Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period

Viral, bacterial and parasitological infections in rats and mice used in biomedical research continue to occur despite improved housing and biosurveillance. The presence of disease in laboratory animals can lead to spurious results for research undertaken in universities, research institutes and the pharmaceutical industry. Here the authors report the results of serological, microbiological, parasitological and molecular tests done on mice and rats from Australasia submitted to a rodent health monitoring laboratory (Cerberus Sciences) from 2004 to 2009. In tested mice, norovirus was the most prevalent virus and ectromelia virus was the least prevalent virus. In tested rats, pneumonia virus of mice was the most prevalent virus and adenoviruses 1 and 2 were the least prevalent viruses. In mice, Helicobacter hepaticus was the most prevalent bacterium, and in rats, Proteus spp. were the most prevalent bacteria. The most common positive helminthological finding in mice and rats was the presence of all pinworms (including Aspicularis spp. and Syphacia spp.). The most common positive protozoan findings in mice and rats were Chilomastix spp. and Trichomonads.     

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.     

Microbiological contamination of laboratory mice and rats in Korea from 1999 to 2003.

To survey the microbiological contamination of laboratory mice and rats in Korea during a 5-year period, we monitored animals housed in mouse and rat facilities with either barrier or conventional systems. At barrier and conventional mouse facilities, the most important pathogen identified was mouse hepatitis virus (MHV), while Mycoplasma pulmonis was the most important pathogen at conventional rat facilities. Interestingly, hantavirus was recovered from both barrier and conventional mouse facilities. The most common protozoon identified was Tritrichomonas muris in mouse facilities and Entamoeba muris in rat facilities. In addition, we found that the microbiological contamination of mice and rats in conventional facilities was severe. These results suggest that conventional facilities should be renovated and monitored regularly to decrease microbiological contamination. We also propose that hantavirus should be monitored in Korea as an important mouse pathogen.     

两种方法对上海及周边地区实验大小鼠螺杆菌携带情况的调查

目的了解上海及周边地区实验小鼠、大鼠螺杆菌携带情况,为我国实验动物等级及监测标准的制定提供参考和依据。方法PCR法共检测了352只小鼠（清洁级101只,SPF级251只）,101只大鼠（清洁级69只,SPF级32只）;ELISA法共检测了88只小鼠（清洁级26只,SPF级62只）,165只大鼠（清洁级84只,SPF级81只）;并对其中88只小鼠、101只大鼠的PCR和ELISA法阳性检测率进行比较。结果PCR法检测小鼠平均阳性率为35．8％（126／352）,清洁级阳性率为51．5％（52／101）,SPF级阳性率为29．5％（74／251）;大鼠平均阳性率为70．3％（71／101）,清洁级阳性率为69．6％（48／69）,SPF级阳性率为71．9％（23／32）;ELISA法检测小鼠平均阳性率为15．9％（14／88）,清洁级阳性率为19．2％（5／26）,SPF级阳性率为14．5％（9／62）;大鼠平均阳性率为52．7％（87／165）,清洁级53．6％（45／84）,SPF级51．9％（42／81）;88只小鼠PCR法阳性检测率为72．7％（64／88）,ELISA法阳性检测率为15．9％（14／88）;101只大鼠PCR法阳性检测率为70．3％（71／101）,ELISA法阳性检测率为49．5％（50／101）。结论上海及周边地区实验大鼠、小鼠中皆存在着不同程度的螺杆菌感染,两种方法阳性检出率比较结果表明回盲部内容物PCR法较检测血清中抗螺杆菌抗体ELISA法更为敏感。     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

