Isolation And Identification Of Gardnerella Vaginalis From Gestational Foxes And Establishment Of Mouse Pathogenic Models

Gardnerella vaginalis（GV）is one of the most important bacteria that can harm foxes,causing them to miscarry and become pregnant.GV was first isolated from vaginitis patients by Leopold in 1953.Animals such as minks,raccoons,horses and pigs are also susceptible to GV.GV seriously hinders the healthy development of fox breeding industry,causing huge economic losses,and also threatens the health of fur animal breeding practitioners and even the wider population.In order to study the prevalence of GV in foxes,this study isolated and identified fox disease materials collected in Zhucheng（Shandong,China）from March to May,2019,and established a mouse pathogenic model of GV to conduct a preliminary study on its pathogenicity.GV isolation was performed on 20 aborted fetuses and 25 female fox vaginal swabs.A total of 14 strains of bacteria were isolated and purified using the conventional method of bacterial isolation and identification,and the samples were crossed and inoculated in the AGAR medium of Colombian blood（containing 5%sterile deflbrinated horse blood）.Individual colonies were selected for microscopic examination after Gram’s staining,and the results showed that the bacterium with variable Gram’s staining was consistent with the morphology of GV.Nine kinds of conventional biochemical identification tubes were used to identify the biochemical characteristics of the isolated bacteria.The results showed that contact enzymes were negative,maltose,mannose and glucose were positive,but xylose,rhamnose,raffinose,sorbitol and starch were not fermented.The result of O-F biochemical reaction was fermentation type,which was consistent with the biochemical characteristics of GV,and GV was preliminarily determined to be isolated.The 16SrRNA gene of the isolated bacteria was amplified by PCR and analyzed by sequencing.The results showed that the16SrRNA gene sequence of the isolated bacteria had more than 99%homology with the GV reference sequence,which further proved that the 14 strains isolated were GV,with a separation rate of 56%（14/25）,indicating that GV was widely present in fox farms in this region.PCR was used to detect 31 potential virulence genes of 14 strains of GV.Results the detection rates of cytolytic Vaginolysin gene,DedA-family protein,rRNA methyltransferase and other genes were 100%,Sialidase A precursor,rib-family surface protein and endothelin-converting enzyme were 64.3%,and the detection rates of LicD protein were 0%.The drug sensitivity of 14 strains of GV to 17 commonly used drugs was detected by disk diffusion method（K-B method）.As a result,GV was highly sensitive to Penicillin,Erythromycin,Tetracycline,Tobramycin,Cefazolin,Ampicillin,Lincomycin,Gentamycin,Chloramphenicol,Streptomycin and intermediate to Amikacin,Kanamycin,Neomycin and Vancomycin,while resistant to Oxacillin,Sulfamethoxazole and Polymyxin B.Based on the drug sensitivity test results and virulence gene carrying status of 14 strains of GV,SD GV1 was selected as the representative strain and mice were used as experimental animals to establish the mouse pathogenic models of GV.36 female 6-weeks-old Kunming mice and 12 male 6-weeks-old Kunming mice were randomly selected and divided into 6groups.The reproductive performance of the mice was tested through the natural mating experiment,and the female mice were observed to have the formation of vaginal plug after put with male mice together,which was regarded as the mating complete.A repeated group was set for each treatment to reduce the experimental error.The female mice in 1² groups were inoculated with GV via vaginal epithelium at a dose of 2.0×10^5.0CFU per mouse,and the female mice were infected with GV.On the 3rd day after the female mice were challenged,2male mice were placed in each cage of female mice,and the female mice were observed once every 8 hours.Before the challenge,2 male mice were placed in the cage to 3⁴ groups,and the formation of vaginal plug was observed once every 8 hours.The female mice with vaginal plug were taken out and fed separately.After 2 days,the female mice with vaginal plug were inoculated via the vaginal epithelium with GV,and the dose was 2.0×10^5.0CFU per mice.5⁶groups were the control group,and PBS was injected.On the 21st,23rd,25th and 27th days after the challenge,1 mouse was taken for autopsy,the vaginal and uterine samples were collected for pathological tissue sections and microscopy.Results within 3⁵ days after the challenge,the mice in the challenge group showed symptoms such as listlessness,inappetence,vaginal exstrophy with feculent secretion,which were consistent with the typical symptoms of bacterial vaginosis.Large amounts of GV were detected in the vaginal secretions of mice.Among the mice inoculated with GV,22 female mice had empty pregnancy,abortion and stillbirth,and one female mouse gave birth to 2 weak pups.Only one female mouse showed normal clinical performance,while the control group showed no abnormal clinical performance.Histopathologic results,mice infected with GV vaginal epithelial lamina propria edema,loose connective tissue,lamina propria numerous neutrophils infiltrating,uterine lamina propria edema,capillary expansion,lamina propria contains a large number of lymphocytes and neutrophils,inflammatory cells infiltration,endometrial epithelial cell falls off,confirmed that the bacteria can be infected mice reproductive system lesions caused by causing them to reproductive barriers.In conclusion,this study shows that GV is widespread in fox farms and has strong pathogenicity,which provides theoretical basis for scientific prevention and control of GV.