To determine whether functional T- and B-cells can affect differentiation and/or proliferation of uterine natural killer (uNK) cells, their numbers in SCID mice (genotype, C.B.-17/Icr-scid/scid) were compared with those of control mice (genotype, C.B.-17/Icr-+/+) on days 8, 12 and 16 of pregnancy. Using biotinylated-Dolichosbiflorus agglutinin (DBA) lectin staining, uNK cells can be readily classified into 4 subtypes, I to IV, from immature to mature types. The number of uNK cells was significantly lower in the decidua basalis of SCID mice than in that of control mice on day 8 of pregnancy. Particularly, the number of uNK cells of immature subtype II was significantly lower in SCID mice than in the control mice. By day 12, however, the uNK cell number in the SCID mice reached the same level as that of the control mice. It is likely that uNK cell differentiation in SCID mice was delayed during the early placentation period due to a lack of functional T and B cells.
To construct a novel live oral vaccine candidate for the prevention of pathogenic Escherichia coli infections in neonatal piglets, an expression and secretion plasmid and an attenuated Salmonella delivery system were used. The individual E. coli genes K88ac, K99, FasA, F41 and intimin adhesins were inserted into pBP244 containing asd, lepB, secA and secB, and these plasmids were transformed into a Salmonella Typhimurium ΔcpxR Δlon Δasd. Forty female BALB/c mice were divided into four groups, A to D (ten mice per group). Groups A and B were administered with the mixture containing all constructs and the S. Typhimurium containing pBP244 only as a control, respectively. Groups C and D were primed and boosted with the mixture and the S. Typhimurium harboring pBP244 only, respectively. Each recombinant adhesin secreted from the individual candidates was confirmed by Western blot analysis. The serum IgG and secretory IgA (sIgA) titers to individual adhesins in all immunized groups were higher than those in control. Furthermore, IgG and sIgA levels in group C were higher than those in group A, and the IgG1 titers were increased in Group C but IgG2a titers were similar or decreased in Group C compared to Group A. In addition, the vaccine strains were not detected in fecal samples of any immunized mice. The novel vaccine candidates are not only highly immunogenic, but also safe for vaccinated mice and environment. In addition, the immune responses can be more efficiently induced through the booster-administration.
Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.
We herein present clinical findings of an Iriomote cat with Hepatozoon felis parasitemia. A male Iriomote cat was captured for ecological analyses three times from January 2010 to January 2011. Although this cat did not show any hematological abnormalities at the time of the first capture, H. felis parasitemia and increased serum creatine kinase levels were detected at the second and third captures. H. felis infection was confirmed by polymerase chain reaction, and amplified 18S ribosomal RNA gene fragments were 100% identical to those of H. felis in leopard cats in Korea. Although the virulence of H. felis in this cat was suggested to be low, this is the first report of an H. felis-infected Iriomote cat with parasitemia.
A 4-year-old, intact male Shiba dog was referred to Yamaguchi University Animal Medical Center, Yamaguchi, Japan, for the following complaints: anorexia, lethargy, intermittent fever, gingival bleeding and abdominal purpura. The dog presented with persistent neutropenia. Histopathological examination of a bone marrow sample revealed round to oval structures that resembled Hepatozoon micromerozoites and formed a "wheel-spoke" pattern. Furthermore, mature neutrophils were observed around these structures. PCR and sequencing using bone marrow aspirate confirmed Hepatozoon canis (H. canis) infection. These findings suggest that the neutropenia observed in this case was associated with osteomyelitis due to H. canis infection. This is the first report of neutropenia associated with H. canis infection. H. canis infection can be included in the differential diagnosis in canine cases of neutropenia in areas where the disease is endemic.
Rutin is one of the flavonoids derived from plants such as buckwheat and is well known as a powerful antioxidant. To determine whether dietary rutin could modulate mucosal immunity, we examined the gene expression of Th1/Th2 cytokines and the receptors in the gut and lung. Aged (18 months old, 18 M) C3H/HeN female mice were orally administered rutin for 10 days. The small intestine and lung were taken and analyzed by real-time PCR for gene expression. Interleukin (IL)-13 and IL-13Rα2 gene expression was significantly low (P<0.05 respectively) in the small intestine of aged rutin-fed mice. Meanwhile, there was no change in interferon γ gene expression between control and rutin-fed mice. IL-13 gene expression was also downregulated in the lung. To examine the mechanism of the inhibitory effect of rutin on Th2 cytokines in aged mice, intestinal nitric oxide synthase (NOS) expression was evaluated. Rutin inhibited inducible NOS (NOS2) gene expression, but not neuronal NOS and endothelial NOS. Gene analysis of cells collected from the small intestine by laser capture dissection revealed that NOS2 expression was significantly inhibited in crypt regions. Thus, rutin might be effective against a Th2-dominant profile through NOS2 inhibition in aged mice.
