| Helicobacter hepaticus(H.hepaticus)is a gram-negative,micro-aerobic,spiral bending bacteria,and worldwide distribution.It can infect a variety of mammals,mainly colonize under the lower digestive tract of the host by a subclinic infection way.It can cause chronic hepatitis,the cecum colitis,hepatocholangeitis and even liver cancer,hepatobiliary adenoma and colon cancer.It can not only seriously endanger animal health,affect the scientific research progress,interfere with the experimental results,but also may endanger public health potentially.So it is one of the important pathogenic bacteria of rodent laboratory animals.Development of a rapid,accurate and suitable detection method for H.hepaticus will be conducive to the microbial quality control and promote the standardization of laboratory animals.The specific 16S rRNA sequence of H.hepaticus could be detected in human hepatitis,liver cancer and other relevant clinical specimens.The clinical outcomes and histopathological characteristics caused by H.hepaticus infection in mice were very similar to those of human chronic liver disease and enteritis.It is prompted that H.hepaticus is closely related to human digestive tract diseases.Therefore,the establishment of a mouse model of H.hepaticus infection will help to further explore the pathogenesis mechanism.It can provide a powerful tool for the study of human-related diseases and a new model for the screening of specific drugs to treat related human diseases.1 Development of a loop-mediated isothermal amplification assay for detection of H.hepaticusIn this study,a rapid loop-mediated isothermal amplification(LAMP)method for detection of H.hepaticus was established,and an epidemiological investigation on rats and mice in laboratory animal production license unit of Shanghai district was carried out with the LAMP method.The developed LAMP method was used to investigate the epidemiology of rats and mice of H.hepaticus natural infection.BALB/c mouse model of H.hepaticus infection was established by challenge.The host responses caused by H.hepaticus infection were evaluated based on immunology,histopathology and intestinal flora structure.Flagellin is highly conserved and is an important virulence factor of H.hepaticus.A set of specific primers including a pair of external primers F3 and B3,an internal primer FIP,BIP and a ring primer LB were designed based on the fla B gene of H.hepaticus and used for development of theLAMP.The results showed that the LAMP assay was only specific for H.hepaticus and negative for other bacteria tested.The detection limit was 86.9 fg/?iL of genomic DNA of H.hepaticus,which was 10 times lower than that in routine PCR assay.Compared with the routine detection methods,the LAMP assay did not depend on the cultivation conditions and the detection instruments.The LAMP method developed here might be used for a rapid detection and surveillance of H.hepaticus and it was suitable for on-site detection2 LAMP detection of H.hepaticus in laboratory rats and mice in Shanghai districtA total of 1492 samples(originated from 54 clean level rats,200 specific pathogen free[SPF]level rats,267 clean level mice and 971 SPF level mice,all the animals were feeding in the barrier system.)were collected and detected for H.hepaticus by LAMP.The results showed that there were different degrees of H.hepaticus contamination in animal facilities in 2014,2016 and 2017,respectively.The contamination rates of the facilities harboring clean level animals were 75.00%,75.00%and 40.00%respectively.The contamination rates of the facilities harboring SPF level animals were 66.67%?23.08%?50.00%respectively.The H.hepaticus total positive rate of animals was 8.65%.From 2014 to 2017,the positive rates were 11.85%,3.51%and 10.71%respectively.In detail,the total positive rate of rats was 5.91%,respectively 2.78%,3.85%,13.24%from 2014 to 2017.The positive rates of clean and SPF rats were 9.26%and 5.00%.The positive rates of mice were 9.21%,respectively 15.69%?3.44%?10.42%from 2014 to 2017.The H.hepaticus total positive rates of clean and SPF mice were 8.24%and 9.47%.For different strains of animals,relatively high positive rates were found in BALB/c,ICR and KM strains.In general,there was different prevalence of infection between rats and mice,while no differences in different years and different levels of tested animals.The investigation added the epidemiological data of H.hepaticus in rodents and provided reference for the quality control of bacteria in laboratory animals.3 Establishment of BALB/c mice model infected with H.hepaticusTo construct a model of H.hepaticus infection,mice were inoculated with 