Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

Canine parvovirus-2 (CPV-2) has been reported worldwide as a major pathogen associated with acute hemorrhagic enteritis. The disease is a major infectious cause of death, particularly in young dogs. The earliest type of CPV-2 was replaced with three main subspecies, CPV-2a, CPV-2b, and CPV-2c, within a few years. Vaccination is carried out regularly, but the emergence of antigenic variants and the influence of maternal antibodies have limited the efficacy of commercial vaccines. New vaccines, such as the subunit vaccine, have been developed for alternative, safe, and effective vaccination. The baculovirus expression vector system (BEVS) is an excellent eukaryotic expression system with a high-level expression of foreign proteins and the ability of post-translational modification. Therefore, it is used widely to produce recombinant protein and subunit vaccines. In this study, the VP2 protein of CPV-2b cloned in the gateway vector system was generated using a baculovirus expression system in Spodoptera frugiperda (SF9) insect cells. Hemagglutination assay (HA) titers (24) were obtained, and the expression was detected in 6-His tagged VP2 and monoclonal antibody (mAb) against CPV-2 by western blotting. The VP2 protein of CPV-2b expressed in this study may provide a basis for a clinical diagnosis and vaccination applications for CPV-2.

Glutamine has been used to treat canine patients with parvoviral enteritis. However, little is known about the effect of L-alanyl-L-glutamine (Ala-Gln) supplementation in dogs with parvoviral enteritis. The objective of this study was to determine whether Ala-Gln supplementation can improve dog survival and ameliorate clinical signs without adverse effects. We conducted a randomized, double-blind, placebo-controlled clinical trial involving 39 client-owned dogs. The dogs were randomly assigned to two groups and administered either an Ala-Gln solution (Dipeptiven, 0.4 g/kg, n = 20) or an equivalent volume of placebo (n = 19) orally twice daily. Of the 39 dogs, 17 were vaccinated (n = 9 in the Ala-Gln-treated group and n – 9 in the placebo group). All dogs received standard treatment while hospitalized. The dogs were monitored according to a clinical scoring system and evaluated diagnostically daily for 11 days. Survival rates in both groups were quantified using Kaplan‒Meier survival curves and statistically compared using the log-rank test. The total score for clinical signs did not differ between the groups, except on day 2. The survival rates differed significantly (p=0.038). Three Ala-Gln-treated dogs (15.0%) died during the study, whereas eight dogs in the placebo group died (42.1%). No adverse effects were found to be associated with Ala-Gln treatment. Oral administration of Ala-Gln improves survival in dogs with parvoviral enteritis without causing adverse effects.

A 9-year-old, intact female, Pungsan dog was referred for purulent vaginal discharge and depression. Abdominal radiographs revealed gas-filled and soft tissue opacity tubular structures in the mid to caudal abdomen. On ultrasonography, fluid-filled uterus and cervix accompanied by hyperechoic foci and reverberation artifacts were identified. Multiple hyperechoic foci were found within the uterine wall, indicating gas content. Escherichia coli was isolated from aerobic and anaerobic bacterial cultures. Radiological differential diagnosis of tubular shaped, dilated gas-filled structure, and gas in the wall of the structure should consider emphysematous pyometra with endometrial pneumatosis in intact female dogs with vulvar discharge.