A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.
In order to compare the stimulation ability of insulin secretion, we determined changes in plasma glucose and insulin concentrations after intravenous administration of various amino acids and essential fatty acids in clinically healthy adult cats. Plasma glucose concentrations were within the normal ranges after injection of amino acids and fatty acids. Plasma insulin concentrations increased rapidly 2 to 4 min after injection of arginine, then decreased to the basal levels at 20 min in all five cats. Insulin peak responses were significantly greater in arginine injections than in normal saline (P<0.01). Areas under the curve (AUC) of plasma insulin concentrations from 0 to 10 min after injection of arginine were significantly larger than after injection of normal saline (P<0.01) and glucose (P<0.05). Increases in AUC of plasma insulin concentration from 0 to 60 min were observed after injection of arginine, leucine, alanine, and fat emulsion. Arginine had a strong insulinotropic effect, and leucine, alanine, and fatty acids had weak ones. Besides, valine, methionine, taurine and glutamine had no stimulant activity of insulin. Given the risk of glucose toxication and required time for testing, the intravenous arginine tolerance test may be useful for estimation of insulin responses in cats.
The double-chambered right ventricle (DCRV) is a rare congenital cardiac disease in dogs, and its detailed epidemiological and morphological features are not clearly understood. By investigating the profile, clinical signs, and characteristics of examination findings of eleven dogs with DCRV by means of a retrospective study, we attempted to clarify the epidemiology and morphology of the condition. The study group consisted of nine males and two females. Breeds included Pug (n=3), Miniature Dachshund (n=1), French Bull-dog (n=1), Shiba (n=1), and Retrievers (n=5). The attachment site of the anomalous muscular bundle was continuous with the cardiac apex in nine dogs, and it was attached to the right ventricle free wall in the other two dogs. In dogs with DCRV, at least one of the following conditions was present concurrently: congenital or acquired tricuspid valve regurgitation (TR), ventricular septal defect, and atrial septal defect. Also, the pressure difference between the two chambers increased over time, and progressive right-sided heart failure was observed. In summary, DCRV occurs in small breeds of dog as well as in large breeds of dog and it may be more prevalent in males. The existence of two types of DCRV in dogs was established. Dog with DCRVs will have a high incidence of concurrent cardiac abnormalities. Concurrent TR may be either congenital or acquired. DCRV is a congenital disorder, but the clinical condition progresses as the dog develops.
Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.
A 28-month-old African hedgehog was referred to our hospital with progressive tetraparesis. On the first presentation, the hedgehog was suspected as having wobbly hedgehog syndrome (WHS) and the animal was treated with medication and rehabilitation. The animal died 22 days after onset. Pathological examination revealed that the animal was involved in astrocytoma between the medulla oblongata and the spinal cord (C1). This report indicates that a primary central nervous system tumor should be considered as one of the differential diagnoses for hedgehogs presenting with progressive paresis, together with WHS.
Hyperbaric air therapy (HBA) is a treatment in which an animal is exposed to air pressurized to about 1.3 atmosphere absolute (ATA). Although HBA has already been administered to humans in medical applications, it has not been reported in clinical veterinary medicine. Therefore, we aimed to determine a safe protocol for dogs. To elucidate oxygen dynamics during HBA, we measured partial pressure of arterial oxygen, oxygen saturation of tissue, and partial pressure of transcutaneous oxygen in dogs. HBA could be performed safely with a protocol of pressurizing speed up to 0.1 ATA/min, maximum chamber pressure of up to 1.3 ATA, and pressure duration of around 45 min per treatment. Under these conditions, tissue was adequately oxygenated during and after treatment.
A 5-day-old hornless goat was referred with dysuria since birth. The scrotum was absent, and a small penis-like structure was seen below the perineal raphe. On the laparotomy, the testicles were found near the inguinal ring- and attached to a uterus-like structure. On histological analysis, the uterus-like structure was blind-end. Germ cells were absent in the testis. The karyotype of this goat was 60, XX and the SRY gene was absent. The goat was homozygous for a DNA deletion responsible for the Polled Intersex Syndrome (PIS). To the authors' knowledge, this is the first report as the clinical case of the PIS-/- goat with urethral atresia.
