The levonorgestrel-releasing intrauterine system (LNG-IUS) is used for contraception and treatment of heavy menstrual bleeding as well as endometrial hyperplasia and early endometrial carcinoma. A 48-year-old woman visited an Internal Medicine outpatient clinic due to significantly elevated CA-125 and CA-19-9 levels in a routine health examination. She had been using LNG-IUS for 3 years. Before LNG-IUS insertion, she suffered from heavy menstrual bleeding and severe dysmenorrhea. Her endometrial sampling and ultrasonographic imaging showed no evidence of endometrial carcinoma at the time of LNS-IUS insertion. After insertion, she complained of neither abnormal uterine bleeding nor dysmenorrhea. She received a routine health check-up every year and showed results within normal range until last year. To rule out pancreatic cancer due to significantly elevated CA-19-9 levels, her physician performed positron emission tomography–computed tomography, which demonstrated increased FDG uptake in the endometrial cavity. We obtained endometrial biopsy and found endometrial carcinoma in her uterus and performed radical hysterectomy with bilteral pelvic lymphadectomy. Permanent pathology confirmed endometrial carcinoma with lymph node metastasis. She received concurrent chemoradiation therapy. We emphasize the necessity of regular follow-ups with ultrasonography and assessment of serum tumor markers for the early detection of endometrial carcinoma, although rare, in women using LNG-IUS, including those without abnormal uterine bleeding.

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 μM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 μM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase β2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and pax6. Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Toxoplasmosis is an important cause of foodborne, inflammatory, as well as congenital abnormalities. There is an urgent need for safe and effective therapies to eliminate or treat this cosmopolitan infectious disease. A medicinal herbal plant, Meliae fructus, has been used to soothe the liver and kills worms in Chinese medicine. In this study, Meliae fructus ethanol extract was examined and screened for its anti-T. gondii activity. For anti-T. gondii activity screening, in vitro study of Meliae fructus extract using tachyzoit of T. gondii RH strain-infected HeLa cells was performed. Further, in vivo anti-T. gondii study using a mouse infection model was conducted. Safety of herbal compounds was evaluated in SD rats by treatment with Meliae fructus extract for 28 days. As a result, selectivity of Meliae fructus ethanol extract was 5.85, which was higher than sulfadiazine selectivity (2.06). We also performed an in vivo study to evaluate the anti-T. gondii activity of Meliae fructus extract in a mouse model. The inhibition rate of Meliae fructus extract was as high as that of sulfadiazine. These results demonstrate that Meliae fructus can successfully cure T. gondii infection and could be a promising native herb treatment for prevention of T. gondii infection.

Characteristics and immuno-modulatory effects of Enterococcus (E.) faecium JS1-8 isolated from Kimchi were investigated for potential probiotic use. We measured their acid, bile, and heat tolerances, adhesion properties in Caco-2 cells, antimicrobial activity against pathogenic bacteria, and bacteriocin-like substance-producing activity. Immuno-modulatory effects of E. faecium JS1-8 were measured by determination of nitric oxide (NO), nuclear factor-κB (NF-κB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) production in RAW 264.7 cells or RAW BLUE cells. JS1-8 survived at pH 2.0 for 2 hr and showed tolerance to 0.3% oxgall bile salt, and it survived after exposure for 5 min at 80°C. JS1-8 showed high antimicrobial inhibition zones to Staphylococcus aureus (460 mm), Listeria monocytogenes (310 mm), Salmonella enteritidis (280 mm), and E. coli (150 mm). Bacteriocin-like substance produced by JS1-8 showed a broad spectrum of activity against Listeria monocytogenes KCCM 40307 and Lactobacillus sake KCCM 40264. Low concentration (1 × 107 CFU/mL) of heat-killed E. faecium JS1-8 induced statistically higher production of NO than Lactobacillus rhamnosus GG (LGG), which is a well-known immuno-modulatory lactic acid bacteria. Low and high (5 × 107 CFU/mL) concentrations of JS1-8 induced statistically higher production of NF-κB than that produced by LGG. We also found that JS1-8 increased production of pro-inflammatory cytokines such as IL-1β and TNF-α in a concentration-dependent manner. As a result, E. faecium JSI-8 could be used as a useful probiotic for controlling pathogens and enhancing host immune responses.

