Establishment and characterization of a canine chondrosarcoma cell line:Mango

In the global progress of bone tumor research,established stable and long-lasting transgenic chondrosarcoma(CSA)cell lines are rare,mainly of murine and human origin,while the establishment of canine CSA cell lines has yet to be reported.This study established a canine CSA cell line to facilitate the basic clinical study of canine CSA.Fifty fve cases of canine osteolytic disease were collected,and more than 10 bone tumor samples from dogs with typical clinical signs were used for primary cell culture.A cell line with stable passaging for more than 100 generations and mouse tumorigenic ability was successfully cultured.According to the clinical characteristics of the dog and the histopathological results of the primary tumor,CSA was diagnosed,and the CSA cell line was designated Mango.Immunohistochemical(IHC)results showed that the immunoreactivity of bone gamma-carboxyglutamate protein(BGLAP),secreted protein acidic and rich in cysteine(SPARC),alkaline phosphatase(ALPL),vimentin(VIM)and S100 were positive.However,the immunoreactivity of pan-cytokeratin(PCK),chromogranin A(CGA),and platelet endothelial cell adhesion molecule-1(CD31)was negative.Immunofuorescence(IF)results showed that the protein expressions in the Mango cell line were consistent with the IHC identifcation of the primary tumor.The Mango cell line’s doubling time was 43.92 h,and the cell formation rate exceeded 20%.There were abnormal chromosome numbers,hetero staining with toluidine blue,and certain calcifcation abilities.It could be passaged stably and continuously without changing the cell morphology and characteristics.In vivo,the cells were successfully injected into the nude mice model with a tumorigenic rate of 100%.The immunophenotype of the xenograft tumor was consistent with that of the primary tumor.Therefore,we efectively established a canine CSA cell line.As a promising cell material,this cell line can be used to construct a tumor-bearing model conducive to the subsequent basic research of canine CSA.Moreover,because of its similarity to human CSA,the animal model of CSA is also indispensable for investigating human CSA.

Molecular characterization of canine parvovirus type 2(CPV2)reveals a high prevalence of the CPV2c genotype among dogs sufering from diarrhea

Canine parvovirus 2(CPV-2)is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies.The genetic variability and antigenic diversity of CPV-2 hinder its efective prevention of infection by vaccination.To investigate the epidemiology and genetic characteristics of CPV-2 in China,rectal swabs from afected dogs were collected from diferent animal clinics in Kunshan from 2022 to 2023.Preliminary detection and capsid gene sequencing of CPV-2 were performed using previously described primers and protocols.The overall detection rate for CPV-2 was 16.5%(33/200).A signifcant association was found between the CPV-2-positivity and clinical signs,age,breed and vaccination status.Sequence analysis revealed the presence of CPV-2c genotypes in all positive samples,which were genetically similar to other Asian CPV-2c strains.Notably,four key mutations(A5G,F267Y,Y324I and Q370R)were detected in all isolates,and one novel mutation(I447M)was detected in three CPV-2 isolates.These mutations in the CPV-2 strains could impact vaccine efcacy and the efectiveness of the virus immune evasion.Surprisingly,no recombination events were observed between the identifed CPV-2c strains and reference strains from China.Our data revealed that amino acid residues 324,426 and 440 of VP2 may under strong selection pressure.This pattern of genetic variation in the CPV-2 lineage warrants continuous laboratory-based surveillance programs in other parts of China to better understand the pattern of seasonal distribution and association between emerging genotypes and the intensity of disease severity.

