The use of 60Co-gamma ray-sterilized calf serum in cell culture

The use of 60Co-gamma-radiation-sterilised calf sera in cell culturing is reported in this paper. Evidence was produced to the effect that 60Co-gamma-irradiation, using a dosage of 3 kGy and a dose rate of 8 kGy/h, of fetal calf serum, neonatal calf serum, and calf serum did not substantially alter the growth-stimulating properties of those sera during 42-day tests. With almost all cell lines and sera used, for all practical purposes, they were identical with the properties of control sera. The following cell lines were used in the experimental programme: one human mammary tumor, MaTu, one human embryonic cell line--E VI, one bat lung cell line--Tb1-Lu, and one human rhabdomyosarcoma--A 204. Growth stimulation was twelve percent below the control value only with Tb1-Lu on Eagle-MEM culturing medium with 3-kGy-irradiation of neonatal calf serum. On the other hand, cell growth was stimulated by 28 percent in A 204 on RPMI 1640 culturing medium, again with 3-kGy-irradiation of neonatal calf serum. Loss of activity by up to 30 percent, depending on the serum used, must be expected from irradiation doses of 10 kGy and 20 kGy which are capable of causing drastic reduction or even complete elimination of serum-borne microorganisms (Bender et al., 1989). Sera irradiated that way would be only conditionally applicable, when it comes to highly vulnerable cell strains.     

Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection

Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.     

The use of gamma irradiation for the inactivation of infectious bursal disease viruses

The maximum dosage of gamma irradiation approved by the U.S. Food and Drug Administration (FDA) for poultry is 3.0 kiloGrays (kGy). This treatment is designed to reduce bacterial contamination on uncooked poultry carcasses and meat products. The possible presence of infectious bursal disease virus (IBDV) on poultry postharvest has prompted some countries to study the risk associated with introducing nonnative strains of the virus from imported commodities. The goal of this study was to determine if this risk could be reduced using gamma irradiation to inactivate IBDV. At the dosage approved by the FDA, the titers of IBDV vaccine strains were reduced between 0 and 1 log10. Titers of the pathogenic IBDV strains tested were not reduced after the 3.0 kGy exposure. Furthermore, titers of pathogenic viral strains were not reduced following exposure up to 5.0 kGy. As the exposure to gamma irradiation increased, the titers of the vaccine strains decreased. At the maximum dosage tested (10 kGy), the 89/03 variant virus vaccine was completely inactivated. Titers of the three classic IBDV vaccine strains were reduced between 1.6-2.0 logs after the 10 kGy exposure; however, these viruses remained viable after this treatment. Gamma irradiation is not an effective intervention to reduce the risk of IBDV introduction via processed poultry.     

辐照技术对宠物食品冻干鸡肉丁品质的影响

试验旨在研究60Coγ射线与电子束辐照对冻干鸡肉丁品质的影响.试验分别进行不辐照、60Coγ-2 kGy、60Co γ-4 kGy、电子束-2 kGy、电子束-4 kGy处理,恒温37℃进行贮存加速试验,测定细菌总数、过氧化值、酸价、碘值、感官、蛋白质和水分.结果 表明:冻干鸡肉丁经辐照后细菌总数≤20 CFU/g;6...     

Effect of radiation on chemical composition and protein quality of cottonseed meal

This study was conducted to compare the effects of electron beam (EB) and gamma ray (GR) irradiation treatments at doses of 10, 20 and 30 kGy on chemical composition, protein quality and protein digestibility of cottonseed meal (CSM). GR irradiation in all doses significantly decreased the crude fiber of samples compared to raw CSM. Free and total gossypol content of CSM was decreased significantly by utilizing both types of irradiation in a dose‐dependent manner. Also, EB irradiation caused decrease in free and total gossypol content more than that of GR irradiation. GR irradiation at doses of 20 and 30 kGy, and EB irradiation at doses of 10, 20 and 30 kGy can significantly decrease protein solubility of CSM compared to that of a raw sample. GR irradiation at a dose of 30 kGy significantly increased apparent digestibility of protein compared to raw and EB irradiation of CSM at a dose of 10 kGy in Leghorn cockerels. Maximum increase in protein digestibility of CSM was observed in GR irradiation at a dose of 30 kGy. In conclusion, the present study showed that EB and GR irradiation reduced gossypol and crude fiber and increased protein digestibility of CSM but had no effect on protein quality of CSM.     

