奶水牛牛分枝杆菌的分离与鉴定

从患疑似结核病的广西奶水牛群中进行牛分枝杆菌的分离和鉴定。共收集了238份临床样品(鼻黏液及牛奶),病料经1.25 mol/mL NaOH溶液预处理后,接种罗氏培养基进行细菌分离。分离菌经进一步纯化后进行抗酸性染色并镜检,镜检后判为阳性的细菌再接种到鉴别培养基上进行鉴别培养,同时,应用PCR方法对镜检为阳性的细菌进行进一步的鉴别检验。结果收集的所有临床样品,经37℃培养后共获得细菌12株。这12株菌经抗酸性染色后镜检都为分枝杆菌阳性,进一步经鉴别培养基鉴定12株菌中有2株为牛分枝杆菌,其余均为非典型分枝杆菌。2株牛分枝杆菌经PCR方法鉴定得以确诊。     

Transfer of chlortetracycline from contaminated feedingstuff to cows' milk

Three groups of four Friesian cows in mid-lactation were fed a compound feedingstuff contaminated with 2, 10 or 300 mg chlortetracycline/kg for 21 days, and were then fed an uncontaminated diet for seven days. A fourth group of four cows was fed an uncontaminated diet throughout the study. Daily pooled milk samples from each cow were analysed by high performance liquid chromatography (HPLC) with a detection limit of 50 microg chlortetracycline/litre. Chlortetracycline was detected in only two milk samples taken from one of the animals fed feed containing 300 mg 300 mg chlortetracycline/kg, and both contained less than the maximum residue limit (MRL) specified by the European Union (100 microg/litre). All the milk samples were also analysed by the Delvotest SP microbiological assay, which has a detection limit of 300 microg chlortetracycline/litre. During the treatment period, this method gave four presumptive false-positive results, because they were not confirmed by HPLC. Selected daily pooled samples from each treatment group were also analysed by the semi-quantitative Charm II radioreceptor assay with a detection limit of 10 microg chlortetracycline/litre. Immunoreactive chlortetracycline was detected only in the animals fed feed containing 300 mg chlortetracycline/kg and several of the results exceeded the EU MRL during the treatment period. No significant treatment effects on animal performance were observed. However, there was a trend towards a higher milk fat concentration (P<0.09) and a lower milk protein concentration (P<0.07) with increasing concentration of chlortetracycline in the diet.     

Isolation of Mycoplasma bovis from bulk milk

The prevalence of mycoplasma in Vermont dairy farms was determined by two surveys conducted in March 1983 and January 1984. Bulk tank milk samples representing 74% and 62% of the herds respectively were cultured. Mycoplasma bovis was detected in 3 of the 2,346 bulk samples collected in the initial survey. The infection rate was 1.3 herds per thousand (95% confidence interval: 0.6-2.0 herds per thousand). No positive cultures were obtained in the second survey.     

乳中早孕因子的分离纯化及性质鉴定

早孕因子(early pregnancy factor, EPF)于1974年首次被澳大利亚学者Morton[1]等人发现的.EPF在动物受精后数小时至数天内即可检出,并在胎儿死亡或排出后很短时间即消失,以此对孕畜进行是否已受孕的超早期诊断,减少空怀率等,亦可作为监测胎儿发育情况的一个有效指标;同时还可用于胚胎移植过程中活胚胎的选择及判定移植是否成功.     

Transfer of sulphamethazine from contaminated dairy feed to cows' milk.

Four groups of four healthy mid-lactation Friesian cows were fed a compound feeding stuff containing either 2, 10 or 250 mg sulphamethazine/kg, corresponding to 0, 2, 10 and 250 per cent of the therapeutic inclusion rate in rations for pigs, at a flat rate of 3 kg twice daily for 21 days, followed by a seven-day withdrawal period. The cows were machine-milked twice daily and pooled milk samples from each cow were analysed by a commercially available microbiological assay with a sensitivity of 100 micrograms/litre and by a high performance liquid chromatography (HPLC) procedure with a limit of detection of 10 micrograms/litre. No sulphamethazine was detected by HPLC in the milk samples taken from any of the cows fed the concentrate containing 2 or 10 mg/kg. The milk samples from all four cows fed the highest concentration of sulphamethazine contained from 21 to 120 micrograms/litre while they were being fed the contaminated concentrate. The cow with the highest concentrations of sulphamethazine was the only one which repeatedly tested positive by the microbiological assay. The concentration of sulphamethazine declined rapidly during the withdrawal period and the drug was not detectable by either method in samples taken from two days after the contaminated feed was withdrawn.     