0.2 ml of reference strain culture by intragastric or intraperitoneal injection.Body weight,leukocyte count,aspartate aminotransferase(AST),alanine aminotransferase(ALT),cytokine expression level were determined at regular intervals post-challenge.The colonization and distribution of H.hepaticus in vivo and histopathology were also evaluated at the same time-points.The results showed that H.hepaticus infection caused significant weight loss and significant increase in white blood cell count in inoculated mice(P<0.01).Serum AST(P<0.001)and ALT(P<0.01)levels were significantly elevated and exhibited a continuous upward trend during the whole experiment.The colonization and tissue distribution results showed that H.hepaticus could be detected in the cecum of all challenged mice in the second week post-inoculation(WPI)H.hepaticus could be detected for the first time in livers of inoculated mice in sixth WPI,and showed 100%positive rate in eighth WPI.H.hepaticus could be detected in gastric tissues of mice from intragastric inoculation group and intraperitoneal challenge group in eighth and 16th WPI,respectively.H.hepaticus could not be detected in the rest tissues of inoculated mice.Thus,H.hepaticus would tend to colonize the intestinal tract of mice initially,and then spread to liver,stomach,etc.The pathological examination showed that there were vacuolar degeneration,steatosis,focal necrosis and inflammatory cell infiltration in hepatocytes of inoculated mice.In stomachs,mucosal epithelial cells experienced obvious papillary hyperplasia and thickening Edema,hyperemia,mucosal degeneration,necrosis,erosion and inflammatory cell infiltration could be observed in different intestinal tissues of inoculated mice.In general,the degree of lesions was positive related to the lenghth of infection process.Cytokine detection results showed that the expression levels of IL-6,TNF-? and IL-1? were significantly up-regulated in mice from the challenge group(P<0.001).The results showed that H.hepaticus could promote the expression of inflammatory factors in the liver cells of BALB/c mice.The establishment of mice model of H.hepaticus infection would provide a powerful tool for further study of pathogenesis mechanism and human-related diseases4 Analysis of intestinal flora structure of mice infected with H.hepaticusIn this study,using Illumina high throughput sequencing of 16S ribosomal DNA V4-V5 region,we identified the microbiota composition of the mice infected by H.hepaticus.The operational taxonomic unit(OTU)clustering,diversity analysis,microbial communities and metabolism prediction were performed.The results showed that the intestinal flora diversity of the inoculated mice was significantly lower than that of the mock mice(P<0.01).The OTU number in the inoculated mice and the mock animals was 89 and 209 respectively,sharing a total of 73 OTUs in both groups.At the phylum level,the sequences of both the inoculated mice and the mock mice were divided into 7 phyla.Bacteroidetes and Firmicutes were the two most abundant bacteria in both groups.There were 5 overlapping phyla in both groups,and 2 unique phyla were found in each group respectively.Verrucomicrobia and Epsilonbacteraeota were found only in the challenged mice,while Tenericutes and Cyanobacteria were found only in the mock mice.At the genus level,a total of 74 genuses were detected in both groups.45 genuses were found in the inoculated mice and 64 genuses were detected in the mock mice.35 genuses were common in both groups,10 genuses were unique to the inoculated mice and 29 genuses were unique to the mock mice.The abundance of bacteroidetes,Muribaculaceae,spirillum,Blautia,Rumini clostridium was relatively high.The result of linear discriminant analysis Effect Size(LEfSe)results showed that only Epsilonbacteraeota exhibited significant difference in the phylum level between two groups,and 31 genuses experienced difference in the genus level between two groups.The abundance of bacteroidetes,clostridium,flavobacteria,ruminococci,klebsiella,enterococcus and helicobacter in the inoculated mice were significantly higher than those in the mock mice.However,the abundance of lachnospiraceae NK4A136 group,oscillibacter,prevotella,enterobacter and rikenellaceae RC9 gut group in the former were significantly lower than those in the latter.Above results indicated that H.hepaticus infection might significantly decrease the enrichment and diversity of intestinal flora in mice.It would lay a foundation for revealing the relationship between H.hepaticus infection and the host intestinal flora responses. |
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