Enzyme-linked immunosorbent assay for detection of Mycoplasma californicum-specific antibody in bovine serum: optimization of assay determinants and control of serologic cross-reactions

An enzyme-linked immunosorbent assay (ELISA) was adapted to detect Mycoplasma californicum-specific antibodies in bovine serum. Cross-reactive antibody was found in the M californicum-positive reference serum when assayed against each of 7 solid-phase antigens of heterologous mycoplasma species. Cross-reactivity was further demonstrated by inhibition of ELISA reactivity to M californicum solid-phase antigen by incubation of sera with antigen suspensions of each heterologous species. Incubation of test sera with a cross-reacting antigen mixture containing equal proportions of the 7 cross-reactive mycoplasmas was used to minimize cross-reactivity in the M californicum-specific ELISA. Specificity of antibody reactivity to M californicum, as measured by ELISA, was determined by enzyme-linked immunosorbance inhibition, in which sera were incubated with M californicum antigen suspensions before determining ELISA reactivity to M californicum solid-phase antigen. Seropositive and suspect sera (n = 55) were obtained from 3 dairies that had bacteriologically verified epizootics of M californicum mastitis. The percentage of inhibition demonstrated in enzyme-linked immunosorbance inhibition was determined for each serum. Inhibition percentages below the 15th percentile (61% inhibition) of this distribution were classified as nonspecific.     

Detection of antibodies to mycoplasmas using an immunoenzyme method

Detection of the antibodies to the species Mycoplasma bovis in the serum and milk of dairy cows coming from a mastitis-infected herd is a good example of utilization of the ELISA immunoenzymologic method in the mycoplasmology. Examining the samples from 75 dairy cows and applying the indirect hemagglutination test, good correlation of the results of the two tests was determined. The antibodies to the species Ureaplasma diversum were demonstrated by the ELISA method both in the bovine serum and in the milk of dairy cows infected slightly with mastitis. We chosen that strain which detected the maximum titres in the selected samples of the sera out of four antigens prepared from various strains of U. diversum. Rabbit sera hyperimmune to 26 strains of the mycoplasmas of various species were used to identify two antigens (after removing the antibodies to the components of the media). Specific reaction was obtained with the antisera to M. hyorhinis and M. arginini.     

桑辐射育种的研究——Ⅳ.辐射在改良叶质和抗病性上的作用

用照射量为6000伦和照射量率为50伦/分的~(60)C_o-γ射线,辐照品种新-之濑嫁接苗,诱导获得了R81—1和R81—2两个突变体.用照射量为5721拉德和照射量率为85拉德/分的~(137)C_x-γ射线,照射桐乡青品种的一年生穗条,诱导获得了另外两个突变体R82—1和R82-2.通过养蚕试验,其中3个突变系R81—1,R81—2和R82—1的叶质,在秋期都显著超过,在春期也大多显著超过标准对照品种荷叶白及其亲本.同样这3个突变系,经连续两年的人工接种桑疫病病菌的鉴定,其抗病性显著强于标准品种荷叶白及其亲本.结果表明,辐射在改良叶质和抗病性方面是有作用的.     

