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.

Enterogenic Stenotrophomonas maltophilia migrates to the mammary gland to induce mastitis by activating the calcium-ROS-AMPK-mTOR-autophagy pathway

Background Mastitis is an inflammatory disease of the mammary gland that has serious economic impacts on the dairy industry and endangers food safety.Our previous study found that the body has a gut/rumen-mammary gland axis and that disturbance of the gut/rumen microbiota could result in‘gastroenterogenic mastitis'.However,the mechanism has not been fully clarified.Recently,we found that long-term feeding of a high-concentrate diet induced mastitis in dairy cows,and the abundance of Stenotrophomonas maltophilia(S.maltophilia)was significantly increased in both the rumen and milk microbiota.Accordingly,we hypothesized that‘gastroenterogenic mastitis'can be induced by the migration of endogenous gut bacteria to the mammary gland.Therefore,this study investigated the mechanism by which enterogenic S.maltophilia induces mastitis.Results First,S.maltophilia was labelled with superfolder GFP and administered to mice via gavage.The results showed that treatment with S.maltophilia promoted the occurrence of mastitis and increased the permeability of the blood-milk barrier,leading to intestinal inflammation and intestinal leakage.Furthermore,tracking of ingested S.maltophilia revealed that S.maltophilia could migrate from the gut to the mammary gland and induce mastitis.Subsequently,mammary gland transcriptome analysis showed that the calcium and AMPK signalling pathways were significantly upregulated in mice treated with S.maltophilia.Then,using mouse mammary epithelial cells(MMECs),we verified that S.maltophilia induces mastitis through activation of the calcium-ROS-AMPK-mTOR-autophagy pathway.Conclusions In conclusion,the results showed that enterogenic S.maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calcium-ROS-AMPK-mTOR-autophagy pathway to induce mastitis.Targeting the gut-mammary gland axis may also be an effective method to treat mastitis.

Genetic vaccination with Flt3-L and GM-CSF as adjuvants: Enhancement of cellular and humoral mmune responses that results in protective immunity in a murine model of hepatitis C virus infection

AIM： To investigate whether transfection of plasmid DNA encoding these cytokines enhances both humoral and cellular immune responses to hepatitis C virus （HCV） in a murine model. METHODS： We established a tumor model of HCV infection using syngenic mouse myeloma cells stably transfected with NS5. Co-vaccination of DNA encoding granulocyte macrophage colony-stimulating factor （GM- CSF） and Flt-3 ligand together with a plasmid encoding for the HCV NS5 protein was carried out. Mice were sacrificed 14 d after the last immunization event with collection of spleen cells and serum to determine humoral and cellular immune responses. RESULTS： Co-vaccination of DNA encoding GM-CSF and Fit-3 ligand together with a plasmid encoding for the HCV NS5 protein induced increased antibody responses and CD4＋ T cell proliferation to this protein, Vaccination with DNA encoding GM-CSF and FIt-3L promoted protection against tumor formation and/or reduction in mice co- immunized with cytokine-encoding DNA constructs, This suggests this strategy is capable of generating cytotoxic T lymphocyte activity in vivo, Following inoculation with plasmid DNA encoding Flt-3L, no increase in spleen size or in dendritic cell （DC） and natural killer cell numbers was observed. This was in contrast to a dramatic increase of both cell types after administration of recombinant Flt3-L in vivo. This suggests that vaccination with plasmid DNA encoding cytokines that regulate DC generation and mobilization may not promote unwanted side effects, such as autoimmunity, splenic fibrosis or hematopoietic malignancies that may occur with administration of recombinant forms of these proteins. CONCLUSION： Our data support the view that plasmid DNA vaccination is a promising approach for HCV immunization, and may provide a general adjuvant vaccination strategy against malignancies and other pathogens.

