Major Pathogenic Bacteria Detection In Goat Diarrhea In Sichuan Province And Biological-characteristics Research Of Shigella Isolated From Goat

Goat diarrhea is a general lamb disease for s with diarrhea as the main clinical symptom.Lambs within 7 days of age show the most serious symptoms and often die.A variety of factors can cause lamb diarrhea,of which the most common diarrhea is bacterial diarrhea,and the main pathogens are pathogenic enterotoxigenic Escherichia coli,Salmonella,Shigella and Proteus mirabilis.A single pathogen can cause lamb diarrhea,however,lamb diarrhea is commonly caused by multiple pathogens.Therefore,the most important thing in lamb diarrhea control is to make accurate diagnosis as early as possible for taking effective prevention and treatment measures.In this study,a multiplex PCR method was established and used for simultaneous detection of four kinds of pathogenic bacteria in goat diarrhea,at the same time,those four bacteria were isolated for further investigation of their drug sensitivities,and some biological characteristics of goat-derived Shigella were also researched.The main findings are as follows:?1?Establishment of multiplex-PCR for detection of four kinds of goat diarrheal pathogenic bacteriaThis study established a multiplex-PCR method for simultaneous detection of four bacteria pathogens causing goat diarrhea.Four pairs of primers were screened based on the k99 gene in ETEC,invA gene in Salmonella spp,ipaH gene in Shigella spp and ureR gene in Proteus mirabilis.The primers and their concentrations,anneling temperature were optimized to determine the best reaction conditions,and the multiplex PCR was used to detect goat diarrhea samples.The results showed that the multiplex PCR amplified the specific bands of the four objective bacteria,and those of other 6 non-objective pathogens were detected negatively.Under the optimized multiplex PCR condition,the detection sensitivity of the multiplex PCR for the four bacteria was 10³ CFU per mL for Enterotixigenic Escherichia coli,10² CFU per mL for salmonella spp,10² CFU per mL for Proteus mirabilis and 10³ CFU per mL for shigella spp,respectively.The repeatability of the method was good.The multiplex PCR method was applied to detect 21 goat diarrhea samples,and the results were consistent with those of bacteria isolation and identification.In this study,the established multiplex PCR method can detect the four kinds of bacterial diarrhea pathogens from goat anal swab,which provides technical support for rapid detection of goat diarrhea pathogens.?2?Multiplex PCR detection,isolation and drug resistance analysis of the four bacteria in goat diarrhea samplesIn this study,total DNA extracted from each of 158 goat diarrhea samples and 68goat healthy samples were used as templates in the multiplex PCR established above.The PCR positive samples were used to isolate and identify the four bacteria,and the antibiotic resistance of the isolated bacteria against 18 antibiotics were tested.The multiplex PCR results showed that the PCR positive rate for Enterotoxigenic Escherichia coli was 69.6%?110/158?,21.5%?34/158?for Salmonella,41.8%?66/158?for Shigella and 17.8%?28/158?for Proteus mirabilis in 158 diarrhea samples respectively.The PCR positive rate for Enterotoxigenic Escherichia coli was22.0%?15/68?in 68 diarrhea healthy samples,11.7%?8/68?for Salmonella,0%?0/68?for Shigella and 11.7%?8/68?for Proteus mirabilis in 68 healthy samples respectively.Enterotoxigenic Escherichia coli were isolated from 93 samples in 158diarrhea feces with the isolation rate 58.8%?93/158?,the isolation rate of Salmonella was 15.2%?24/158?,that of Shigella twas 34.2%?54/158?,and that of Proteus mirabilis was 10.0%?15/158?.Enterotoxigenic Escherichia coli were isolated from 11samples in 68 healthy samples with the isolation rate 16.1%?11/68?,Salmonella11.7%?8/68?,Proteus mirabilis 8.8%?6/68?,and no Shigella was isolated in healthy samples.It can be seen from the above results that either PCR detection rate or the bacteria isolation rate of diarrhea samples were higher than those of healthy samples,andThe detection rate and separation rate of Enterotoxic Escherichia coli and Shigella in diarrhea samples were significantly higher than those of healthy samples.It can be concluded that Enterotoxigenic E.coli and Shigella are the most important pathogens causing bacterial diarrhea in goats in parts of Sichuan.Antibiotic sensitivity analysis showed that all of the four kinds of pathogenic bacteria were sensitive to Cefotaxime,spectinomycin,amikacin,cephradine,while the four isolated bacteria were resistant to the other 14 antibiotics in certain degrees and showed multiplex drug resistance.?3?Biological characteristic research of Shigella from goatTotal 54 Shigella strains were isolated from 54 fecal samples of goats and identified by specific PCR of Shigella,which were used to identify their serotypes and detect their virulence genes by PCR,as well as research the pathogenicity of ascendant Shigella strains to BALB/c mice,which will provide the information for the prevention and control of the disease caused by Shigella.The 10 virulence genes,including sat,sen,setA,pic,sigA,icsP,ial,iusA,iutA and ipaH of Shigella were detected by PCR,and the pathogenity to BALB/c mice of the isolated Shigella which were the dominant serovar and owned the total 10 virulence genes was further researched.According to the results of serovars and virulence genes,4 isolated Shigella which were the dominant serovar Shigella Flexneri 2b strains were selected and studied their pathogenicity to BALB/c mice by oral infection.The serotyping results exhibited that the 54 Shigella isolates were identified as Shigella Flexneri,incuding 4 Shigella Flexneri 1a isolates?4/54?,11 Shigella Flexneri 1b isolates?11/54?,28 Shigella Flexneri 2b isolates?28/54?,and 11 Shigella Flexneri 3b isolates?11/54?.All 54 Shigella isolates carried the three virulence genes of sat?sen?ipaH.The carrying rates of other virulence genes of setA?pic?sigA?icsP?ial?iusA and iutA were 73.6%?65.2%?77.8%?82.0%?79.1%and 79.1%,respectively.Each strain carried at least 4 virulence genes,and four shigella flexneri 2b isolates carrying10 virulence genes,which could cause death of the no-antibiotics treated BALB/c mice at the doses of 1.7×10⁸ CFU.Furthermore,the LD₅₀0 of the Shigella flexneri 2b strain SWUN553 was 1.04×10⁶ CFU to BALB/c mice.In histopathological examination,there appeared the degeneration,necrosis,inflammatory cell infiltration and other serious damage in the heart,liver,spleen,lung and colon of the experimental mice.The ileum submucosa has a slight inflammatory cell infiltration,which suggested that the colon might be the invasion portal of the Shigella flexneri 2b strain SWUN553.This study illustrated parts of the biological characteristics of the isolated Shigella from goat in Sichuan province,which provided the scientific basis for the prevention and control of the disease caused by Shigella.