Skin is the outermost organ and acts as a barrier between the organism and environment. Skin protects the organism from environmental insults, such as chemicals, pathogens, and UV light. Much of the protective function of skin is dependent on the epidermis, a multi-layered epithelium that is composed of various cell types such as keratinocytes and melanocytes. Keratinocytes produce protective components through a sophisticated differentiation process. Disturbance of keratinocyte differentiation is related to several skin diseases such as psoriasis and atopic dermatitis. In this study, we prepared extract of combined medicinal plants (ECMP) consisting of Taraxacum platycarpum H. Dahlstedt, Heartleaf Houttuynia, Glycyrrhiza uralensis Fischer, and root bark of Ulmus davidiana. We demonstrated that ECMP enhanced keratinocyte differentiation and barrier functionality using an in vitro cell culture system and in vivo animal test. Treatment of cultured keratinocytes with ECMP resulted in induction of keratinocyte differentiation, as evidenced by increased differentiation markers such as involucrin, loricrin, and filaggrin. In line with these results, ECMP decreased proliferation of keratinocytes cultured in vitro. ECMP applied topically to tape-stripped mouse skins accelerated reduction of transepidermal water loss (TEWL), indicating fast recovery of barrier function. Immunohistochemistry showed that ECMP increased the filaggrin level in tape-stripped mouse skins. These results suggest that ECMP may be applicable for keratinocyte differentiation-related skin diseases.

Salsola komarovi Iljin is a halophyte and herbaceous annual native to the sand dunes and beaches of Japan, northern China, Sakhalin, and Korea. The plants have been known as an ecologically important species for enhancing formation of sand dunes in Korea. The purpose of this study was to examine the anti-gastric ulcer effect of Salsola komarovi Iljin halophyte in an HCl-ethanol-induced gastritis model. SD rats (7-weeks-old) were divided into normal (I, n=10), control (II, 60% HCl-ethanol + water, n=10), 60% HCl-ethanol + Ranitidine 300 mg/kg (III, n=10), 60% HCl-ethanol + Salicornia herbacea L. 500 mg/kg (IV, n=10), 60% HCl-ethanol + 50% alcohol extract of Salsola komarovi Iljin 500 mg/kg (V, n=10), and 60% HCl-ethanol + water extract of Salsola komarovi Iljin 500 mg/kg (VI, n=10) groups. Salsola komarovi Iljin significantly suppressed gastric lesions and ulcers in the 60% HCl-ethanol-induced gastric model. Especially, 500 mg/kg of 50% alcohol extract of Salsola komarovi Iljin showed significant inhibitory effects against gastritis. Especially, 50% alcohol extract of Salsola komarovi Iljin 500 mg/kg showed a significantly inhibitory effect, which was more potent than that of 300 mg/kg of Ranitidine. In histopathological analysis of the animal model, Salsola komarovi Iljin attenuated gastric ulcer formation. Our results suggest that Salsola komarovi Iljin has inhibitory effects against gastritis and gastric ulcers and could be developed as a new anti-gastric ulcer agent.

Canine atopic dermatitis (CAD) is an allergic skin disease with characteristic clinical features associated with immunoglobulin E (IgE) antibodies. Identification of the causative allergens is the diagnostic goal, which is essential to treat and manage CAD patients. CAD is commonly associated with environmental allergens surrounding the patients. For this reason, it is important for diagnostic tests to select allergens that are related to the environment of each country and each province. There are two main allergen-specific tests, serological IgE test (SAT) and intradermal skin test (IDT). SAT did not show direct cutaneous reaction but did show serological reaction against allergens. However, SAT is simpler and more convenient than IDT in small animal practice. In this study, we selected domestically prevalent allergens for SAT, including 60 food allergens and 60 inhalant allergens, and tested eight dogs tentatively diagnosed with CAD based on Favrot’s criteria. Furthermore, IDT was performed on four dogs from the SAT group for comparison of SAT and IDT, and the results were very similar. In SAT, four types of mites (Bloomia tropicalis, Glycophagus domesticus, Euroglyphus maynei, and mite mixture 1 Korea; house dust mites), four types of molds (Botrytis cinerea, Alternaria alternata, mold fungi mixture 11, mold fungi mixture), and one type of pollen (tree pollen mix 3 Korea) induced a reaction in more than half of dogs tested. In IDT, all four dogs reacted positively to Dermatophagoides farinae, and three reacted positively to Dermatophagoides pteronyssinus and house dust. The mean agreement rate between SAT and IDT in this study was 76.3%. This is the first trial to apply local allergens for SAT in Korean veterinary medicine, and it might play an important role for diagnoses and management of animal allergic diseases.