The Dog as a Risk Factor in Transmission of Visceral Leishmaniasis: A Review

Visceral leishmaniasis (VL) is a serious public health problem in Brazil and worldwide. Despite the euthanasia of dogs serologically positive, such action has not solved the endemic in Brazil. A risk area for VL involves the presence of the vector, the occurrence of canines and the record of human cases. The factors that have favored the persistence and spread of VL in Brazil are related to the predatory action of man on the environment, to the migratory movements and rural exodus, and a close coexistence of man and animals. Thus considering the epidemiological chain of VL, one fact seems clear: we do not know yet the true extent of the participation of the dog in the infectious cycle of VL. The clinical disease is an important indicator of the extent of the problem in an endemic area. The immunological events are complex and involve resistance and susceptibility to canine visceral leishmaniasis (CVL). One aspect in CVL is that many symptomatic dogs underwent xenodiagnosis not infect the vector. In such cases the pattern recognition receptors CD11b+, TLR2+, and NO present higher values for dogs with results in immunohistochemistry of skin and xenodiagnosis negative (IMH-/XENO-) than dogs with immunohistochemistry in skin and xenodiagnosis positive (IMH+/XENO+), suggesting that innate immunity modulates the competence of the dog to infect the vector. The organic response in CVL varies from individual to individual and within the same individual, with a strong evidence of being organ-specific. Only 20% of asymptomatic dogs have parasites in the skin and 15% are able to recover from clinical signs and eliminate the parasites spontaneously. In this review, we analyze the epidemiology of visceral leishmaniasis and clinical, immunological and pathological conditions that can contribute to understanding the role of dogs in transmission of visceral leishmaniasis.

Moderate quantity of lard mixed with sunflower oil attenuate lipid accumulation in mice

Lard,a fat rich in saturated fatty acids(SFAs),is regarded as a risk factor for metabolic diseases.In the present study,effect of different lard blended with sunflower oil diets on lipid accumulation in adipose tissue,liver,and serum by mouse model was researched.Body weight,body fat percentage,cross-sectional area of adipocytes,liver triglycerides(TGs),and oil red stained area in mice liver of lard blend sunflower oil(L-SFO)group were significantly lower than those of sunflower oil(SFO)group,whereas no significant differences were observed between mice of lard and L-SFO groups.Serum TG and free fatty acid levels were significantly lower in L-SFO group than in other two groups.Furthermore,data showed that sunflower oil decreased contents of hormonesensitive lipase and carnitine palmitoyl transferase 1(CPT-1)and increased fatty acid synthase activity in liver tissue.A mixture of lard and sunflower oil rather than only sunflower oil or lard might promote body fat loss and reduce lipid accumulation in adipose tissue,serum,and liver by promoting hydrolysis of TG,increasingβ-oxidation of fatty acids.These data suggested that mixing lard and vegetable oil(e.g.sunflower oil)for cooking,or alternate using lard and vegetable oil could be beneficial for reducing body fat.

Effect of mixing lard with soybean oil in cooking on hepatic antioxidative ability and renal metabolic activity

A simulate daily oriental dietary pattern(a blend of lard and soybean oil)was performed in this research to investigate influence on liver and kidney function.Sixty mice were randomly divided into 6 groups with diets of different fat added oils respectively for 12 weeks.Malondialdehyde and uric acid contents in mice fed with blended oil were significantly lower than in those fed only with soybean oil and lard due to the improved activities of antioxidant enzymes.Daily use of a blend of lard with soybean oil significantly increased antioxidant capacity,reduced lipid peroxidation of liver and serum uric acid production,thus protected liver and renal function.It also suggests that the oriental dietary pattern might reduce the risk of gout.