实验树鼩肠道螺杆菌属细菌特征分析

近年来,树鼩(Tupaia belangeri)作为一种新型的实验动物被广泛应用于生物医学研究的各个领域。本研究组前期的研究结果显示,螺杆菌属(Helicobacter)是树鼩肠道微生物群落中相对丰度最高的一类细菌,但其具体的细菌种类和结构特征仍然不清楚。因此,本研究将开展实验树鼩肠道中螺杆菌属细菌的分布种类和特征分析,为后续实验研究工作提供资料。通过系统采集72只树鼩的粪便样本,提取核酸后采用巢氏PCR法应用属特异性引物扩增螺杆菌属特异性片段,再分别采用7个种特异性引物对属特异性阳性样本扩增螺杆菌种特异性片段,包括肝螺杆菌(H.hepaticus)、家鼠螺杆菌(H.muridarum)、胆汁螺杆菌(H. bilis)、啮齿类螺杆菌(H. rodentium)、弯曲螺杆菌(Flexispira rappini)、鼩螺杆菌(H. suncus)和盲肠螺杆菌(H. typhlonius)。属特异性引物扩增阳性但种引物扩增阴性的样本进行核酸序列测定和BLAST比对分析,确认其最终所属的螺杆菌种类。结果显示,72份树鼩粪便样本中,属特异性引物扩增阳性有18份,总体阳性率为25.0%。其中,盲肠螺杆菌阳性8株、胆汁螺杆菌阳性6株,其余8份阳性样本经过测序和BLAST比对分析后确认为同性恋螺杆菌(H. cinaedi)阳性5株、猫螺杆菌(H. felis)阳性2株和猕猴螺杆菌(H. macacae)阳性1株。有4份树鼩粪便样本出现同时携带盲肠螺杆菌和胆汁螺杆菌的情况。将螺杆菌属细菌携带结果与实验树鼩的性别和年龄组进行比较分析后发现,不同性别间以及不同年龄组间,属或种阳性样本情况均无差异(P 0.05)。本研究结果表明,实验树鼩具有较高的螺杆菌携带率,且不分性别和年龄,主要以盲肠螺杆菌、胆汁螺杆菌和同性恋螺杆菌为主。     

Comparison of Helicobacter spp. genetic sequences in wild and captive seals, and gulls

Helicobacter species are widely distributed in the gastrointestinal system of humans and many animal taxa. Investigations of natural infections are essential to elucidating their role within the host. The feces of fur seals Arctocephalus pusillus doriferus and sea lions Neophoca cinerea from 3 separate captive populations, as well as a wild colony from Kangaroo Island, Australia, were examined for the occurrence of Helicobacter spp. The feces from several wild silver gulls Larus novahollandiae were also investigated. As detected by PCR, 18 of 21 samples from captive and 12 of 16 samples from wild seals were positive for Helicobacter spp. Three species were identified in these animals. Whilst one possibly novel type was identified from wild fur seals, the majority of wild and captive individuals had the same species. This species also occurred in more than 1 seal type and in silver gulls, and shared a 98.1 to 100% identity to other Helicobacter spp. from harp seals and sea otters. A similar sequence type to species identified from cetaceans was also detected in several captive seals. This study reports for the first time the presence of Helicobacter spp. in wild and captive seals and demonstrates the diversity and broad-host range of these organisms in the marine host.     

微生物法检测实验动物血清中的抗生素残留

目的建立实验动物血清中抗生素残留检测方法,保证微生物质量检测结果的准确。方法采用微生物抑制法,用嗜热脂肪芽孢杆菌作为指示菌,对1640份实验动物血清进行抗生素残留检测。并与Premi@Test抗生素检测试剂盒进行比对。结果在受试样品中,检测出小鼠、大鼠、豚鼠、兔和犬的血清中存在抗生素残留,阳性率分别为27.2%、16.4%、39.9%、23.7%和47.6%。抽取82份血清和18份阴阳对照品,与Premi@Test检测的结果相比较差异不显著（X2=16.680,P〉0.05）。血清中抗生素的残留与DHL琼脂平皿的菌落生长有一定相关性（X2=9.939,P〈0.05）。结论本研究采用嗜热脂肪芽孢杆菌作为指示菌的微生物抑制法,能够对动物血清中的抗生素进行检测,具有良好的敏感性和重复性,成本低,操作简便。为实验动物体内抗生素残留检测和微生物学检测结果的准确性提供依据。     

Microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study

Over recent years, the use of individually ventilated cage (IVC) rack systems in laboratory rodent facilities has increased. Since every cage in an IVC rack may be assumed to be a separate microbiological unit, comprehensive microbiological monitoring of animals kept in IVCs has become a challenging task, which may be addressed by the appropriate use of sentinel mice. Traditionally, these sentinels have been exposed to soiled bedding but more recently, the concept of exposure to exhaust air has been considered. The work reported here was aimed firstly at testing the efficiency of a sentinel-based microbiological monitoring programme under field conditions in a quarantine unit and in a multi-user unit with frequent imports of mouse colonies from various sources. Secondly, it was aimed at determining biocontainment of naturally infected mice kept in an IVC rack, which included breeding of the mice. Sentinels were exposed both to soiled bedding and to exhaust air. The mice which were used in the study carried prevalent infectious agents encountered in research animal facilities including mouse hepatitis virus (MHV), mouse parvovirus (MPV), intestinal flagellates and pinworms. Our data indicate that the sentinel-based health monitoring programme allowed rapid detection of MHV, intestinal flagellates and pinworms investigated by a combination of soiled bedding and exhaust air exposure. MHV was also detected by exposure to exhaust air only. The IVC rack used in this study provided biocontainment when infected mice were kept together with non-infected mice in separate cages in the same IVC rack.     

