Establishment of specific pathogen-free rabbit colonies with limited-flora rabbits associated with conventional rabbit flora, and monitoring of their cecal flora.

In the present study we attempted to establish specific-pathogen-free (SPF) rabbit breeding colonies with two groups of limited-flora (LF) rabbits, both ex-germfree rabbits, and their offspring. Two groups of LF rabbits associated with cecal flora of conventional (CV) rabbits produced in a previous study [Exp. Animals, submitted], were transferred to individual barrier rooms and some of the LF rabbits were accommodated in isolators to maintain the basic flora for SPF rabbits. The composition of the cecal flora of LF rabbits was stable for a long period; bacteroides remained predominant and clostridia dominant. From the SPF rabbits, different types of bacteria, e.g., enterobacteriaceae and streptococci, which could not be isolated in the isolator were detected at a low population level at an early stage in the establishment of the SPF colonies, but the basic composition of the cecal flora was mainly bacteroidaceae and clostridia and did not change over a long period, and the floral composition became similar to that of CV rabbits. The fertility and weaning rates of the SPF rabbits were satisfactory for a SPF rabbit colony. In addition, these SPF colonies were free of more than one year rabbit-specific pathogens.     

Acholeplasma cavigenitalium sp. nov., isolated from the vagina of guinea pigs.

A mollicutes isolated from a guinea pig vagina was shown to be serologically distinct from previously recognized Mycoplasma and Acholeplasma species. Colonies isolated from 10 different guinea pigs were cloned and examined in detail. These strains were closely related and had the following properties: guanine-plus-cytosine content of 36 mol%, no requirement for sterol, and aerobic growth. Glucose was not metabolized, and arginine and urea were not hydrolyzed. Strain GP3 (= NCTC 11727) is the type strain of a new species, Acholeplasma cavigenitalium.     

Nutritional and sanitary statuses alter postweaning development of caecal microbial activity in the rabbit

The postweaning development of caecal microbial activity was studied in the rabbit according to the sanitary status (conventional "C" vs. specified pathogen-free "SPF") and the nutritional status (standard-fibre "SF" vs. deficient-fibre "DF" diet). The two diets were distributed ad libitum from weaning (28 days) to 70 days of age, respectively, to 80 C and 72 SPF rabbits. From 28 to 42 days, the volatile fatty acids concentration in the caecum (tVFA) of C rabbits was 50 mM/L and increased by 46% between 42 and 56 days, without interactions with the diet effect. In parallel, the bacterial fibrolytic activity decreased for xylanase and CMCase (-32% and -60%, respectively, P<0.05), while pectinase activity decreased more regularly from 28 to 70 days (-28%, P<0.05). At weaning, tVFA was similar among C or SPF rabbits, while at 70 days, it decreased by 23% for SPF and increased in C group (+31%). Cellulasic and hemicellulasic activity of bacteria were two to three times lower, respectively, in SPF rabbits compared to conventional ones. No interaction was detected between sanitary and nutritional status at 70 days of age for the caecal fermentative activity. With the FD diet, tVFA decreased by 10%, while butyrate proportion increased by 37% (at 70 days), whatever the sanitary status. In 70-day-old rabbits (C or SPF group), pectinasic activity was reduced by 30% when rabbits were fed the FD compared to the SF one.     

SPF级与普通级新西兰兔血液学参数的比较

目的对普通级新西兰兔进行剖宫产,获得SPF兔,比较SPF兔和普通级兔血液学参数。方法采取剖宫产手术培育成无菌兔,接种正常寄生菌使之SPF化。耳缘静脉采血,自动生化分析仪测定SPF兔、普通级兔血液学参数。结果经检测SPF兔符合国家SPF兔标准;SPF兔和普通级新西兰兔白细胞数、血红蛋白、红细胞容积、中性粒细胞百分比、嗜酸性粒细胞百分比、嗜碱性粒细胞百分比、中性粒细胞数、单核细胞数、嗜酸性粒细胞数、嗜碱性粒细胞数、总蛋白、球蛋白、白蛋白/球蛋白、谷酰转肽酶、葡萄糖、钙、高密度脂蛋白胆固醇等参数差异极显著（P〈0.01）;血小板数、淋巴细胞百分比、肌酐差异显著（P〈0.05）;红细胞数、平均红细胞体积、平均血红蛋白含量、平均血红蛋白浓度、红细胞体积分布宽度、平均血小板体积、单核细胞百分比、淋巴细胞数、白蛋白、丙氨酸转氨酶、天冬氨酸转氨酶、总胆红素、碱性磷酸酶、尿素氮、尿酸、磷、总胆固醇、甘油三酯、低密度脂蛋白胆固醇、磷酸肌酸激酶、乳酸脱氢酶差异不显著（P〉0.05）。结论不同等级微生物环境对新西兰兔血液学指标有显著影响。     

Isolation of acholeplasmas and a mycoplasma from vegetables.