A 7-year-old castrated male mixed-breed dog was presented with a complaint of acute pain. The dog had suffered from isolated seizures for two years. Magnetic resonance imaging (MRI) of the brain revealed a smooth brain surface due to lack of gyri and sulci formation of the cerebrum and thick cortical grey matter. Additionally, ventriculomegaly and an arachnoid cyst were noted. Multiple spinal cord compressions induced by intervertebral disc protrusion were observed on a cervical MRI. Based on these findings, the dog was diagnosed as having lissencephaly concurrent with intervertebral cervical disease. After therapy for seizure and cervical pain, the clinical signs were completely resolved. To the author's knowledge, this is the case report to diagnose lissencephaly in a mixed-breed dog.
Most F1-Dh/+ male mice resulting from a cross between inbred DDD strain females and DH-Dh/+ strain males exhibit growth retardation and die during the neonatal period. The lethality is caused by a combination of three independent gene loci, namely the Dh locus on chromosome 1, Grdhq1 locus on the X chromosome, and a putative Y chromosome-linked locus in some strains. Among these loci, Grdhq1 was previously mapped to a distal region of the X chromosome using progeny from♀(♀DDD × ♂DH-+/+) F1 × ♂DH-Dh/+ mice. In this study, fine mapping of Grdhq1 was performed using progeny of ♀(♀DDD × ♂CAST/EiJ) F1 ♂DH-Dh/+ mice. Contrary to expectation, Dh/+ male pups carrying the DDD allele at DXMit135 (genetic marker nearest to Grdhq1) survived to weaning. The presence of modifier loci that suppressed the lethality by impeding the action of Grdhq1 was suggested; therefore, a genome-wide scan was performed in the surviving Dh/+ males. As a result, a significant modifier locus was identified on proximal chromosome 11. This in turn suggested that Grdhq1 was located more distally than we had expected; that is, the actual location of Grdhq1 appeared to be near and/or distal to the Mid1 locus. Thus, the results revealed that the neonatal lethality in (DDD × DH-Dh/+) F1-Dh/+ males was caused by the fourth gene locus on chromosome 11 in addition to the above-mentioned three gene loci on chromosomes 1, X, and Y.
In the present study, we tried to detect DNA for ribosomal RNA genes of piroplasma parasites from the liver or blood of 43 Japanese serows (Capricornis crispus) in Iwate Prefecture of Japan by polymerase chain reaction. Approximately 500-bp amplicons were obtained in 35 (81.4%) of the 43 samples by amplification for V4 hyper-variable regions of the 18S rRNA gene, and the amplicons were considered to be DNA of Theileria species. The complete nucleotide sequences (1,700 or 1709 bp) of the 18S rRNA gene were determined in 20 samples and were divided into 5 genotypes that were phylogenetically separated into two different lineages showing a polyphyletic relation. The Theileria DNAs of the two different lineages were considered to be those of distinct species.
In the present study, we tried to detect protozoan blood parasites from the liver or blood of 141 Japanese sika deer (Cervus nippon) in Iwate Prefecture of Japan by polymerase chain reaction. Approximately 500-bp amplicons were obtained in 76 (53.9%) of 141 samples by amplification for V4 hyper-variable regions of the 18S rRNA gene, and the amplicons were considered to be DNA of Theileria species. The complete nucleotide sequences (1701-bp) of the 18S rRNA gene were determined in 25 samples and were divided into 8 genotypes that were phylogenetically separated into two distinct lineages showing a monophyletic relation. The two lineages of Theileria were detected in different rates (12 and 88%) from sika deer in Iwate Prefecture.
The seroprevalence of Toxoplasma gondii was examined in adult women and domestic animals used for meat products from the Mymensingh District, Bangladesh. Cattle, goats and sheep showed a high seroprevalence (12, 32 and 40%, respectively), while the sera from all fifteen women examined in the same area were seronegative. Considering that primary infection in women during pregnancy can cause abortion and congenital defects, accidental ingestion of T. gondii infected meat products from domestic animals represents a risk factor for adult women living in the same area.
Bumped kinase inhibitors (BKIs) target analog-sensitive kinases, which the genomes of mammals rarely encode. Previously, we demonstrated that a BKI effectively suppressed the in vitro replication of Toxoplasma gondii, the causative pathogen of toxoplasmosis, by targeting T. gondii calcium-dependent protein kinase 1 (TgCDPK1) (Eukaryotic Cell, 9: 667-670). Here, we examined whether the BKI 1NM-PP1 reduced parasite replication in vivo. A high dose of 1NM-PP1, by intraperitoneal injection, just before the parasite inoculation effectively reduced the parasite load in the brains, livers, and lungs of T. gondii-infected mice, however, a low dose of 1NM-PP1 with oral administration didn't change the survival rates of infected mice.