Rederivation of Helicobacter hepaticus-infected Mongolian gerbils by Caesarean section and cross-fostering to rats and mice

The Mongolian gerbil serves as an animal model for a wide range of diseases. As these animals are extensively used for the study of Helicobacter pylori-induced gastritis, naturally occurring infections with rodent Helicobacter species in gerbils are a possible source of interference in studies of H. pylori-associated disease. The gerbil stock at the Central Animal Facility in Hannover was persistently infected with H. hepaticus. The aim of this study was to derive Helicobacter species-free Mongolian gerbils. Therefore, germfree gerbil pups were obtained by Caesarean section and the pups were transferred to female rats and mice with recently delivered litters. In total, four Ztm:NMRI mice, four Ztm:SPRD rats and one DA/Ztm rat that originated from a specified pathogen-free area were selected to serve as foster mothers. With this approach, it was possible to obtain Helicobacter-free gerbils. Rearing by mice was more successful than by rats, as six of nine gerbils were reared by mice, but only one of 29 gerbils was reared by rats.     

Efficacy of three microbiological monitoring methods in a ventilated cage rack

The use of individually ventilated caging (IVC) to house mice presents new challenges for effective microbiological monitoring. Methods that exploit the characteristics of IVC have been developed, but to the authors' knowledge, their efficacy has not been systematically investigated. Air exhausted from the IVC rack can be monitored, using sentinels housed in cages that receive rack exhaust air as their supply air, or using filters placed on the exhaust air port. To aid laboratory animal personnel in making informed decisions about effective methods for microbiological monitoring of mice in IVC, the efficacy of air monitoring methods was compared with that of contact and soiled bedding sentinel monitoring. Mice were infected with mouse hepatitis virus (MHV), mouse parvovirus (MPV), murine rotavirus (agent of epizootic diarrhea of mice [EDIM]), Sendai virus (SV), or Helicobacter spp. All agents were detected using contact sentinels. Mouse hepatitis virus was effectively detected in air and soiled bedding sentinels, and SV was detected in air sentinels only. Mouse parvovirus and Helicobacter spp. were transmitted in soiled bedding, but the efficacy of transfer was dependent on the frequency and dilution of soiled bedding transferred. Results were similar when the IVC rack was operated under positive or negative air pressure. Filters were more effective at detecting MHV and SV than they were at detecting MPV. Exposure of sentinels or filters to exhaust air was effective at detecting several infectious agents, and use of these methods could increase the efficacy of microbiological monitoring programs, especially if used with soiled bedding sentinels. In contemporary mouse colonies, a multi-faceted approach to microbiological monitoring is recommended.     

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.     

Microbiological monitoring of Guinea pigs reared conventionally at two breeding facilities in Korea.

In this study, microbiological monitoring of guinea pigs reared conventionally in two facilities was performed twice in 2004, with a three-month-interval between surveys. This study was based on the recommendations of the FELASA Working Group, with some modifications. In serological tests in the first survey, some animals from facility A showed positive results for Encephalitozoon cuniculi, Sendai virus, pneumonia virus of mice (PVM), and Reovirus-3 (Reo-3); facility B showed a positive result only for E. cuniculi. The results of the second survey were similar to the first, except for the presence of Sendai virus; all animals from the two facilities were Sendai virus-negative in the second experiment. No pathogenic bacteria were cultured in the organs of any of the animals in the first survey. However, in the second survey, Bordetella bronchiseptica was cultured from the lung tissue of two 10-week-old animals from facility A. Chlamydial infection was examined by the Macchiavello method, but no animal showed positive results. Tests using fecal flotation or the KOH wet mount method showed no infection of endoparasites, protozoa, ectoparasites, or dermatophytes in any animal in both surveys. However, in the histopathological examination, an infection of protozoa-like organisms was observed in the cecum of some animals from facility A. The present study revealed that microbiological contamination was present in guinea pigs reared conventionally in two facilities in Korea, suggesting that there is a need to improve environmental conditions in order to eradicate microbial contamination.     