Influenza A viruses (IAVs) are members of the family Orthomyxoviridae and genus Orthomyxovirus. Avian and mammalian species are the host of IAVs, which includes humans and dogs. Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory diseases in dogs. This study monitored the antigen and antibody against CIV in dogs in the Republic of Korea (ROK) from 2016 to 2021. One thousand and seventy-two nasal swabs and 1,545 blood samples were collected from animal hospitals and animal shelters. Five nasal swabs in 2017 and seven in 2018 from stray dogs were positive for CIV according to RT-PCR. The prevalence of H3N2 CIV ranged from 9.5% to 24.8%, according to the hemagglutination inhibition (HI) assay. On the other hand, none of the serum samples from 2018 to 2021 showed seropositivity against the avian H5, H7, and H9 viruses. The HI titers for H3N2 ranged from 16 to 512. The distribution of HI titer 16–32 was 57.6% in seropositive samples. The pet dogs were vaccinated against CIV, but the stray and military dogs were unvaccinated. In 2017 and 2018, the seroprevalence of CIV in stray dogs was higher than in the other years, and viral RNA was detected in nasal swabs. It may mean previous exposure of stray dogs to CIV. With the increasing number of pet dogs and the close contact between humans and dogs, canines could serve as an intermediate host for transmitting IAVs to humans. Therefore, continuous surveillance of CIV is needed for public health and the potential emergence of novel zoonotic viruses.

This case report describes satisfactory correction of deep and large canine corneal ulcerations by application of bidirectional corneo-conjunctival transposition (CCT). A 12-year-old spayed female Maltese dog with a large corneal descemetocele, perforation, and blepharospasm of the right eye was referred to Chungbuk National University Veterinary Teaching Hospital. More than half of the thickness of the cornea was damaged, and the ulcer was progressive. On ophthalmic examination, menace response and dazzle reflex were absent. No corneal melting was observed. As the patient had large and deep corneal ulcers, traditional one-sided CCT was not sufficient to cover the wound lesion. To increase corneal transparency after recovery, we decided to perform bidirectional CCT from 12 o’ clock on the dorsal side to 7 o’ clock on the ventral side. The dog was medicated with topical eye drops, ofloxacin, atropine, and moxifloxacin before surgery. Debridement with a diamond burr was then performed around the descemetocele. Five weeks after surgery, the dazzle reflex was restored as the blood vessel receded from the cornea to the conjunctiva. Eight weeks after surgery, corneal transparency and corneal stability were gradually restored, but not completely. Bidirectional CCT provides structural support and helps corneal wound healing in large canine corneal ulceration.

Canine parvovirus type-2 (CPV-2) is a major etiological agent causing gastrointestinal enteritis in domestic and wild carnivores. Since the emergence of CPV-2 in the late 1970s, subtypes CPV-2a, CPV-2b, and CPV-2c have spread worldwide. CPV-2 prevalence differed according to region and season. This study aims to investigate the prevalence of CPV-2 infection in Korea. Samples were collected from 536 dog feces in animal shelters and 225 necropsied intestinal tissues of dog carcasses submitted in the Animal and Plant Quarantine Agency (APQA) for diagnostic purposes from 2016 to 2020 in Korea. Among the 761 samples, 181 (23.8%) were positive for the following subtypes: CPV-2a (n=138), CPV-2c (n=16), CPV-2b (n=14), and CPV-2 (n=2). Feline parvovirus (n=2) and co-infection with CPV-2a and CPV-2c (n=1) were also detected. There was no significant difference in the regional distribution of CPV-2 in Korea, which is prevalent in winter. This result shows the prevalence of CPV-2 according to various environments in Korea and will be useful in establishing an effective prevention strategy against CPV-2 that reflects the situation in Korea with continuous monitoring.

The canine parvovirus (CPV) causes clinical signs, such as severe enteritis, dehydration, diarrhea, vomiting, leukopenia, and hair loss, which may lead to death. Vaccination is still the most important approach, as no specific treatment exists to prevent CPV. Monoclonal antibodies are valuable tools to study the pathogenic mechanisms of CPV and develop effective diagnostic reagents and pharmaceuticals. In this study, two monoclonal antibodies (MAbs) against CPV-2a were obtained through hybridoma technology by fusing myeloma cells and B cells from the spleens of mice immunized with CPV type 2a (CPV-2a). Two MAbs (CPV-330 and CPV-620) were studied on the reactivity of vaccine (CPV-2a) and field strains (CPV-new 2a, -2b, and -2c) by indirect immunofluorescence (IFA), hemagglutination inhibition test (HI), virus neutralization test (VN), and inhibition of virus growth test. Two MAbs showed similar antibody titers for HI and VN. On the other hand, CPV-330 inhibited the viral replication in Crandell-Rees Feline Kidney (CRFK) cells better than CPV-620. These CPV MAbs may provide valuable biological reagents to study the CPV pathogenic mechanisms and work as therapeutic antibodies.