Immunohistochemistry was performed to assess whether oxidative stress and/or denatured proteins play roles in the pathogenesis of canine degenerative myelopathy (DM). Two Pembroke Welsh Corgi (PWC) dogs with a homozygous mutation (c.118G>A) in the canine superoxide dismutase 1 (SOD1) gene were examined. The pathological features of the dogs were consistent with those of previous cases of DM in PWC. In the spinal lesions, diffuse SOD1 expression was observed in the neurons while no inclusion-like aggregates had formed, which disagreed with the findings of a previous study. A unique inducible nitric oxide synthase (iNOS) staining pattern in reactive astrocytes and a significant increase in ubiquitin immunoreactivity in the spinal lesions were also observed. These findings indicate the involvement of oxidative stress and the accumulation of ubiquitinated proteins in the pathogenesis of canine DM, whereas the role of SOD1 remains unclear.
Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.
A 10-month-old cat was diagnosed with congenital subvalvular aortic stenosis. To resolve its hypoxia, oxygen therapy was administered a couple of times a week during two months. The oxygen partial pressure in the chamber was maintained between 30 and 35%, and the time for one procedure was 12-24 hr. The animal died due to severe respiratory failure. At necropsy, the lungs were voluminous and had a rubbery texture. Histologically, large type II pneumocytes with occasional atypia had diffusely proliferated within the lungs. Interstitial fibrosis was not observed, although some alveolar septa were thickened along with fibrinous exudates and neutrophilic infiltration. The histology of these lesions was consistent with diffuse alveolar damage (DAD), which might have been partially due to oxygen toxicity.
A cerebral vascular hamartoma was identified in the frontal lobe, striatum and thalamus of the right side of the brain of a male, 7-year-old Shih Tzu. Histologically, the lesion consisted of thin-walled vessels, which showed various sizes and occasionally contained fibrin thrombi. These vascular walls were composed of a single layer of fibromuscular tissue lined by flat endothelium with various amount of collagen, but devoid of large coat of smooth muscles and elastic tissue. Immunohistochemically, the lining endothelial cells were positive for von Willebrand Factor antibody. Neuropil between the vessels was stained with Klüver-Barrera stain, and positive for synaptophysin and GFAP antibodies. Based on these findings, the lesion was diagnosed as vascular hamartoma, which might resemble venous malformation in humans.
On the assumption that animals of wild feline species died in the field, caudal epididymal sperm were cryopreserved following storage of the feline epididymides at 20°C for 0-24 hr, and their qualities were observed. Compared to the qualities at 0 hr, no significant differences were noted following 12 hr of storage at 20°C. On comparison of the qualities between caudal sperm cryopreserved after 24 hr storage at 4°C and after 12 hr at 20°C followed by 12 hr storage at 4°C, no significant differences were noted. These findings suggest that the cryopreserved sperm collected from epididymides of dead animals might be useful for artificial insemination if cryopreservation was performed within 12 hr exposure to ambient temperature.
Psittacid herpesvirus type 1 (PsHV-1) was isolated from a captive galah (Eolophus roseicapillus) in Japan that was suspected of having Pacheco's disease (PD), an acute fatal disease in psittacine birds. PsHV-1 has been classified into four genotypes based on the UL16 gene sequence. In the present study, we investigated the genetic and pathogenic characteristics of the isolated virus, FOY-1, compared with a reference strain, RSL-1. The FOY-1 strain was classified into PsHV-1 genotype 2. The FOY-1 strain was found to be less pathogenic to budgerigars than RSL-1, which was classified as genotype 4 in an in vivo study. This is the first report regarding the classification of originally isolated PsHV-1 in Japan and its characterization by animal infection experiment.
We previously reported that rabies virus strain CE(NiM), but not the parental Ni-CE strain, killed mice after intracerebral inoculation. CE(NiM) and Ni-CE are genetically identical except for two amino acids at positions 29 and 95 in the M protein. In this study, to identify which residue determines the pathogenicity, we examined pathogenicities of two Ni-CE mutants, CE(NiM29) and CE(NiM95), which were established by replacement of an amino acid residue at position 29 or 95 in the Ni-CE M protein with the corresponding residue of CE(NiM), respectively. We found that CE(NiM95), but not CE(NiM29), killed mice, indicating that the amino acid at position 95 in the M protein is the pathogenic determinant.