Mycobacterium bovis lipids: virulence and vaccines

The objective of this study was to determine the amount and infectivity of porcine circovirus type 2 (PCV2) shed in nasal, oral and fecal secretions following experimental infection. Fecal, oral and nasal swabs and blood were collected at regular intervals until 69 days post-inoculation (DPI) from five PCV2-experimentally inoculated pigs (Trial 1). To assess the infectivity of the PCV2 present in excretions, secretions, and on a hypodermic needle, 26 PCV2-na?ve pigs (Trial 2) were inoculated with various samples obtained from Trial 1 pigs. In Trial 1, PCV2 DNA was detected in all sample types by 69 DPI. There were no differences in the amount of PCV2 DNA present in different sample types over time. In Trial 2, intraperitoneal inoculation with contaminated fecal, nasal and oral samples; intranasal inoculation of nasal secretions; and feces fed to na?ve animals resulted in viremia and seroconversion. Viremia and microscopic lesions were noted in one animal injected using a contaminated needle. In conclusion, experimental PCV2 exposure results in a long term infection. PCV2 is shed in similar amounts by nasal, oral and fecal routes and is infectious to na?ve pigs confirming that multiple routes of transmission are likely important in spread of PCV2 between pigs.     

Molecular epidemiologic analysis of Mycobacterium bovis isolates from Mexico

OBJECTIVE: To assess phylogenetic relationships among Mycobacterium bovis isolates by use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting and to relate genetic profiles of isolates to epidemiologic characteristics. ANIMALS: 400 cattle with tuberculosis. PROCEDURE: Mycobacterium bovis was isolated from various organs of cattle slaughtered in 6 geographic regions of Mexico. Most cattle were adult Holsteins from large herds that did not participate in a tuberculosis control program. Four random primers and 2 selected primers were used in RAPD-PCR fingerprinting of 88 isolates. Pairwise genetic distance between isolates was obtained and subjected to cluster analysis with bootstrapping to test for levels of support. RESULTS: 98 different fragments were obtained; there was broad genetic diversity among isolates, and each isolate had a unique RAPD-genotype, including those originating from the same herd. Clustering by geographic location, affected organ, or severity of lesion was not detected. Linkage disequilibrium analysis suggested that M. bovis was highly clonal and that mutations develop at a rapid rate among isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Use of RAPD-PCR could not differentiate M. bovis isolates by epidemiologic characteristics or identify common sources of infection.     

中国荷斯坦牛乳尿素氮浓度昼夜变化规律研究

随机收集了8头处于泌乳期的中国荷斯坦奶牛24h内乳样,对乳中尿素氮浓度的昼夜变化情况进行研究。结果发现,同一奶牛个体在不同的时间点乳尿素氮浓度差异较大。因此,单个时间点的乳液尿素氮浓度代表性较差,有必要收集全天乳样。     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

A mathematical model for Mycobacterium bovis excretion from tuberculous cattle

An analysis was carried out of available information from a series of experiments on the excretion of M. bovis from infected cattle. The analysis indicated that an inverse exponential relationship exists between 'dose' of organisms given and the delay before excretion commences. This relationship was represented mathematically. Available field data supported the relationship and indicated that in natural bovine tuberculosis excretion of M. bovis begins around 87 days after infection occurs. It is also suggested that the data supports the concept of single nuclei infections in cattle.     

Population structure of Mycobacterium bovis isolates from cattle in Mexico

The molecular fingerprints of 878 isolates of Mycobacterium bovis collected from cattle between 2009 and 2010 in different regions of Mexico were used in this study. One hundred and ninety-four spoligotypes were observed in total with a high degree of heterogeneity. Sixty-four percent of the isolates grouped into just nine spoligotypes, and 27% fell into only two spoligotypes: SB0673 and SB0669; 149 were orphan spoligotypes. The two predominant spoligotypes were found in almost all states in Mexico, especially in central Mexico, where there is a high concentration of dairy cattle; however, some spoligotypes were closely associated with restricted geographical areas. The hypothetical evolutionary relationship among spoligotypes was estimated using the spoligoforest program in the spolTools webpage. Four trees with connected components and nine unconnected nodes were found. The biggest tree had SB0140 strain as a root, suggesting this as the oldest strain in the tree. However, the relationship of this spoligotype with SB0673 and SB0669 was weak. The discriminatory power of spoligotyping for this M. bovis sample of isolates was 0.94, and the recent transmission index (RTI) 0.83, suggesting a high rate of recent transmission of some strains of M. bovis in the population. This parameter indicates that new measures are required to stop the dissemination of tuberculosis in cattle.     