Skin protects the body by mediating various immune responses against exogenous substances including bacteria, fungi, and viruses, in addition to its predominant role as a physical barrier. Despite the significant protection offered via various mechanisms, bacterial infection of the skin is one of the most common skin diseases in veterinary medicine. This study demonstrated the structural and immunological changes in the skin during infections with Pseudomonas aeruginosa and Staphylococcus pseudintermedius using skin explants from four healthy beagles. Skin structure was generally well preserved in uninfected controls, but defects in skin structure, including injury of keratinocytes and dermal–epidermal junctional disruption, were identified when skin explants were exposed to P. aeruginosa and S. pseudintermedius. On exposure to P. aeruginosa, marked linear cleft formation was noted along with acantholysis along the basal layer after 24 hours of culture. In addition to the defects in the skin structure, mRNA expression levels of the AMPs cBD103 and S100A8 were decreased, which was confirmed by immunohistochemical staining. Taken together, these results suggest that our ex vivo canine skin model is a research tool for investigating bacterial skin infections in dogs.

The prevalence of cancer in companion dogs is growing nowadays with the increasing worldwide population of domestic dogs. Since there is a less established standard of care in veterinary medicine, investigational treatments, such as the development of biomarkers can be considered as a therapeutic intervention for early diagnosis. Despite the enormous efforts that have been invested in the search of biomarkers, still, there is a need for easy detection of significant biological markers for predicting canine cancers at an early stage. In this study, we have analyzed the expression pattern of previously reported 46 canine cancer-associated candidate genes in blood specimens using real-time qPCR. We hypothesized that analysis of gene expression in blood would provide preliminary evidence of local or systemic immunogenic response which further contribute to the easy and early diagnosis of canine cancer from blood specimen as an analytical tool. The datasets included a total of 22 blood samples collected from different breeds of dogs diagnosed with cancer and five from healthy normal dogs. RT-qPCR analysis was performed by employing the SYBR Green PCR mix to assess the expression of these 46 genes in a total of 27 samples. From our result, a total of nine genes (ROS1, C1QA, CD48, IL1b, TLR2, IL2R, CHI3L1, CTSS, and TLR7) were found to be significantly up-regulated (p < 0.05 and p < 0.01) in the cancer samples compared to non-cancer samples. The relative expression level of ROS1, C1QA, CD48, IL1b, TLR2, IL2R, CHI3L1, CTSS, and TLR7 genes was 5.74, 4.78, 3.94, 2.94, 2.57, 2.53, 2.50, 2.04, and 2.57, respectively, in cancer samples compared to non-cancer samples. Thus, our results reveal several highly expressed cancer genes that can be therapeutic target genes for further testing in canine cancers.

Natural killer (NK) cells have cytotoxic effects on tumor cells and viral pathogens. NK cell-derived exosomes (NK-exosomes) also express typical NK cell markers and cytotoxic molecules, therefore, exert anti-tumor and immune homeostatic activities. In this study, canine NK-exosomes separated from cytotoxic NK cell supernatant carried specific markers such as CD81, Alix, and Perforin 1. We examined the anti-tumor effects of NK-exosomes in an experimental murine model using the canine mammary carcinoma cells, REM134. REM134 cells were xenografted of mammary fat pad of mice. CD133, Bmi-1, MMP-3, IL-6, TNF-α, and PCNA are useful as a molecular marker for tumorigenesis and metastasis. The treatment of canine NK-exosomes inhibited tumor growth and significantly (p<0.01) downregulated the expression of Bmi-1, MMP-3, IL-6, TNF-α, and PCNA in REM134-treated mice. Also, the expression of CD133, potent cancer stem cell marker, was significantly downregulated in the canine NK-exosomes-treated mice compared with that of the tumor group. Collectively, these results suggested that canine NK-exosomes has a potential capacity for regulation of cancer progression and metastasis against canine mammary carcinoma.

