Establishment And Application Of Monitoring Methods For Five Kinds Of Pathogens In SPF Rats And Mice

Objective: Laboratory animals are the basis for life science research and an important supporting condition for the production and study of new drugs. According to the disparities of carrying microorganisms and parasites, laboratory animals can be classified into four grades: normal animal, clean animal, specific pathogen free animal (SPF animal) and sterile animal. SPF animals are internationally recognized standards of laboratory animals and suitable for all the scientific researches. The important projects of scientific researches and GLP lab in our province can not reach to the international standards unless SPF animals are applied. The quality control of microorganisms and parasites is the key to evaluate whether the laboratory animals reach SPF level. Accordingly, a complete set of detection system should be established to ensure that laboratory animals have no pathogens which should be excluded. SPF animals should neither carry all the pathogens that exist in normal animals and clean animals, nor another six bacteria, eleven viruses and two parasites.Over the years, the detection of pathogens in laboratory animals relied mainly on the traditional isolation and identification methods which were time-consuming or low specific. Compared with these methods based on the morphological analysis, the genotype of pathogens is more specific and accurate, and will not be affected by external factors (such as temperature change, chemical drugs, etc). Consequently, molecular biological technologies, the analytical methods towards the genotype of pathogens, have been increasingly applied in the identification and classification of pathogens and phylogenetic studies of laboratory animals because of their so many advantages, such as highly ?sensitive and specific, rapid, simple, the ability of detecting bacteria that have been dead or difficult to culture and so on. This topic aimed at the establishment of detection methods for five bacteria (Staphylococcus aureus, Streptococcus pneumonia, beta-hemolytic streptococci, Klebsiella pneumonia, aeruginosus Bacillus) which should be excluded in SPF animals and the application of these methods to the detections of rats and mice in Laboratory Animal Center of Hebei Province, thus in order to evaluate the feasibility and maneuverability.Methods:1 Traditional methodsAppropriate reference strains were inoculated on the corresponding selective mediums, the colonial characteristics were observed and a single colony was purely cultured on 2nd day. To the single colony that was purely cultured: KIA test was used for lactose and glucose utilization, acid and gas production; bacterial micro-biochemical test for biochemical reactions; semi-solid momentum test for power of bacteria; Gram stain for morphology; plasma coagulase test for coagulase production of Staphylococcus aureus; oxidase test for oxidase production of Pseudomonas aeruginosa; 42℃test for growth of Pseudomonas aeruginosa in high-temperature environment.2 Extraction of bacterial genomic DNA and determination of DNA's concentrationTIANamp bacteria DNA kit was used to extract the genomic DNA of the above five bacteria and E. coli. The whole extraction steps were following the kit's instructions. DNA's concentrations of Staphylococcus aureus and Streptococcus pneumonia were determined.3 PCR amplification and determinations of specificity and sensitivitySpecific primers designed according to nuc gene of Staphylococcus aureus and pbp2B gene of Streptococcus pneumonia were used for PCR amplification in order to identify these two reference strains.Determination of specificity: specific primers of Staphylococcus aureus and Streptococcus pneumonia were respectively used for PCR amplification of six bacteria.Determination of sensitivity: 10-fold serial dilutions of Staphylococcus aureus and Streptococcus pneumonia were respectively used for PCR amplification of six bacteria.4 Detection of animals in Laboratory Animal Center of Hebei ProvinceThe tracheal secretions and ileocecal contents of laboratory animals were inoculated on the corresponding selective mediums, a single colony purely cultured was detected by the traditional identification methods, while the genomic DNA of bacteria was extracted and specific primers were used for PCR amplification. Finally a conclusion was made that whether some pathogen was carried by the laboratory animals according to all the test results.Results:1 Klebsiella pneumoniae: pale pink colonies, produce acid and gas on the KIA medium, citrate utilization, urease, lysine decarboxylase, glucose and lactose utilization were positive, non-powered.2 Staphylococcus aureus: Golden colonies were generated on SP agar plate; colonies on blood agar plate were surrounded byβhemolytic rings; G+ cocci, grape-like; both mannitol fermentation test and plasma coagulase test showed positive results. Only the PCR product of S. aureus showed a 270bp size, clear and identifiable DNA band. When various genomic DNAs of bacteria were mixed as a template for PCR, a positive signal could be detected only in the group containing the genomic DNA of S. aureus. 0.3ng was the lower limit of template needed to obtain a detectable PCR product.3 Streptococcus pneumonia:αhemolysis, G+ double-cocci. Only the PCR product of Streptococcus pneumonia showed a 682bp size, clear and identifiable DNA band. When various genomic DNAs of bacteria were mixed as a template for PCR, a positive signal could be detected only in the group containing the genomic DNA of Streptococcus pneumonia. 30pg was the lower limit of template needed to obtain a detectable PCR product.4 Beta-hemolytic streptococcus:βhemolysis, G+, salicin, sucrose, mushroom sugar and lactose utilization were positive.5 Pseudomonas aeruginosa: produce green pigment, G-, xylose, citrate utilization, gelatin liquefaction, oxidase test and 42℃growth test were positive, powered.6 After test, a KM mouse in Laboratory Animal Center of Hebei Province carried Staphylococcus aureus, which was confirmed by PCR assay. Any of the five pathogens was not found in other tested animals.Conclusions:1 Every test result derived from the traditional identification methods generally conforms to .2 The results by PCR are consistent with that by traditional methods in detecting Staphylococcus aureus and Streptococcus pneumonia. Comparing with traditional methods, PCR may be an effective supplement owing to its rapidness, simplicity, high specificity and sensitivity.3 KM mice detected by traditional methods and PCR can not reach the SPF level, and the results from the two methods are identical.