Spontaneous Mammary Carcinomas in Female Dogs:Association between the Immunohistochemical Degree of Aggressiveness of Tumors,Intensity of DNA Damage and Residues of Pyrethroids

Diagnosis and biological behavior of breast cancer of female dog represent one of the biggest challenges facing the Veterinarian in recent years. Due to its exponential growth and the degree of aggressiveness, the exact cause of this tumor is probably multifactorial and it is believed that may suffer influence from environmental factors. Among the suspected environmental contaminants are the pyrethroids. Aiming to investigate the participation of pyrethroids in tumorigenesis in female dogs, a study was conducted using 50 female dogs, 22 were positive for simple breast carcinoma (Group I), 18 with a diagnosis of complex breast carcinoma (Group II) and 10 negative (Group III) for breast cancer. In order to detect DNA damage, the Comet assay was performed on mammary samples of these animals, which also had samples submitted to the technique of High Performance Liquid Chromatography (HPLC), which aimed to quantify the concentration of pyrethroids. The results of HPLC of each animal were compared with those obtained by the Comet assay analysis of variance and the means were compared by the test groups “Student T” at the significance level of p ￡ 0.05. Despite presenting correlation between the amount of DNA damage and tumor aggressiveness, no statistical differences were found in the DNA damage of different histologic types of breast carcinoma. As for pyrethroids, even these were detected in 22% of tumor tissues and peritumoral fat, there was no difference in DNA damage between cells exposed and not exposed to environmental contaminant.

Transmission of ceftazidime-avibactam-resistant Escherichia coli among pets,veterinarians and animal hospital environment

Ceftazidime-avibactam (CZA) is a recently approved combination synthetic β-lactamase inhibitor used in human clinical medicine. Cases of CZA resistance in humans have already been reported, but limited research has investigated CZA resistance in pets. This study explored the prevalence and transmission of CZA-resistant Escherichia coli (CZAREC) among pets, their owners, veterinarians, and the environment in animal hospitals. A total of 5,419 clinical samples were collected from dogs and cats, along with samples from the environment (n = 5,843), veterinarians (n = 557), and pet owners (n = 368) in animal hospitals. From these samples, 760 Escherichia coli (E. coli) isolates were obtained, out of which 60 were identified as CZAREC. These included 34 isolates from the environment (9.14 %, n = 372), three from veterinarians (8.11 %, n = 37), and 23 from animals (6.82 %, n = 337). No CZAREC isolates were found in pet owners. The predominant sequence types of CZARECs were ST156 (n = 20), ST410 (n = 19) and ST101 (n = 7). Bayesian analysis revealed six clusters comprising 47 isolates from the hospital environment, pets, and veterinaries, displaying genetic relatedness of less than 100 core genome single nucleotide polymorphisms (cgSNPs) between any two isolates in each cluster. Some CZAREC isolates with high genetic similarity persisted in the same animal hospital for four to six months. Moreover, discriminant analysis of principal components indicated that most isolates from different hosts shared a genetic source in the human/dog/cat merged cluster. Overall, evidence of CZARECs transmission was found among pets, the environment, and veterinarians in animal hospitals. The findings emphasize the importance of monitoring CZARECs in the veterinary clinical setting to ensure the health of both pets and humans.

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Re-emergence of canine Leishmania infantum infection in mountain areas of Beijing

Canine Leishmaniasis(CanL)is an endemic infectious disease in China,causing visceral Leishmaniasis(VL)and result-ing in important public health problem.However,in the last 3 y,endemic trends have changed considerably and spa-tial-temporal aggregation areas have shifted from northwestern to central China.Although Beijing was an endemic area for CanL in the last century,this disease has not been reported in Beijing since control programs were imple-mented in the 1950s.In the present study,PCR and immunochromatographic(ICT)were used to estimate prevalence of Leishmania infection in domestic dogs living in Beijing,a VL re-emergencearea.In total,4420 canine blood samples were collected at vet clinics in 14 districts of Beijing.Overall prevalence(percentage of dogs seropositive and/or PCR positive)of CanL infection in Beijing was 1.22%(54/4420).However,prevalence of CanL in the western mountain areas was 4.68%(45/961),significantly higher than that(0.26%,9/3459)of the plains.In addition,multilocus sequence typing(MLST)of seven enzyme-coding genes was used to examine phylogenetic relationships of CanL strains.Forty-one Leishmania infantum isolates were well separated from the other strains and divided into five major clades(A to E)by MLST analysis.All clades were closely related to strains from Sichuan Province and Gansu Province.A phylogenetic tree,based on the MLST,revealed that L.infantum in Beijing was genetically related to strains from western endemic of Mountain type VL in China.In conclusion,CanL has re-emerged in Beijing,and almost 5%of dogs living in Beijing’s mountain areas were infected with L.infantum.The phylogenetic tree based on MLST effectively distinguished species of Leishmania and reflected geographical origins.Because dogs are considered a natural reservoir,comprehensive control measures including surveillance,phylogenetic analyses and management should be implemented to mitigate or eliminate Leishmaniasis.