An 8-year-old, spayed female Maltese dog was presented with a one-month history of erythema, swelling and alopecia of periocular region with pruritus. The skin lesions were first detected at the age of three years, but this was the first time that symptoms had appeared in the eyes. Physical examination revealed markedly swollen and erythematous eyelids and conjunctiva including Meibomian glands. In addition, periocular alopecia and tears were identified. Slit lamp microscopy revealed erythema of conjunctiva and swelling of Meibomian glands. The Schirmer’s tear test was normal. Impression smear cytology of eyes revealed sterile neutrophils and corneal epithelial cells. There were no virus or bacterial infections in the eyes. On skin examination, generalized erythema was detected but there were no other skin lesions. A case of allergic blepharoconjunctivitis associated with canine atopic dermatitis was diagnosed based on history taking and skin examination. Treatment included cetirizine, cyclosporine, prednisolone and Forus eye drops®. The owner was instructed to wear an Elizabethan collar around his dog's neck at all times and restrict walking. And the diet was changed to hypoallergenic dog food. Medications and environmental restrictions significantly reduced erythema, edema and swelling of meibomian glands. Pruritus was also decreased. Six weeks later, the edema lesions of eyes disappeared.

A one-year-old, intact male Maltese was referred with dehydration, anorexia, and marked hyperglycemia. The dog had been managed due to meningoencephalitis of unknown etiology (MUE) for three months. The dog had been treated with long-term prednisolone administration. Diabetic ketoacidosis (DKA) was identified based on the blood chemistry and venous gas analyses, and intensive treatments including insulin administration were initiated. On further examinations, there was no any other disease that contributed to the occurrence of DKA. Insulin resistance resulted from the administration of prednisolone was highly suspected, but the agent could not be tapered due to managing MUE. Following resolution of DKA, the dog was discharged with life-long insulin and prednisolone therapy. Over the next two years, the dog continued to be routinely re-evaluated and was managed with permanent insulin therapy (0.8–1.4 units/kg SC 12 hourly) and medications including prednisolone (0.4–1.1 mg/kg PO 12 hourly). Because MUE severely progressed, the dog was euthanized by owner’s request. Histopathologic examination of pancreas obtained by post-mortem revealed that both endo- and exocrine pancreas was within normal limit. The case described herein showed the risk of ketoacidosis as well as hyperglycemia after long-term prednisolone administration in a dog without pancreatic islet pathology.

Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSCexo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 μg/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 µg/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

Canine parvovirus (CPV) remains a leading infectious cause of death in canines, especially in young puppies. Though vaccination is being carried out regularly, immunization failures occur, and puppies may be exposed to infection. Virus-like particles (VLPs) act like a subunit vaccine, mimicking the structure of authentic viruses. Therefore, VLPs have the potential to be used as vaccine candidates. Since Viral Protein 2 (VP2), a major structural protein of CPV, is the crucial antigen for CPV, the purpose of this study was to produce a recombinant VP2 of new canine parvovirus-2a using the baculovirus expression system in SF9 insect cells. The results revealed that recombinant VP2 assembles to form VLPs with antigenic properties similar to those of natural CPV, the recombinant VLP can produce a hemagglutination assay (HA) titer (1:210) in SF9 cells. Expression of the recombinant 6-His-tagged VP2 in SF9 cells was confirmed by western blotting. These findings suggest that the recombinant VP2 expressed in this study could be used as an efficient subunit vaccine against CPV infection.