Preparation of anti-canine interleukin-31 receptor alpha polyclonal antibody and evaluation of its therapeutic efect in canine atopic dermatitis

Canine atopic dermatitis(CAD)is a prevalent genetically susceptible infammatory and pruritic allergic skin condition afecting not only the health of dogs but also the quality of life of their owners.Interleukin-31(IL-31)and interleukin-31 receptor alpha(IL-31RA)are essential for the development of pruritus in primates and mice.Hence,it is expected that inhibiting IL-31RA will be an efective approach to alleviate pruritus.The purpose of the study was to produce anti-canine IL-31RA polyclonal antibodies(anti-IL-31RA pAbs)and evaluate their efcacy in inhibiting house dust mite(HDM)-evoked pruritic responses.Dogs were immunized with antigens formed by IL-31RA recombinant short peptides coupled to BSA to produce anti-IL-31RA pAbs.The CAD model was developed by using HDM allergen stimulation,and the efects of IL-31RA pAbs on the reduction of pruritus in CAD model dogs were examined.The Canine Atopic Dermatitis Extent and Severity Index(CADESI)-4 and pruritus Visual Analog Scale(pVAS)were utilized to evaluate pruritic responses,and skin tissue samples were collected from the inguinal area for pathological assessment of skin infammatory cell infltration.The results showed that anti-IL-31RA pAbs with high titers(1:128,000)and specifcity were efectively produced.In the CAD model group,the severity of skin damage,pruritus score,infammatory cell infltration and level of infammatory factors were considerably elevated.Anti-IL-31RA pAbs relieved pruritic behavior and dermatitis in dogs compared to placebo-treated dogs.In conclusion,anti-IL-31RA pAbs efectively suppressed CAD in vivo and are anticipated to be an efective novel treatment for pruritic skin disorders such as CAD.

Update on Canine and Human Rabies in a Rabies Endemic Situation in the Republic of Guinea

Each year, rabies kills nearly 60,000 people worldwide. Asia and Africa are the most affected. It is a public health problem because of the number of stray dogs in these nations. In Guinea, rabies is endemic and cases are often under-reported. The objective of this study was to determine the prevalence of rabies among human and animal exposures in Guinea. This was a retrospective analytical study from 2019 to 2021 conducted in the country’s human and veterinary health divisions and, primarily focused on rabies épidémiologique surveillance. The selection was based on human contact with an animal causing behavioural disorders, as well as animals put under observation following aggression on humans. The data were analysed using Epi Info version 21 software. Qualitative variables were expressed as proportions with 95% CIs, quantitative variables as mean ± standard deviation. The chi2 test and the Fischer exact test were used to compare proportions. A total of 85 veterinary facilities were visited and 100 animals registered, of which 93% were reported cases of exposure to canine rabies. One thousand five hundred and thirty-one (1531) people exposed to rabies were recorded between 2019 and 2021. During the period, the prevalence of human rabies was 0.533% [0.042 - 0.064] at 95% CI and that of canine rabies was 0.55% [0.039 - 0.061] at 95% CI. The biting animal was the dog in 97.2% of cases. Subjects over 45 years of age (57.7%) were the most affected. The prevalence of canine rabies was 0.5% compared with 0.533% for human rabies between 2019 and 2021. This low prevalence does not reflect the true extent of the phenomenon in Guinea, as few people consult health structures in the event of dog bites. Regular surveillance of the human and dog population is necessary for better rabies control in Guinea.

Therapeutic Effects of an Alpha-Casozepine and White Fish Muscle Hydrolyzed Complementary Feed on Canine Separation-Related Disorders and Quality of Life of Dogs and Their Pet Owners

Background: Separation-related disorders are prevalent in dogs and can result in problem behaviours such as inappropriate elimination, destructive behaviour, and distress vocalizations which occur when an owner is absent or perceived as absent. Numerous natural products are marketed for managing anxiety in dogs;however, few, if any, of these products have documented evidence of efficacy in veterinary behaviour. Purpose: This open, multicentric clinical trial aimed to assess the efficacy of a new supplement containing alpha-casozepine and fish muscle hydrolysate on the separation-related problem in dogs and the impact on the quality of life of dogs and their pet owners. Material and Methods: Fifty-one dogs with clinical signs compatible with separation-related problems were included. The dogs were treated with a complementary feed (Zylkene Plus, Vetoquinol, France) for 30 days. Separation-related behaviour assessment (SRP), emotional disorders assessment (EDED) and the quality of life (QoL) of the pets and their pet owner were assessed at baseline and the end of the study by the veterinarians. In addition, owners were asked to rate their dog’s SRP weekly after enrolment using the same questionnaire that the veterinarian used at the beginning of the study. Results: There was a statistically significant improvement in the SRP, EDED, and QoL scores (P Conclusion: The results of this study support the hypothesis that the combination of alpha-casozepine and fish muscle hydrolysate is a promising and effective option to reduce the frequency and severity of separation-related behaviour in dogs and improve the quality of dogs and their owners.