The isolation of Mollicutes from food has not been reported. To isolate Mollicutes in the presence of high levels of unwanted bacteria, we first incubated fresh vegetables in liquid culture media containing lysozyme, ampicillin, and thallous acetate. Culture fluids were than separated from the vegetable samples, subjected to one freeze-thaw cycle, and passed through a filter of 0.4-micron porosity. Filtered samples were cultured in SP4 medium and in a conventional medium containing horse serum. With this procedure 21 acholeplasma isolations representing three species were obtained from endive, broccoli, and kale. Of 35 food samples tested, 11 were positive for acholeplasmas; acholeplasmas isolated from 6 of these samples were recovered only in SP4 medium. In seven single vegetable samples, two or more Acholeplasma spp. were isolated. A. laidlawii was isolated from all three vegetables and A. axanthum was found in broccoli and kale. Four isolates were serologically identified as A. oculi. Mycoplasma verecundum was the only Mycoplasma species recovered. Several isolates could not be typed serologically, as they reacted with antisera to both A. morum and A. hippikon. these isolates may include new Acholeplasma spp.     

Establishment of a SPF colony of human apo(a) transgenic rabbits by frozen-thawed embryo transfer.

In the present study, embryo transfer was performed using frozen-thawed embryos to establish a SPF colony of human apolipoprotein (a) (apo(a)) transgenic rabbits. Apo(a) transgenic rabbits were kept under conventional condition and were infected with Bordetella bronchiseptica. Embryos at the morula stage were collected and stored in liquid nitrogen. After thawing, the in vitro survival rate was 84.6%, and 125 morphologically normal embryos were transferred to 6 SPF recipient rabbits. Four rabbits became pregnant and 23 live pups were born. PCR and Western blot analyses revealed that 9 of 23 pups were transgenic and expressed apo(a) protein. Microbiological tests showed all rabbits were free from infections. We succeeded in establishing a SPF colony of apo(a) transgenic rabbits. These rabbits are now maintained under a barrier system and are available for medical research.     

Acholeplasma multilocale sp. nov., isolated from a horse and a rabbit.

Acholeplasma strains were isolated from the nasopharynx of a horse (strain PN525T [T = type strain]) and the feces of a rabbit (strain B1). One clone of strain PN525T and one clone of strain B1 were examined in detail. These clones were indistinguishable from each other and were serologically distinct from the previously described Acholeplasma and Mycoplasma spp. The strains had the following properties: guanine-plus-cytosine content of 31 mol%; sterol was not required for growth, which occurred under both aerobic and anaerobic conditions; glucose was metabolized; and arginine was hydrolyzed. Strain PN525 (= NCTC 11723) is the type strain of a new species, Acholeplasma multilocale.     

腮腺炎病毒抗血清的制备

以制备适用于疫苗生产检定中病毒鉴别试验和外源因子检查的高效价腮腺炎病毒抗血清为目的。用腮腺炎病毒接种SPF鸡胚尿囊腔,培养收取病毒尿液免疫SPF鸡,采集抗血清。腮腺炎病毒接种Vero细胞,培养病毒抗原经PEG沉淀,超速离心法纯化后免疫家兔采集抗血清。比较两种免疫方法所得病毒抗血清效价。结果显示SPF鸡抗腮腺炎病毒血清中和抗体GMT为1:1716,兔抗腮腺炎病毒血清中和抗体GMT为1:732。两种动物抗血清均适用于疫苗生产相关检定。免疫SPF鸡制备的病毒抗血清无特定病原及抗体污染,是毒种外源因子检测和疫苗鉴别试验的理想试剂。免疫SPF鸡制备病毒抗血清的程序简单,结果易于验证,有利于生物试剂标准化。     