Viral protein 2 (VP2), which is the structural protein of parvovirus, can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. VP2 of canine parvovirus (CPV) is responsible for neutralizing antibodies in immunized animals. In this study, VP2 protein of canine parvovirus-2c was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells. The results show that VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and a high hemagglutination (HA) titer (1:27). The recombinant 6-His-tagged VP2 protein with a molecular mass of about 65 kDa was detected by anti-His antibody and anti-PPV serum. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of CPV and in the vaccination against CPV.

Canine inflammatory mammary carcinoma (CIMC) similar to human inflammatory breast cancer is a very aggressive, metastatic type of cancer. Previous studies have introduced a new type of tumor angiogenesis called vasculogenic mimicry that may play an important role in the progression of inflammatory mammary cancer. The purpose of this study is to investigate the development process of vessels by neoplastic cells in CIMC. Patient dog, 14-year old Shit-Tzu female, had a hard and somewhat movable dark-reddish mammary tumor, sized 6.2 cm in diameter. Bloody dark turbid exudate was released from the tumor. In addition to histological examination, immunohistochemistry for pancytokeratin, VCAM-1, MECA- 32, TWIST-1, and Ki-67 was respectively performed using the ABC method. Histologically, the inflammatory mammary carcinoma was characterized by tubular solid tumor emboli within the lymphatic vessels surrounded by desmoplastic fibrous connective tissue. Some of the neoplastic cells were transforming into elongate or spindle shapes and forming small vessel-like structures in the solid tumor mass. The neoplastic cells were immunoreactive for VCAM-1 and MECA-32, but showed low immunoreactivity for Ki-67. Immunoreactive neoplastic cells for VCAM-1 and MECA-32 suggested the possibility that the neoplastic cells transform into endothelial cells of vessels by epithelial-mesenchymal transition, further supported by serial morphological changes identified by histological investigation and immunohistochemistry for TWIST-1. The high capacity of the neoplastic cells forming the vasculatures in CIMC explains the high ratio of metastasis to other regions, even though Ki-67 index was not so high.

Probiotics improve the immune system. However, the effects of its lactic acid bacteria on atopic dermatitis relief and inflammation improvement is not fully understood. Recently, one of the probiotics, Lactobacillus helveticus HY7801 (HY7801), was found to have an anti-inflammatory effect. In this study, we investigated the effects of HY7801 on atopic dermatitis-induced animal models. After four weeks of oral administration, the group treated with HY7801 showed amelioration of the atopic dermatitis compared to the group receiving placebo. In the HY7801 treated group, the epidermal hyper-proliferation and collagen deposition were inhibited compared to the placebo group, and the secretion amount of the inflammatory factors, such as TNF-α, IL-4 were reduced. In conclusion, these results suggest that HY7801 acts as a functional probiotic via amelioration of the atopic dermatitis such as a decrease of epidermal hyper-proliferation, and collagen deposition and anti-inflammatory effects.

Although canine brucellosis has been known to be an important re-emerging zoonosis, the pathophysiological mechanisms of Brucella canis infection remains clues to be solved. Different culture models, single and co-culture models, were constructed with canine epithelial cells, D17 and macrophage, DH82 to investigate the induction of immune responses in in vivo B. canis infection. Expression of genes related with induction of immune responses, Th1, Th2 and Th17, was compared in the two different models after the bacterial infection. In this study, expression of cytokine genes, IL-1β, IL-5, IL-6, IL-10, IL-23, and TNF-α was quantified in the DH82 at different time points using RT-qPCR in the two different culture systems after the infection. Cytokine genes related with Th1, IL-1β and TNF-α and Th17, IL-6 and IL-23 were expressed with time-dependent manners in the both systems (p<0.05). However, increase of Th2-related cytokine genes expression was not detectable in the both systems by comparison with control. The expression of Th1 and Th17 related cytokine genes was earlier in single cell culture than those in co-culture model (p<0.05). In general, amounts of the expressed genes were shown higher in single cell model than those in co-culture models. This study indicate that Th1 and Th17-associated immune responses are central to B. canis infection in dogs. In addition, it suggests a specific role of epithelial cells in the B. canis infection in vivo, which should resolved in the further study.