Detection of Antinuclear Antibodies in Canine Serum by Immunoperoxidase in MDCK and Indirect Immunofluorescence in HEp2 Cells

Antinuclear antibodies are found in animals suffering from Systemic Lupus Erythematosus (SLE) and some other diseases, their presence in the blood is determined by antinuclear antibody (ANA) test using indirect immunofluorescence (IF) with HEp2 cells as a substrate. In this work, an immunoperoxidase (IP) assay was developed to evaluate the ANAs in canine sera, using canine kidney cell lines (MDCK) and compared with a commercial immunofluorescence test on Hep2 cells for this system, a fluoresceinated anti-canine Ig antibody was standardized. The study was performed on 50 sera from dogs submitted to the laboratory with different clinical diagnoses of autoimmune-associated diseases. The procedures on both cells were unified to perform comparisons of the reactions, direct sera or at different dilutions were added to a monolayer of permeabilized MDCK cells, followed by a peroxidized anti-canine IgG conjugate, and a substrate for the IP reaction. The same sera were tested on the commercial IF assay on Hep2 cell system. In 22/50 cases, the presence of LE cells in peripheral blood was determined. A high correlation was found in the detection of antinuclear antibodies between both cell lines and techniques, however there were differences in the reaction patterns in the nucleus and cytoplasm between cell lines. The diffuse nuclear pattern observed in MDCK cells was more related to the presence of high percentages of LE cells in peripheral blood. The differences found in the results were possibly associated with the presence of homologous antigens between the MDCK cells and the dog. In addition, the methodology and standardization for the use and interpretation of a reference serum was developed to unify the interpretation criteria in the laboratory.

Antiarrhythmic Efficacy of Neferine Assessed by Programmed Electrical Stimulation in a Canine Model of Electropharmacology

An ishemic ventricular tachyarrhythmias canine model was established in open-chest dogs subjected to programmed electrical stimulation (PES)for 5-8 days after acute myocardial infarction. The electrophysiologic effects of neferine (Nef) and procainamide (PA) were observed in this model. With routine methods of PES,ventricular tachycardia (VT)and ventricular fibrillation (VF) could be reproducibly initiated. Both drugs lengthened the QTc interval (P【0.01) and effective refractory period(ERP)of normal and ischemic ventricular myocardia (NERP and IERP) respectively (P【0.01), decreased the dispersion of ERP in ischemic myocardium and the dispersion of ERP in left ventricle (P【0.01), and increased the diastolic excitability threshold of normal and ischemic ventricular myocardia (P【0.01). The two compounds prevented the PES-induced VT or VF (Nef group P【0.01, PA group P【0.05) and ischemia-induced VF (P【0.05). The results indicated that neferine and procainamide may be effective in preventing the onset of reentrant ventricular tachyarrhythmias after myocardial ischemic damage in dogs.

Serological survey on canine coronavirus antibodies in giant pandas by virus neutralization test

In order to survey the infectious situation of canine coronavirus (CCV) in giant panda population, a virus neutralization test detecting specific antibodies against CCV in giant panda抯 sera was established by using two-fold dilutions of serum and 100 TCID50 of the virus. The 62 sera samples of giant pandas, which were gathered from zoos and reserve region of Sichuan Province, China were detected. The neutralization antibody titer of 1:4 was recognized as the positive criterion, 8 sera samples were detected to be positive, and the positive rate was 12.9%. The titers of neutralizing antibody ranged from 1:8 to 1:32. It was the first comprehensive investigation on neutralization antibodies against CCV in giant panda population in China. The results of study showed that the infection of CCV in giant panda population was universal, which has posed a threat to the health of giant panda. Therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against CCV infection.