Recombinant thymosin β4 (rTβ4) has been reported to migrate and promote vascularization, wound-healing, and hair growth in a mouse hindlimb ischemia model of peripheral vascular disease. C57BL/6 mice (11-weeks-old) were anesthetized and an ischemic model was made by cutting the right aorta femoralis. The ischemic group was intraperitoneally administered with saline (300 μL/mouse) and the muscular administration group received rTβ4 (150 μg in 300 μL of saline) or rTβ4 (150 μg in 300 μL saline) to the abdominal cavity at 3-day intervals for 21 days. Myoatrophy of the ischemic group was observed compared to the normal control group. Generation of adjacent vessels was carried out in the rTβ4 administration group compared to the ischemic group. The biopsy results showed significant fibrosis around the muscular undersurface and perimysium in the musculus quadriceps femoris of the ischemic group, whereas partial fibrosis was observed in the perimysium and endomysium in the rTβ4 administration group. Immunostaining indicated that expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-1 (VEGF-1), and endothelial nitric oxide synthase (eNOS) in the rTβ4 group were higher than those of the ischemic group. Western blotting showed that expression levels of HIF-1α, VEGF-1, and eNOS in the rTβ4 group were higher than those of the ischemic group. In conclusion, rTβ4 increases expression levels of HIF-1α, VEGF-1, and eNOS, resulting in angiogenesis.

A 12-year-old spayed female Pomeranian (weighing 2.4 kg) was referred with primary complaints of acute dyspnea, cough, and lethargy. Diagnostic imaging studies found degenerative mitral valve cusps, chordae tendinae rupture, severe mitral regurgitation (5.45 m/s of peak velocity), and marked left atrial and ventricular dilation. The dog was diagnosed as having degenerative mitral valve disease (DMVD) with ISACHC stage IIIa heart failure. Her clinical condition was stabilized after administration of cardiac medication (e.g. diuretics and pimobendan). Ten months later, the dog was referred back to the clinic due to a sudden worsening of clinical signs. Echocardiographic study found pulmonary hypertension in addition to DMVD. After medication was adjusted, clinical signs were stabilized in 2 weeks. The patient was returned after 4 months for cardiac recheck and there was no obvious worsening of clinical signs. Incidental finding of a left-to-right atrial septal defect from rupture of the atrial septum secondary to marked left atrial dilation by DMVD was noted by echocardiography. To diminish left atrial volume overload, the frequencies of both furosemide and pimobendan were increased (i.e. from q 12 hr to q 8 hr) in addition to adding spironolactone (1 mg/kg q 12 hr). Based on diagnostic findings, this case was re-diagnosed as acquired atrial septal defect secondary to rupture of the atrial septum with advanced stage DMVD. The dog was then stabilized and is currently being regularly monitored.

A 2-year-old intact female pomeranian dog presented dyspnea, labored breathing, cough, exercise intolerance, machinery heart murmur, and precordial thrill. A left-to-right patent ductus arteriosus (PDA) was diagnosed based on two-dimensional echocardiography, thoracic radiography, electrocardiography, and blood work. An angiography was performed to accurately evaluate the size and shape of the duct. An interventional approach for transcatheterial occlusion of the PDA was achieved using an Amplatz® Canine Duct Occluder (ACDO), which is a commercially available ductal occluding device. Due to the limited size of the dog’s femoral artery, a device smaller [125% of minimal ductal diameter (MDD); recommended size: 150~200% of MDD] than recommended was mounted. After placement of the ACDO, precordial thrill and continuous heart murmur disappeared, and the patient was discharged the next day after stabilization with O2 supply. Upon follow up examination, dyspnea, labored breathing, cough, exercise intolerance, and cardiomegaly were improved with no complications after the procedure. The ACDO was well maintained in position. This case represents successful clinical application of the Amplatz® Canine Duct Occluder to achieve closure of a PDA using a slightly smaller device than the recommended size.

Guillain-Barre´ syndrome (GBS) is an acute inflammatory demyelinating polyneuropathy most commonly characterized by rapidly progressive, essentially symmetric weakness and areflexia. This study examined clinical symptoms of clinical variants of GBS through a cerebrospinal fluid (CSF) study, nerve conduction (NCV) study, treatment, and prognosis. There were 16 children with GBS who visited our hospital from January 2011 to December 2013. Guillen-Barre´-like syndromes with transient synovitis were noted in three children. Clinical variants of GBS with acute demyelinating encephalomyelitis were observed in one child. Previous infections were noted in 16 children with Guillen-Barre´-like syndrome. There were ascending infections in 16 cases. Fifteen children showed symmetric infections, and one showed asymmetric infection. In NCV, slow waves were noted in two cases. We treated using intravenous immunoglobulin (IVIG) in four cases, IVIG with steroid in two, cases and supportive care in 10 (62.5%) cases. Five children treated with IVIG and 10 with supportive care management were completely improved.Our study suggests that supportive care is effective as a treatment for clinical variants of GBS. Further study is necessary for more patients.