SPF大鼠感染金黄色葡萄球菌原因的调查分析

目的连续监测某实验动物饲养场SPF大鼠携带的金黄色葡萄球菌Staphylococcus aureus(SA)情况,结合该饲养场环境、人员等检测结果,查找污染源,为保障和提高实验动物质量提供依据。方法2011—2017年依据《实验动物金黄色葡萄球菌检测方法(GB/T 14926.14-2001)》对SPF大鼠进行SA监测,并采集饲养环境和饲养人员的样本进行检测。对所有分离的SA菌株应用金黄色葡萄球菌A蛋白(SPA)基因分型和脉冲场凝胶电泳(PFGE)分型,分析污染源。结果35份SPF大鼠检出16株SA,检出率45.71%;18份饲养环境和饲养人员样本检出2株SA,检出率11.11%。SPA分型可分成5个型别,T2360为优势型别,主要由2013年和2017年SPF大鼠中的菌株组成。PFGE有5种带型,SA4为主要带型。饲养环境中SA的PFGE带型与2017年SPF大鼠中的完全一致。结论饲养环境中存在的SA与SPF大鼠感染的SA具有紧密联系。     

Differential fluorescence labelling with 5-dimethyl-aminonaphthalene-1-sulfonyl chloride of intact cells and isolated membranes in Salmonella typhimurium and Acholeplasma laidlawii

For fluorescence labelling intact cells and isolated cell envelopes (membranes) from Salmonella typhimurium and Acholeplasma laidlawii were treated with mixed dansylchloride-lecithin-cholesterol vesicles. This kind of dansylation, which has been supposed to be specific for cell surface proteins, produced fluorescent protein pattern after SDS-polyacrylamide gel electrophoresis only when isolated envelopes were dansylated. Acid hydrolysis of fluorescent cell envelopes of Salmonella typhimurium yielded O-dansyltryosine and epsilon-N-dansyl-lysine besides the free sulfonic acid and unidentified compounds. However, no fluorescent proteins were detectable in cell envelopes isolated from dansylated intact bacteria from Salmonella typhimurium. In accord Acholeplasma laidlawii showed only fluorescence from proteins with a molecular weight higher than 100000.     

H9N2亚型AIV排毒及形成气溶胶的研究

为研究H9N2亚型AIV排毒及形成气溶胶规律,将SPF鸡饲养于正负压隔离器中,采用AGI-30收集器(All Glass Impinger AGI-30)和气管泄殖腔棉拭子在攻毒后不同时间收集空气、气管和泄殖腔样品,并利用HI、Dot-ELISA和RT-PCR检测样品.结果发现攻毒后第4天开始形成气溶胶,并持续到第43天,实验证明H9N2亚型AIV不仅可以在呼吸道和泄殖腔复制,而且可以形成气溶胶.气管泄殖腔棉拭子在接种后第3天开始排毒,至第7天攻毒鸡全部分离到病毒.排毒时间可持续到第45天.但泄殖腔的排毒量明显低于气管的排毒量,这也说明H9N2型禽流感主要通过呼吸道排毒.     

Binding of lectins to membranes of mycoplasmas from aging cultures

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.     

Studies on Tyzzer's disease in rats.

An outbreak of an epidemic disease occurred in a specified-pathogen-free (SPF) breeding colony of rats. The clinical signs and the post-mortem findings were characteristic for Tyzzer's disease. The causative agent, Bacillus piliformis, was demonstrated microscopically in ileum, liver and myocardium, and transmitted to mice where its pathogenicity appeared to be similar to that of another strain isolated from mice. B. piliformis from spontaneously-infected rats was demonstrated by indirect immunofluorescence technique. By means of the same technique it was found that the fluorescence antibody titre obtained of the individual sera from spontaneously-infected mice, rats and rabbits was the same, whether the antigen employed was organisms isolated from rats or mice. By testing sera from healthy rats in 3 different colonies by use of immunofluorescence technique, antibodies were found in several sera.     

Heterogeneity in the physical state of the exterior and interior regions of mycoplasma membrane lipids

The electron paramagnetic resonance spectra of spin-labeled fatty acid in intact mycoplasma cells and isolated membrane preparations have been compared. With Mycoplasma hominis and Acholeplasma laidlawii preparations, the freedom of motion of the spin-label was higher in labeled intact cells than in labeled isolated membranes but no differences could be detected between the labeled intact cells and membranes isolated from the labeled intact cells. It is proposed that the higher freedom of motion of the spin-label in the intact cells is due to a higher fluidity of the outer half of the lipid bilayer of mycoplasma membranes rather than to alterations in the structure of the membrane upon isolation.     