Detection of mycoplasmas in eye swab specimens of cattle

In the present investigation 262 conjunctival swabs were taken from 178 cattle and examined for mycoplasmas. The isolation was possible from 111 swabs. Mycoplasmas were found in eyes with clinical symptoms of IBK (in 64 of 148 swabs investigated = 43.2%) as well as in healthy eyes (in 47 of 114 swabs investigated = 41.2%). Consequently a correlation between clinical findings and isolation of mycoplasmas could not be observed. Unfortunately 60 of 111 isolates could not be subcultivated after storage at -20 degrees C. Using the indirect immunofluorescence test 41 of the 42 surviving isolates were identified as M. bovoculi which before has not been isolated in the Federal Republic of Germany. One isolate was determined as A. laidlawii. The 17 M. bovoculi strains investigated for their biochemical reactions showed the same characteristics like the reference strain M. bovoculi M 165/69. In repeating examinations mycoplasmas could be isolated 5 times after one month and 14 times after 6 months. Cattle younger than 2 years were more often infected with mycoplasmas (62.5%) than older animals (19.4%). No difference, however, could be observed in the clinical manifestations of IBK between younger and older animals. Mycoplasmas were more frequently isolated in autumn (43.6%) than in spring (21.4%) and summer (29.3%). In most of the animals examined both eyes were colonized by mycoplasmas. No spiroplasmas could be detected in the 262 conjunctival swabs investigated.     

不同辐照剂量和氛围下 60 Co-γ射线辐照蚕丝的电子自旋共振分析

在不同辐照剂量、不同辐照氛围下用60Co-γ射线辐照蚕丝纤维,通过电子自旋共振波谱(ESR)检测蚕丝纤维产生的自由基浓度,分析不同辐照条件对蚕丝纤维性能的影响。结果表明:在0~250 kGy剂量范围内,随辐照剂量的增加,60Co-γ射线辐照蚕丝产生的自由基浓度增加,但辐照剂量过大会使蚕丝纤维发生裂解而影响其性能,故辐照剂量应不超过50 kGy;与同剂量空气下辐照蚕丝纤维产生的自由基相比,在真空下辐照蚕丝纤维产生的自由基浓度增加,且主要为氨基己酸自由基,但随着辐照剂量的增加,真空和空气下辐照蚕丝纤维产生的自由基浓度差异变小。     

Attachment of Mycoplasma bovoculi to bovine conjunctival epithelium and lung fibroblasts

A specialized tip structure in some mycoplasmas facilitates their attachment to host cells. Mycoplasma bovoculi strains FS8-7 and M165/69 did not have specialized membrane structure and did not exhibit capsule when stained with ruthenium red and examined by use of transmission electron microscopy. The organisms attached in vitro to bovine lung fibroblasts, with no apparent specialized structure. Attachment to conjunctival epithelium in vivo was observed (after death) in a calf infected with M bovoculi. Close association between M bovoculi and the host cells was noticed. Mycoplasmal cells pretreated with hyperimmune rabbit serum and labeled with protein A-gold complex had gold particles randomly distributed around the membrane. Gold-labeled monoclonal antibodies, M25.5 and M7.3, which were directed against 2 surface antigens of M bovoculi, also were distributed randomly on the mycoplasmal surface as seen in results of double-labeling experiments.     

Serologic response of vaccinated cattle to strains of Moraxella bovis isolated during epizootics of keratoconjunctivitis.

In studies to determine whether there were antigenic differences between strains (isolates) of Moraxella bovis, the sera from vaccinated calves were tested with isolates of M bovis while the calves were experiencing epizootics of infectious bovine keratoconjunctivitis (IBK). Before the epizootics of IBK, the calves were intramuscularly vaccinated with a formalin-killed autogenous M bovis bacterin. During the epizootics, the eyes were examined by cultural technique, and isolates which were obtained were categorized by catalase activity, source (diseased or nondiseased eyes), and reactivity with the various sera. The serum reactivity of the isolates was compared with that of the vaccinal strain. The vaccinal strain and 8 of the 1 5 selected isolates obtained during the 1974 epizootic were catalase negative. Seven of the 15 isolates from the 1974 epizootic and all of the selected isolates from the 1975 epizootic were catalase positive. A significantly higher (P less than 0.01) percentage of calf sera were serologically reactive with the vaccinal strain and other catalase-negative isolates (45.0%) than with catalase-positive isolates (34.8%). The results, although not definitive, suggest that there may be antigenic differences among strains of M bovis. These differences should be considered when cattle are vaccinated against IBK under natural conditions of exposure.     