The morphology of the lingual papillae in a female Bengal tiger (Panthera tigris tigris) was examined by scanning electron microscopy (SEM). The tongue was 22.3 cm in length and 7.1 cm in width. Numerous filiform papillae were distributed over the entire dorsal surface of the tongue. SEM examination of the tongue revealed two types of mechanical papillae, i.e. filiform and conical papilla, and two types of gustatory papillae, i.e. fungiform and vallate papilla, on the dorsal surface of the tongue. Each filiform papilla consisted of one primary papilla and several secondary papillae. The filiform papillae on the anterior part of the tongue were divided into one primary and 6~14 secondary papillae. Unlike other mammalians, however, secondary papillae in the mid-part of the tongue showed pineal-like papillae. In the posterior part of the tongue, secondary papillae were rare or absent. Fungiform papillae were surrounded by filiform papillae and densely distributed on the lingual surface. There were two vallate papillae on the borderline between the lingual body and root of the tongue. A vallate papilla contained two secondary papillae inside the grooves. Conical papillae were located in the area of the vallate papillae and covered the posterior part of the tongue root. No foliate papillae were seen on both margins of the posterior part of the tongue. Our results indicate that the structure on the lingual papillae of the Bengal tiger is somewhat different from that of other mammals.

To investigate the association between maternal iron status at the third trimester and fetal birthweight, maternal serum iron, ferritin, total iron-binding capacity (TIBC), and complete blood count values were measured at 36-weeks gestation. Delivery database on mothers who delivered babies at Chungbuk National University Hospital between January 2008 and March 2013 was extracted. A total of 353 uncomplicated term babies were analyzed using hierarchical regression and ANCOVA. Maternal age (standardized regression coefficient β=0.115, P<0.05), height (β=0.108, P<0.05), BMI (β=0.210, P<0.001), and gestational age (β=0.298, P<0.001) were significantly associated with birthweight. However, birthweight was not associated with maternal iron parameters. After adjusting for maternal age, height, BMI, and gestational age, babies born to mothers with lower mean values of hemoglobin, hematocrit, and serum ferritin were heavier than those born to mothers with higher values. Babies born to lower hemoglobin (11 g/dL) mothers were heavier than those born to higher hemoglobin (12 g/dL) mothers. However, birthweight was not significantly different between mothers with 10 g/dL or 13 g/dL of hemoglobin. Comparing birthweight according to 30 ug/dL of serum iron, 360 ug/dL of TIBC, 15 ng/mL of serum ferritin, and 10% transferrin saturation, babies born to mothers of the lower group were heavier than those born to mothers of the higher group. Therefore, maternal serum iron status at the third trimester seems to not be associated with birthweight.

Pruritus is one of the most important symptoms of allergic inflammatory skin disease. Conjugated linoleic acid (CLA) has been reported to have preventive effects against allergic inflammation. The objective of this study was to determine whether or not oral administration of CLA suppresses pruritus induced by compound 48/80 (composed of N-methyl-p-methoxy phenethylamine with formaldehyde) in mice, and if so, whether or not this effect is associated with serum histamine and prostaglandin (PG) E2 levels. Liquid CLA mixture (36.25% 9c-11t CLA, 36.95% 10t-12c CLA, 1.12% 9c-11c, and 1.94% t9-t11 CLA) was emulsified in 0.5% carboxymethyl cellulose (CMC) sodium salt and orally administered to mice at doses of 200 mg/kg once per day for 3 days. Similarly, disodium chromoglycate (DSCG), an antipruritic substance, was administered orally at the same concentrations as the negative control. Compound 48/80, a pruritus-inducing reagent, was subcutaneously injected 30 minutes after final administration of CLA. Scratching behavior of mice was counted just after compound 48/80 injection. Serum histamine and PGE2 concentrations were evaluated individually. Mice administered with CLA showed reduced frequency of scratching behavior compared to those without CLA. Antipruritic activities in CLA-treated and DSCG-treated groups were 48.5% and 26.8%, respectively. CLA and DSCG also diminished serum concentrations of histamine and PGE2 compared to compound 48/80 alone, respectively. This result suggests that dietary CLA has an antipruritic effect by down-regulating serum histamine and PGE2 levels for relief of compound 48/80-induced scratching behavior in mice, which will be useful in allergic pruritus as a preventive medicine.