Phenotypic Characterization Murine Sarcoma TG-180 Immunophenotypical Characterization Murine Sarcoma TG-180

The sarcoma is the generic nomenclature for neoplasm of mesodermal cells, which express in man and animals. Silent growth requires early diagnosis technique for identifying their proteins. The experimental model in vivo murine sarcoma 180-TG (TG-180), is widely used in research to provide the stimuli of infectious and neoplastic antigens. In this case, the technique of immunohisto-chemistry helps identify the expressions of tumor cell variants. The objective of the research was to characterize immunoexpression murine sarcoma TG 180, by immunohistochemistry, antibodies AE1/AE3, vimentin, CD3, CD 45, CD79α and S100A4. For this, murine sarcoma TG-180, was implanted subcutaneously in 20 mice “Swiss”, male, 30 days old, 28 g for 10 days. Samples were taken and subjected to immunohistochemistry, via use of HistoMouse-MA™? kit. There was specifically labeled S100A4 and vimentin antibodies, indicative of poorly differentiated neoplasms fibroblasts. In fact, the model is established by identifying the origin of the cell, once identified, chemotherapeutic tests can also be performed. Neoplasia like these, when installed in man and animals, depending on the degree of aggressiveness requires treatment protocol varied between surgery and chemotherapy or combination of treatments.

Eosinophilic gastroenteritis in basset hound dog

An 1-year-old male Basset Hound dog was evaluated for chronic intermittent vomiting, hematemesis, and melena which had been ongoing for several months. The histopathologic examination revealed that all layers of the small intestine were thicker than normal. The lamina propria of the mucosa, including the villi, exhibited a prominent cellular infiltrate which consisted of numerous eosinophils and an increased numbers of plasma cells in addition to the normal lymphocytic component. The muscularis mucosa was invaded, and in some places disrupted, by eosinophils, which also infiltrated into the submucosa and muscularis propria. This report describes the pathological findings of a case of eosinophilic gastroenteritis (EG) in a dog.

Low prevalence of colistin-resistant Escherichia coli from companion animals,China,2018-2021

China banned colistin as growth promoter for animals in the year of 2017.A decrease of colistin-resistant Escherichia coli(COREC)and mcr-1-positive Escherichia coli(MCRPEC)were observed in livestock(pigs and chickens)and humans after the ban policy.However,the prevalence of COREC among Chinese companion animals after the ban policy has not been investigated.Here,we recovered 771 E.coli isolates from the China Antimicrobial Resistance Surveil-lance Network for Pets(CARPet)surveillance system(19 provinces/municipalities)from 2018 to 2021.We identified 12 COREC from eight dogs and four cats,among which one feline and three canine isolates were MCRPEC.The prevalence of COREC and MCRPEC in pets from 2018-2021(1.1%-2.2%and 0.8%-1.1%)were lower than those from 2012-2016(7.1%-17.8%and 6.1%-14.3%).The phylogenetic analysis revealed that the four MCRPEC isolates displayed genetic diversity,while one canine isolate exhibited only 26 SNPs difference with one human MCRPEC isolate in the same city,suggesting the exchange of MCRPEC isolates between companion animals and humans.In three MCRPEC isolates,mcr-1 was located on an IncI2 plasmid,which exhibited 99.5%-99.9%nucleotide sequence identity with plasmid pHNSD133-MCR from E.coli of chicken origin.In the remaining MCRPEC,mcr-1 was chromosomally located flanked by intact ISApl1 elements forming a unit of ISApl1-mcr-1-pap2-ISApl1.Despite the low prevalence of COREC and MCRPEC observed in companion animals after the ban policy,the association of pet-derived MCRPEC and mcr-carrying plasmids with those from humans and farm animals suggest that annual surveillance of colistin resistance in bacteria of pet origin is essential.