牛分支杆菌热休克蛋白65基因的分子克隆和表达

以牛型分枝杆菌Vallee菌株基因组DNA为模板,应用PCR方法扩增热休克蛋白65(Hsp65)基因,PCR产物经纯化与EcoRⅠ/SaⅠ双酶切后与经同样处理的原核表达载体pGEX-6P-1进行连接,转化感受态细胞BL21中,筛选和构建了原核重组表达质粒pGEX-H65,核苷酸序列测定验证其正确性.以1 Mol/LIPTG诱导,进行SDS-PAGE电泳.结果表明,Hsp65基因表达的融合蛋白GST-H65裂解为两部分;即64 kD和22 kD,两个蛋白分子量相加与预测的86 kD(HsP60 kD GST26 kD)相符.牛分支杆菌热休克蛋白HSP65的成功表达为其功能的研究打下基础.     

新疆牛分枝杆菌MIRU-VNTR基因分型的初步研究

为了解牛分枝杆菌的基因型特点,初步建立适合新疆地区的MIRU-VNTR基因分型方法,本研究利用24个MIRU-VNTR位点,对30株牛分枝杆菌新疆分离株进行基因分型研究。结果显示,在24个MIRU-VNTR位点中,有9个位点(ETR-A、B、D、E;MIRU24;QUB11a、11b、1895、26)具有多态性,可将30株菌分为7个基因型,分型指数为0.611。而国际通用的5个ETRs位点(ETR-A～E)可将该30株菌分为4个基因型,分型指数为0.712。多态性位点中多态性指数较高的5个位点(ETR-B、ETR-D、MIRU24、QUB11a、QUB26)具有与9个位点相似的分型能力,分型指数为0.715。研究表明,新疆地区与其他地区或国家的牛分枝杆菌在基因分型方面存在差异。5个多态性较高的位点均具有良好的分型能力,表明MIRU-VNTR基因分型方法可以作为新疆地区牛分枝杆菌分子流行病学研究的辅助方法。     

Iodine concentration in the blood serum of milk cows from Saxony as well as in cows' milk and milk products (baby food)

Within the framework of investigations into the metabolism of Saxon dairy-cows, more than 3300 analyses of blood serum total iodine concentrations have been carried out between 1997 and 2003. The total iodine concentration proved as suitable parameter for the recognition of the iodine supply situation of the milk cows. While to first two years 28,5 % of the samples were rated below the recommended minimum level (0,04 mg/l), thus indicating deficiencies, the total iodine concentration then rose demonstrably, even indicating an oversupply in some herds (> or = 0,20 mg/l). The iodine concentration in 117 independently tested milk samples (official food monitoring) confirmed this situation. An adequate iodine supplementation in feedstuffs is of great significance for the bovine metabolism as well as for the iodine concentrations in milk and therefore the iodine intake of the population. Of the negative consequences of an uncritically excessive iodine supplement particularly for high milk-yield cows one warns. This can lead to precariously high iodine concentrations in milk and dairy products. Further tests of blood total iodine levels in dairy herds on selective animals as well as analysis     

Molecular epidemiology of Mycobacterium bovis: usefulness in international trade

Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.     

Mycobacterium bovis isolated from a dusky langur with granulomas in the intestine

Tubercles were seen in the spleen of a male dusky langur (Presbytis obscurus) on laparotomy. Subsequently, tuberculous lesions in the intestine, lungs, and a hilar lymph node were observed on necropsy of the monkey. Histologic examinations of these tissues revealed granulomas, and acid-fast bacilli were observed within granulomas in replicate sections that were stained with auramine-O. An acid-fast organism was isolated and identified as Mycobacterium bovis. Guinea pigs and rabbits inoculated intraperitoneally with the organism developed granulomas in the lungs, liver, and spleen. Lesions did not develop in chickens inoculated with the culture.     

Isolation of Mycobacterium bovis from the prepuce of a herd bull.

A tuberculous lesion was found in the prepuce of a 5-year-old tuberculin-positive herd bull at necropsy. Microscopic examination revealed granulomas with Langhans'-type giant cells; Mycobacterium bovis was isolated. The bull had been used in artificial insemination procedures and for natural breeding.     

水貂源牛分枝杆菌的分离与荧光PCR鉴定

目的探讨水貂源结核杆菌复合群的检测方法,并进行病原学调查。方法利用7H10培养基分离水貂结核病的病原,采用荧光探针PCR方法进行检测。结果经鉴定分离的病原菌为致病性牛分枝杆菌。结论通过荧光探针PCR能够直接对结核杆菌复合群进行亚种鉴定,该方法具有快速、敏感、特异性强的优点,能够减少动物检疫时间。