Rearing of germfree rabbits and establishment of an SPF rabbit colony]

Baby rabbits hysterectomy-derived from conventional Japanese white rabbits were reared under aseptic condition by feeding with 4 types of artificial diets. Rabbit milk for the artificial diet was obtained from conventional dams at 7--25 days after delivery. The artificial diets was given by stomach tube twice a day. The total volumes of diet given (ml per day) were Y = 2.3X + a (1 to approximately 14 days of age), Y = 32.2 + a (l5 to approximately 25 days of age), or Y = (32.2 + a) - 37.5 (X - 25) (26 to approximately 34 days of age), (X = age in days, a = volumes fed at 0 day of age). After 14 days of age young animals were also fed freely sterilized commercial pellets and weaned at 35 days of age. Out of 155 rabbits, 130 were aseptically reared till 36 approximately 40 days of age, and no difference on weaning rate was seen between the 4 groups of rabbits. Thereafter, they were exposed to a barriered room of SPF rabbitry outside the isolators. The best growth was seen in animals given by artificial diet containing rabbit milk at 40%. The SPF breeding colony of rabbits was found to be free of Pasteurella pneumotropica, Bordetella bronchiseptica, Salmonella spp., Eimeria spp., Encephalitozoon cuniculi and Acaritic otistis.     

Isolation ofAcholeplasma oculi from human amniotic fluid in early pregnancy

Acholeplasmas have been isolated from a variety of animals, insects, and plants, but onlyAcholeplasma laidlawii has previously been found in humans. We have isolatedAcholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associatingA. oculi with various diseases in livestock, the prevalence and significance ofA. oculi in humans has not been determined.     

DNases of Acholeplasma spp

One strain from each of seven species of Acholeplasma was examined for the presence of DNases. Six of the strains were found to be DNase positive when assayed with DNA-methyl-green-containing agar, indicating that this method can be used to differentiate the seventh strain, Acholeplasma axanthum, from the remaining Acholeplasma spp. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA revealed DNases in the cell extracts of all seven strains and in the supernatant growth medium of five of the strains. The electrophoretic patterns were highly characteristic for each strain and can be used for the rapid identification of different strains of Acholeplasma.     

Sensitivites in vitro to antimicrobial drugs of bovine mycoplasmas isolated from respiratory and genital tracts

A total of 155 Mycoplasma strains were examined for sensitivity to nine antibiotics and four nitrofurans by the agar dilution method. They consisted of 69 strains of Mycoplasma bovirhinis, 33 strains of M. bovigenitalium, 49 strains of Acholeplasma laidlawii and four strains of A. modicum isolated from the nasal secretions, tracheas and lungs of calves manifesting respiratory symptoms and from bovine genital tracts collected at a slaughterhouse. As a result, furamizole and mitomycin C showed the strongest growth-inhibiting effect on all the strains. They were followed in this effect by kitasamycin tartrate, spiramycin adipate, tylosin tartrate, tetracycline-HCl and chloramphenicol. Furthermore, these five drugs were followed in the effect by furazolidone, nitrofurantoin and sodium nifurstyrenate. Fradiomycin sulfate and kanamycin sulfate showed only little effect on all the strains. Erythromycin lactobionate showed a strong growth-inhibiting effect on the Acholeplasma strains, but not on the Mycoplasma strains. There were some cross resistant strains of the Acholeplasma species to the effects of the macrolides.     

Characterization and Identification of Caprine,Genital Mycoplasmas

A total of 55 mycoplasma strains, isolated from the vagina of goats, were examined. Three strains, being arginine positive and glucose negative, could not be finally classified. Five isolates were identified as Acholeplasma laidlawii. Fourty-seven strains were phosphatase positive, glucose and arginine negative. Nine of these formed “film and spots” on standard growth medium, and reduced tetrazolium aerobically. Serological examination identified 7 as M. agalactiae, while 2 were M. bovis. The remaining 38 isolates did not reduce tetrazolium aerobically, and did not produce “film and spots.” on standard growth medium. All these except one were identified as M. bovigenitalium by immunofluorescence. Their relationship to group 11 otf Al-Aubaidi is discussed.     

Mycoplasma spermatophilum, a new species isolated from human spermatozoa and cervix

A mycoplasma isolated from human spermatozoa and a human cervix was shown to be serologically distinct from 98 previously recognized Mycoplasma and Acholeplasma spp. Six mycoplasma colonies were cloned and examined in detail for morphology, growth, and biochemical characteristics; five of these were from sperm samples and one was from a cervix. These strains were closely related and had the following properties: guanine-plus-cytosine content of 32 mol%, requirement for sterol, and anaerobic growth. Glucose was not metabolized, and arginine and urea were not hydrolyzed. Strain AH159 (= NCTC 11720) is the type strain of a new species, Mycoplasma spermatophilum.