China antimicrobial resistance surveillance network for pets(CARPet),2018 to 2021

China Antimicrobial Resistance Surveillance Network for Pets(CARPet)was established in 2021 to monitor the resist-ance profiles of clinical bacterial pathogens from companion animals.From 2018 to 2021,we recovered and tested 4,541 isolates from dogs and cats across 25 Chinese provinces,with Escherichia coli(18.5%)and Staphylococcus pseudintermedius(17.8%)being the most predominant bacterial species.The Enterobacterales were highly susceptible to tigecycline,meropenem,colistin,and amikacin(70.3%-100.0%),but showed moderate resistance to ampicillin,ceftriaxone,doxycycline,florfenicol,levofloxacin,enrofloxacin,and trimethoprim-sulfamethoxazole(29.3%-56.7%).About 66.3%of Acinetobacter spp.were resistant to florfenicol,with relatively low resistance to another 11 antibiot-ics(1.2%-23.3%).The Pseudomonas spp.showed high susceptibility to colistin(91.7%)and meropenem(88.3%).The coagulase-positive Staphylococcus spp.showed higher resistance rates to most antimicrobial agents than coagulase-negative Staphylococcus isolates.However,over 90.0%of Staphylococcus spp.were susceptible to linezolid,dapto-mycin and rifampin,and no vancomycin-resistant isolates were detected.E.faecium isolates demonstrated higher resistance rates to most antimicrobial agents than E.faecalis isolates.Streptococcus spp.isolates showed low resistance to most antimicrobial agents except for doxycycline(78.2%)and azithromycin(68.8%).Overall,the tested clinical isolates showed high rates of resistance to commonly used antimicrobial agents in companion animals.Therefore,it is crucial to strengthen the monitoring of bacterial resistance in pets.By timely and effectively collecting,analyzing,and reporting antimicrobial resistance dynamics in pets,the CARPet network will become a powerful platform to provide scientific guidance for both pet medical care and public health.

Catalpol ameliorates LPS-induced endometritis by inhibiting inflammation and TLR4/NF-κB signaling

Catalpol is the main active ingredient of an extract from Radix rehmanniae,which in a previous study showed a protective effect against various types of tissue injury.However,a protective effect of catalpol on uterine inflammation has not been reported.In this study,to investigate the protective mechanism of catalpol on lipopolysaccharide(LPS)-induced bovine endometrial epithelial cells(bEECs)and mouse endometritis,in vitro and in vivo inflammation models were established.The Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR),western blot(WB),and immunofluorescence techniques.The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factorα(TNF-α),interleukin(IL)-1β,and IL-6,and chemokines such as C-X-C motif chemokine ligand 8(CXCL8)and CXCL5,both in bEECs and in uterine tissue.From the experimental results of WB,qRT-PCR,and immunofluorescence,the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group.The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase(MPO)activity.The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.

Erratum to: Catalpol ameliorates LPS-induced endometritis by inhibiting inflammation and TLR4/NF-κB signaling

Erratum to:J Zhejiang Univ-Sci B(Biomed&Biotechnol)201920(10):816-827 https://doi.org/10.1631/jzus.B1900071The original version of this article unfortunately contained a mistake.In p.823,Figs.8 c and 8 d were incorrect,and the obvious pathological changes were mistakenly placed in the picture.