additionally, myelin pallor and a decrease in NF-positive signals were observed on day 14. Microgliosis and astrogliosis were also detected. Observations similar to the globus pallidus, except for edema, were noted in the internal capsule. In the corpus callosum, a mild decrease in NF-positive signals, microgliosis, and astrogliosis were observed. These results suggest that after transient hypoglycemic coma, edema and/or degeneration occurred in the white matter, especially in the globus pallidus, internal capsule, and corpus callosum in the early stages.
To clarify the prevalence of canine intracranial tumors in Japan, a retrospective study was performed using data on 186 canine intracranial tumors. Of 186 cases, 159 cases (85.5%) were primary and 27 cases (14.5%) were secondary intracranial tumors. Among primary intracranial tumors, meningioma (50.9%) was the most common, followed by glial tumors (21.4%) and primary intracranial histiocytic sarcoma (12.6%). These 3 tumors were most frequently found in middle-aged to elderly dogs without any sex predilection. Regarding glial tumors, the incidence of oligodendroglial tumors (79.4%) was higher than that of astrocytic tumors (17.6%). A significant breed predisposition (P<0.05) was observed for meningioma in Rough Collie, Golden Retriever, Miniature Schnauzer, and Scottish Terrier; for glial tumors in Bouvier de Flandres, French Bulldog, Newfoundland, Bulldog, and Boxer; for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi, Siberian Husky, and Miniature Schnauzer. The high incidence of oligodendroglial tumors in dogs and the breed predisposition for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi have not been reported in previous epidemiological studies on canine tumors. Since the incidence of intracranial tumors was vary among dog breeds, the present results demonstrate the uniqueness of the canine breed population in Japan.
We examined a 10-year-old cow in which about half of the liver was displaced by malignant tissue consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Cytokeratin (CK) 18 and 7 were expressed in the latter. Metastasis was present in the hepatic, pancreaticoduodenal and mediastinal lymph nodes, where malignant cells had hepatocellular features, but more pleomorphic and atypical than in the primary lesion. Areas composed solely of CC cells or less-differentiated HCC cells were observed. In contrast, well-differentiated HCC cells were almost always admixed with the other two types, and may have had the ability to transform into CC cells and to dedifferentiate into less-differentiated cells. This report suggests that CK18 is an excellent marker for biliary differentiation in cattle.
A 1-month-old rabbit, imported as a pet by a distributor, died suddenly in the quarantine period in Japan due to suppurative pleuropneumonia. A bacterial isolate from its right lung was identified as Pasteurella multocida serotype A: 11. The isolate was classified as ST204 using the RIRDC scheme of multilocus sequence typing, suggesting that the isolate was genetically related to European isolates of the same sequence type listed in the PubMLST database and not to four other isolates that originated from past imported rabbits. In the immunohistochemical assay, an antiserum recognizing the somatic serotype 11 antigen generated from chicken could specifically detect P. multocida, indicating that the antiserum for somatic serotyping was useful for immunohistochemical diagnosis of rabbit pasteurellosis.
This study assessed the effects of retroperitoneal carbon dioxide (CO2) insufflation on cardiopulmonary variables and intra-abdominal pressure (IAP) in mechanically ventilated dogs in sternal recumbency with the abdomen unsupported, following placement of a positioning kit and towels under the pectoral and pelvic regions. General anesthesia was induced in eight healthy adult male Beagles. A Swan-Ganz catheter was placed in the pulmonary artery via the jugular vein for cardiac output measurements. A Foley urethral catheter was placed to monitor transvesical IAP. A 10 mm balloon blunt-tip trocar was inserted into the retroperitoneal space. With a fixed respiratory rate and tidal volume by mechanical ventilation, insufflation pressure was sequentially increased from 0 to 10 mmHg in 5 mmHg increments, followed by desufflation. All variables were measured before insufflation, 5 min after the establishment of each insufflation pressure, and after desufflation. At 10 mmHg, the IAP was nearly equal to insufflation pressure. Cardiopulmonary function was not compromised at any point, although the cardiac index (CI), heart rate, mean arterial pressure (MAP), and mean pulmonary arterial pressure increased within normal ranges. End-tidal CO2 concentration, arterial CO2 partial pressure, and oxygen delivery index (DO2I) increased, whereas pH decreased, at 10 mmHg. CI, MAP, and DO2I did not recover to baseline after decompression. Thus, retroperitoneal CO2 insufflation up to 10 mmHg is well tolerated by mechanically ventilated dogs positioned in sternal recumbency with the abdomen unsupported, although sympathetic changes may occur with an insufflation pressure increase.
H9N2 is widespread among poultry and humans. Though this subtype is not lethal to either species, it can cause considerable financial losses for farmers and threaten human health. In this study, 10 new H9N2 avian influenza viruses (AIVs) produced by reassortment were isolated from domestic birds in Liaoning Province between March 2012 and October 2014. Nucleotide sequence comparisons indicate that the internal genes of one of these strains are highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions occurred in the HA and NA proteins separately, indicating that all 10 of these isolates may have an enhanced ability to infect mammals. A cross-hemagglutinin inhibition assay conducted with two vaccine strains that are broadly used in China suggests that antisera against vaccine candidates cannot completely inhibit the new isolates. Two of the 10 newly isolated viruses could replicate in respiratory organs of infected BALB/c mice without adaption, suggesting that these isolates can potentially infect mammals. The continued surveillance of poultry is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV and the need for more extensive AIV surveillance.
A serologic investigation of Brucella infection was performed in 7 species of cetaceans inhabiting along the coast of Japan. A total of 32 serum samples were examined by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis antigens. One serum sample from five melon-headed whales (Peponocephala electra) was positive for B. abortus. No serum sample showed positive for B. canis. The ELISA-positive melon-headed whale serum demonstrated a strong band appearance only against B. abortus antigens in Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in melon-headed whale population and the bacterial antigenicity is more similar to that of B. abortus than B. canis.