Construction of Recombinant Pseudorabies Virus Expressing Canine Distemper Virus H Gene and Analysis on Its Biological Characters

[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

ELISA Detection of Antibody against Canine Distemper Virus in Fox

Canine distemper is one of the most important common viral epidemics in farmed foxes. In this study, the whole blood samples of foxes that had been immunized with canine distemper vaccine were collected from a farming area in Changli County. The antibody against canine distemper virus in the 85 prepared serum samples were detected with a double-antibody sandwich ELtSA method. The results showed among the 85 samples from the 4 farms, 81 samples were antibody-positive, and only 4 samples had no antibody against canine distemper. The overall antibody level was higher in that farming area. It is indicated canine distemper vaccination and vaccination program have gotten attention from most of the local farmers, and the immune effect of canine distemper vaccine is better.

Generation of a Canine-origin Neutralizing scFv Against Canine Parvovirus

Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.

Histopathological Observation of Canine Distemper in Raccoon Dogs

In this study, histopathological changes in the heart, liver, spleen, lung, kidney and other organs of raccoon dogs died of canine distemper were observed. According to the results, the lung of infected raccoon dogs exhibited severe hemor- rhage and emphysema; the liver was congested and degenerated; the spleen was enlarged; the exhibited severe hemorrhage and hyaline degeneration. Multiple organs of infected raccoon dogs were congested and hemorrhaged with tissue damage, in- flammatory cell infiltration and a series of pathological changes. This study laid a solid foundation for clinical diagnosis and treatment of canine distemper in raccoon dogs.

Effects of a bioartificial liver support system on acetaminophen induced acute liver failure canines

AIM To evaluate the safety and efficacy of the bioartificial liver support system in canines with acute liver failure (ALF). METHODS Nine canines with acute liver failure by acetaminophen induced received TECA Ⅰ bioartificial liver support system (BALSS) from Hong Kong TECA LTD Co. Blood was perfused through a hollow fiber tube containing (1 2)×10 10 the porcine hepatocytes. In contrast, another 10 canines with acute liver failure by Acetaminophen received drugs. Each treatment lasted 6 hours. RESULTS BALSS treatment resulted in beneficial effects for acetaminophen induced ALF canines with survival and with the recovery of the liver functions and tissues, and plasma ammonia decreased from 135 9μmol/L ± 17 5μmol/L to 65 7μmol/L ± 22 0μmol/L , 32 5μmol/L ± 8 8μmol/L , GPT from 97 8U/L ± 8 7U/L to 64 8U/L ± 11 9U/L , 19 0U/L ± 6 3U/L , GOT from 103 0U/L ± 16 7U/L to 75 7U/L ± 19 6U/L , 26 5U/L ± 5 0U/L , and AKP from 158 3U/L ± 12 1U/L to 114 5U/L ± 19 8U/L , 43 8U/L ± 5 6U/L during and after the treatment. In contrast, 10 ALF canines in both the drug and control groups died 1 or 2 days after treatment. CONCLUSION TECA 1 artificial liver support system is safe and efficacious for canines with acute liver failure.

Current Status of Canine Rabies in China

Abstract The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal role of dogs in the spread of rabies indicates that controlling and preventing canine rabies could be a key step in eradicating human rabies in China. The primary aims of this review are to discuss the properties and pathogenesis of the rabies virus, the clinical signs and diagnosis of canine rabies, threshold host density and vaccination of dogs, and the prevention and control of canine rabies in China.