The objective of this study was to investigate the antimicrobial effects of carvacrol (CV) against Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) strains in milk. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CV against S. aureus and E. coli O157:H7 were determined. In addition, bactericidal kinetics and antimicrobial activity of CV against the aforementioned pathogens in milk over a period of 2 weeks were investigated. CV exhibited antibacterial activity against both foodborne pathogens tested. The MIC and MBC of CV against S. aureus were 15.0 and 20 mg/mL, respectively, whereas those against E. coli O157:H7 were 16.0 and 32 mg/mL, respectively. In time-kill assays, CV at MBC reduced the number of S. aureus and E. coli O157:H7 in milk to undetectable levels within 24 hr. The antibacterial effects of CV persisted for 14 days without any loss of activity. Results of this study suggest that CV has a potential antibacterial activity against foodborne pathogens such as S. aureus and E. coli O157:H7 in milk.

Cytokeratin (CK) comprises the intermediate filament cytoskeleton of epithelial cells. Patterns of CK expression can be regarded as a specific marker for epithelial differentiation status. The aim of this study was to identify CK expression on tongues of Korean native goats ranging from 60-day-old fetuses to newborns during prenatal development using immunohistochemistry. The tongues of fetuses were removed from 2- to 4-year-old female Korean native goats by caesarean section performed under general anesthesia. Immunohistochemistry was performed to assess CK expression patterns on developing goat tongues using serial paraffin-embedded sections. Light zones signifying CK immunoreactivity in dorsal lingual epithelia were weakly positive in 60-day-old fetuses. In 90-day-old fetuses, deep areas in dorsal lingual epithelia were strongly positive for CK expression and superficial areas were moderately positive. In 120-day-old fetuses, light zones of lingual epithelia in the vallate papilla were strongly positive for CK expression, whereas ducts of von Ebner’s glands were moderately positive. In neonates, taste buds were positive for CK expression, whereas non-taste epithelial cells and von Ebner’s glands were negative. These findings indicate that goat tongues have different patterns of CK expression during development and provide a morphological basis for studies on the biological mechanism of epithelial differentiation.

Ischemic stroke is the most common type of stroke in humans. The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) in a canine model of stroke. Ischemic stroke was induced by using prepared autologous thrombus. The dogs were placed in lateral recumbency on the operation table and the cervical area of each dog was sterilized by using alcohol. After making a cervical incision, the common carotid artery and internal carotid artery (a branch of the common carotid artery that supplies an anterior part of the brain) were exposed. A 200 μL injection of the autologous thrombus prepared 24 hr prior to surgery was delivered with a 20 gauge venous catheter through an internal carotid artery. After successful delivery of the autologous thrombus, the venous catheter was removed, and the cervical incision was sutured. Neurologic signs including generalized seizures, tetraparesis, and altered mental status, were observed in all 3 dogs after induction of ischemic stroke and the signs manifested immediately after awakening from anesthesia. T1- and T2-weighted images and fluid-attenuated inversion recovery (FLAIR) images of the brain were acquired 1 day before and 1 day after surgery. On the day following ischemic stroke induction, MRI revealed multifocal lesions in the cerebral cortex and subcortex such as T1 hypointensity, T2 hyperintensity, FLAIR hyperintensity, and diffusion-weighted hyperintensity in all 3 dogs. Upon postmortem examination, ischemic lesions were found to be consistent with the MRI findings and they were unstained with 2% triphenyltetrazolium chloride. Histologic features of the earliest neuronal changes such as cytoplasmic eosinophilia with pyknotic nuclei were identified. Neuropil spongiosis and perivascular cuffing were also prominently observed at the infarcted area. The present study demonstrated the features of MRI and histopathologic findings in canine ischemic stroke models.

Canine herpesvirus (CHV) is a member of the alphaherpesvirus subfamily, which can cause severe hemorrhagic diseases in neonatal pups as well as mild or subclinical respiratory infections in adult dogs. We examined the effects of cold stress on disease progression of CHV, an alphaherpesvirus, in neonatal puppies. Eight puppies were challenged intranasally with CHV suspension and divided into a cold stress treatment group and a hyperthermal group. Four pups were left uninoculated as controls and divided into cold and hyperthermal groups. In the challenged cold treatment group, all pups showed CHV-related disease within 5 days; pathological changes were observed in organs of puppies showing clinical symptoms. Grossly, numerous petechial red foci were scattered throughout lungs, kidneys, livers, and intestines of all CHV-infected puppies exposed to cold stress. Most puppies showed typical clinical signs and macroscopic lesions, and CHV infection was confirmed by isolation of the virus. However, in the challenged hyperthermal group, only one of the pups showed mild symptoms of CHV-induced disease. None of the puppies in the uninoculated group showed abnormal signs, although they were exposed to cold stress. These findings indicate that cold stress can cause rapid disease progression of CHV, an alphaherpesvirus.