Examination of cattle with respiratory diseases for Mycoplasma and bacterial bronchopneumonia agents

A total of 247 mycoplasma strains was isolated from 435 lungs, tracheobronchial secretions and nasal swabs originating from cattle with symptoms of bronchopneumonia. Mycoplasma (M.) bovis was found 89 times (36%) and was the most common mycoplasma species in the lungs. M. bovirhinis, M. bovigenitalium, M. spec. and Acholeplasma (A.) laidlawii were isolated 158 times (64%). Among these mycoplasmas M. bovirhinis was the most widespread species (114 isolations). In 55 cases (62%) M. bovis was associated with Pasteurella or Actinomyces (A.) pyogenes. The other mycoplasma species were found in 67 cases (42%) together with these bacteria. Without mycoplasmas Pasteurella and A. pyogenes occurred in 33 of the probes investigated (21%). Beside mycoplasmas Haemophilus (H.) somnus was isolated from 16 of 162 tracheobronchial secretions investigated. The results confirm earlier suppositions that in most of the cases bronchopneumonia of cattle is a multifactorial event, frequently associated with mycoplasmas--especially M. bovis.     

Mycoplasmas: use of polyvalent antisera for identification by indirect immunofluorescence.

It would be an advantage, under many circumstances, to be able to make use of polyvalent antisera in the process of identifying mycoplasmas. As the indirect immunofluorescence test is sufficiently sensitive and also generally accepted as being rather specific, this technique was chosen to investigate whether polyvalent antisera are applicable in routine identification of mycoplasmas. Three polyvalent sera were used, each consisting of 9 or 10 rabbit antisera raised against 29 of the more common species of the genus Mycoplasma. Twenty-six field strains were examined. One strain did not react with any of the 3 polyvalent antisera although it was later identified as M. bovigenitalium. The remaining 25 strains reacted with 1 and only 1 of the polyvalent antisera and were subsequently identified by immunofluorescence utilizing monospecific antisera. Strains of the following species were identified: M. arginini, M. bovigenitalium, M. bovis, M. bovoculi, M. canis, M. capricolum, M. cynos, M. edwardii, M. hominis, M. hyorhinis, M. molare, M. mycoides subsp. mycoides and M. opalescens. It is concluded that polyvalent antisera may be used in identification procedures and thereby permit the use of a limited number of monospecific antisera without preceding biochemical testing.     

Classification and identification of ovine and caprine Mycoplasmas.

The purpose of this investigation was to give a survey of the classification of ovine/caprine mycoplasmas as a basis for the identification of strains isolated from sheep and goats. A total of 13 strains representing 13 species and/or serogroups were biochemically examined, and serological cross-titrations were performed using metabolism inhibition, growth inhibition and immunofluorescence. Serogroup 6 (Al-Aubaidi) was found to be identical with Mycoplasma capricolum.The results of identification of 57 isolates, sent to the reference centre from different countries, are given.On the basis of the above investigations and a comparison of some of the classification systems described in the literature, it is concluded that the following species have been isolated from goats and/or sheep: M. agalactiae, M. arginini, M. bovis, M. capricolum, M. conjunctivae, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. ovipneumoniae, M. putrefaciens, Acholeplasma granularum, A. laidlawii and A. oculi. In addition, both Ureaplasmas and strains representing 6 serogroups (groups 5, 7, 10 and 11 of Al-Aubaidi and groups 17 and 18 of Cottew) have been isolated. These serogroups ought to be finally species-classified as soon as possible. kw|Keywords|k]ovine/caprine mycoplasmas; k]classification; k]identification     

Les immunostimulants orientent la reponse locale au virus de l''I.B.R. vers la production d''Interferon

Immuno-adjuvants like MDP or levamisole inoculated in the calf through general route increase the respiratory mucosa response to the irradiation inactivated IBRV.