Biotinylated dextran amine anterograde tracing of the canine corticospinal tract

In this study, biotinylated dextran amine （BDA） was microinjected into the left cortical motor area of the canine brain. Fluorescence microscopy results showed that a large amount of BDA-labeled pyramidal cells were visible in the left cortical motor area after injection. In the left medulla oblongata, the BDA-labeled corticospinal tract was evenly distributed, with green fluorescence that had a clear boundary with the surrounding tissue. The BDA-positive corticospinal tract entered into the right lateral funiculus of the spinal cord and descended into the posterior part of the right lateral funiculus, close to the posterior horn, from cervical to sacral segments. There was a small amount of green fluorescence in the sacral segment. The distribution of BDA labeling in the canine central nervous system was consistent with the course of the corticospinal tract. Fluorescence labeling for BDA gradually diminished with time after injection. Our findings indicate that the BDA anterograde tracing technique can be used to visualize the localization and trajectory of the corticospinal tract in the canine central nervous system.

Development of a Polyclonal Antibody-based AC-ELISA and Its Comparison with PCR for Diagnosis of Canine Parvovirus Infection

A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Transcellular transport characteristics of huperzine alone or in combination with ginkgolide B across Caco-2 and Madin-Darby canine kidney cell monolayer

Objective:To study the various processes involved in transcellular transport(TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal(MDCK)cell monolayer.Methods:The transepithelial passage was assayed in the apical-to-basolateral(AP to BL) direction and opposite direction(BL to AP) in both cell lines.The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography(HPLC).The passage rates of huperzine A and ginkgolide B were calculated.Bi-directional TT(absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer.Results:TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time.The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B.Conclusions:The compound preparations of HA in combination with CB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics,and improve the bioavailability through BBB.

Short-term results in canines of novel stent-graft design for chimney technique in TEVAR

Introduction:Parallel stent-stent grafting is a major endovascular technique used to preserve the supra-aortic branches during thoracic endovascular aortic repair(TEVAR)of aortic pathologies involving the aortic arch.The short-and mid-term results of this technique are satisfactory;however,endoleak remains a major concern.Thus,here we designed a new chimney stent-graft to decrease the endoleak rate.Aim:To testify the feasibility and safety of the new chimney stent-graft system in a canine model.Material and methods:Six Labrador retrievers were used.Pre-operative data were collected and all operations were performed under general anesthesia.The main and chimney stent-grafts were implanted through the abdominal aorta and left subclavian artery approaches,respectively.Completion digital subtraction angiography(DSA)was performed to confirm the immediate outcomes.All dogs were fed separately for 6 months and sacrificed after aortic angiography.The thoracic aorta and the main and chimney stent-grafts were harvested for histopathologic examination.Results:No complications were found in follow-up DSA.All branch arteries were patent.In?ammatory responses were observed around the stent-grafts in 3 experimental animals,and slight hyperplasia was observed in the surrounding tissues compared with the normal vessels.There was no mural thrombus in the stent,endothelial cells were noted on the inner surface of the stent,and thrombus was formed in the outer skirt and gutter area.The histopathologic examinations revealed similar results to those of gross necropsy observations.Conclusions:This study demonstrated the feasibility and safety of the Longuette stent-graft and the first to report a revised stent-graft specific for chimney technique.

Three-dimensional Localization of Impacted Canines and Root Resorption Assessment Using Cone Beam Computed Tomography

Summary： The purpose of this study was to develop a new way to localize the impacted canines from three dimensions and to investigate the root resorption of the adjacent teeth by using cone beam computed tomography （CBCT）. Forty-six patients undergoing orthodontic treatments and having impacted canines in Tongii Hospital were examined. The images of CBCT scans were obtained from KaVo 3D exam vision. Angular and linear measurements of the cusp tip and root apex according to the three planes （mid-sagittal, occlusal and frontal） have been taken using the cephalometric tool of the InVivo Dental Anatomage Version 5.1.10. The measurements of the angular and linear coordinates of the maxillary and mandibular canines were obtained. Using this technique the operators could envision the location of the impacted canine according to the three clinical planes. Adjacent teeth root resorption.of 28.26 % was in the upper lateral incisors while 17.39% in upper central incisors, but no lower root resorption was found in our samples. Accurate and reliable localization of the impacted canines could be obtained from the novel analysis system, which offers a better surgical and orthodontic treatment for the patients with impacted canines.