湖南省2006～2010年实验动物微生物质量监测结果分析

目的了解湖南省实验动物微生物学质量,为实验动物实行科学管理提供重要依据,保证实验动物质量,确保实验结果准确和实验动物科技事业工作者的职业健康与安全。方法在相关单位生产繁殖群以单纯随机抽样原则采样,按照GB 14922.2-2001、GB/T 14926-2001和GB/T14926.21-2008进行实验动物微生物检测。结果 SPF级大鼠批次合格率为73.68%,SPF级小鼠批次合格率为77.78%,普通级新西兰兔批次合格率为60.61%,普通级豚鼠批次合格率为100%。结论我省2006～2010年实验动物微生物学质量前期呈现下降趋势,后期得到较大改善。要继续采取措施加强实验动物管理,消除实验动物种群微生物感染。     

Occurrence of Helicobacter species other than H. hepaticus in laboratory mice and rats in Sweden

The aim of this study was to determine which Helicobacter species other than H. hepaticus colonize laboratory mice and rats in Sweden. We analyzed 63 intestinal samples from mice and 42 intestinal samples from rats by partial 16S rDNA sequence analysis. Previously these samples had been found positive for Helicobacter species but negative for H. hepaticus in a polymerase chain reaction screening assay at the National Veterinary Institute in Sweden. H. ganmani, H. typhlonius, H. rodentium, an uncharacterized Helicobacter species ('hamster B'), and a possibly novel species were detected in mice. The possibly novel species was most closely related to H. apodemus strain YMRC 000216 (98.3% sequence similarity). Two different Helicobacter species were detected in rats: H. ganmani and H. rodentium. H. ganmani colonization of rats has not previously been reported.     

Monitoring a mouse colony for Helicobacter bilis using a Helicobacter-genus-specific nested PCR

Although Helicobacter infections of laboratory mice are usually subclinical, they may interfere with in vivo experiments and thus may lead to misinterpretation of data. As such, it is important to provide a means to unequivocally identify infections with murine Helicobacter spp. In the present study, a nested polymerase chain reaction (PCR) was established and shown to be 10 to 100 times more sensitive than the single-step PCR commonly used for routine diagnosis of Helicobacter spp. Experimental infection of Helicobacter-free mice demonstrated that faeces, caecum, colon and rectum but not liver are equally suitable for the detection of H. bilis. However, use of faecal pellets is advantageous since detection of H. bilis is possible one week after infection and analysis of faeces instead of tissues avoids euthanasia of animals. Furthermore, it generates representative data for all animals housed in the same cage and analysis can be repeatedly performed. Use of samples from breeding pairs but not offspring provides representative information about the Helicobacter status of a mouse colony. Both C3H/HeJ and C57BL/6 mice appear to be susceptible to H. bilis and persistent infection was observed during the 20-week experimental period. Analysis of pooled faecal pellets by nested PCR seems to be the most sensitive approach for H. bilis monitoring of the given breeding colony.     

Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections

Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals.
Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus , and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns.
Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H . bilis , in 91% for H. hepaticus, and in 82% for H. ganmani . The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium .
Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.     

从粪便样品中检测动物冠状病毒的分子技术研究

目的 感染冠状病毒的动物向环境排毒主要是通过粪便,建立直接从粪便样品对动物冠状病毒进行检测的分子技术具有重要的公共卫生学意义。方法 通过计算机模拟和实验方法对已报道的2对针对冠状病毒pol基因的通用引物的通用性进行了验证。不经传统的病毒分离．直接从环境样品中提取病毒RNA,通过一步法RT-PCR进行检测,并通过分子杂交和Nested PCR扩增结合TaqMan探针实时荧光检测的PCR技术．提高对冠状病毒检测的灵敏度和准确度,并对猪、禽冠状病毒感染的临床样品进行分析检测。结果 2对引物可以覆盖所有已知的冠状病毒,包括SARS,RT-PCR产物通过测序可以确定冠状病毒种类;实时荧光定量NestedPCR有很高的灵敏度,可以灵敏地检出所有供试的阳性样品,而荧光增量实时监测可以排除凝胶电泳检查的假阳性。结论 该研究为从环境中普查和鉴定冠状病毒提供了可靠的技术方法。     

我国商业化SPF级小鼠病原体污染分析

目的按现行国标《实验动物微生物学检测方法》和《实验动物寄生虫学检测方法》对我国商品化的无特殊病原体（specific pathogen free,SPF）小鼠进行微生物状况检测,为生产高质量的实验动物提供依据。方法对五家主要实验动物生产单位生产的ICR、KM、C57BL/6、BALB/c及BALB/c-nu品系的SPF级小鼠进行随机抽检。检测抽检小鼠所携带的微生物和寄生虫情况。结果在抽检的SPF级小鼠中,病毒污染主要包括呼肠孤病毒Ⅲ型（Reo-3）、小鼠肺炎病毒（PVM）和多瘤病毒（POLY）;主要细菌污染为肺炎克雷伯杆菌、金黄色葡萄球菌和绿脓杆菌。这些微生物病原体中,除条件性致病菌绿脓杆菌外,其他病原体对小鼠本身和实验研究均具有一定的影响。