We have shown from the 2nd day of the inoculation an interferon activity in the nasal mucus. This interferon partially destroyed at pH2 and at 56°C, persists in the respiratory fluids for one week. It is never found in the sera of the animals.

This inactivated virus given through nasal route does not stimulate antibody production in the local system or in the serum.

The dose of levamisole administered is critical: 5 mg/kg are the minimal quantity required to give a favourable answer whereas the 2.5 mg/kg normally used are without effect.

The interferon presence in the respiratory fluids gives a satisfactory interpretation of the good resistance to respiratory infection obtained in 500 calves with this treatment: inactivated virus by nasal route and immuno-adjuvant by intradermic or intramuscular route.     

Mycoplasma as a cause of canine urinary tract infection

Mycoplasmas were isolated from 60 specimens of urine obtained by cystocentesis from 41 dogs (23 males and 18 females) with urinary tract infection. Mycoplasmas were isolated in pure culture from 41 (68%) of the specimens, and were isolated in conjunction with one or more bacterial species from 19 (32%) specimens. Clinical signs of urinary tract infection were noted in 20 of 31 dogs in which mycoplasmas were isolated in pure culture, and numbers of WBC in the urine sediment were above the reported normal range in 22 of 25 urine specimens from those 20 dogs. Twenty-four of 29 mycoplasma isolates were found to be Mycoplasma canis, 4 were found to be M spumans, and 1 was identified as M cynos.     

Mycoplasma bovis: disease,diagnosis, and control

Mycoplasma bovis is a major, but often overlooked, pathogen causing respiratory disease, mastitis, and arthritis in cattle. It is found worldwide and has spread into new areas, including Ireland and parts of South America, in the last decade. In Europe, it is responsible for at least a quarter to a third of all calf pneumonia although this may be an underestimate as few laboratories regularly monitor for mycoplasmas. Like all mollicutes, M. bovis is inherently refractory to certain groups of antibiotics because it does not possess a cell wall; furthermore evidence is accumulating that strains of M. bovis are becoming resistant to antibiotics, including tetracycline, tilmicosin and spectinomycin, traditionally used for their control. No vaccines are presently available for the control of M. bovis infections.     

不同辐照剂量对蜂球囊菌杀灭效果的影响

本试验以蜂球囊菌(Ascosphaera apis)为研究对象,探究不同辐照剂量对蜂球囊菌的杀灭效果,以期明确有效杀灭蜂球囊菌的最低辐照剂量,为Co60γ射线辐照蜜蜂饲料(特别是蜂花粉)时的辐照剂量选择提供依据。利用细菌纯化技术从患白垩病意蜂幼虫体内分离纯化得到蜂球囊菌,并结合形态学、乳酸酚棉兰染色及5.8SrDNA序列分析技术进行鉴定;同时制备不同梯度浓度的蜂球囊菌孢子悬液加入到蜜蜂幼虫饲料中,以饲喂方式侵染3日龄意蜂幼虫,确定半数致死浓度(LC50);蜜蜂幼虫饲料中添加上述确定LC50的孢子,以不同辐照剂量处理,并通过侵染幼虫试验,确定有效杀灭蜜蜂饲料中孢子的最低辐照剂量。结果表明,通过形态学和分子生物学鉴定,从患白垩病的意蜂幼虫体内分离纯化得到真菌即为蜜蜂白垩病的病原——蜂球囊菌;蜂球囊菌对人工饲养条件下的意蜂幼虫的LC50为9.5×104个/mL;当Co60γ射线辐照剂量为7.0kGy时,幼虫患病率与未辐照组差异显著(P0.05),由此确定对添加半数致死浓度的蜂球囊菌孢子饲料的最低有效辐照剂量为7.0kGy。