Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination.

The collection of biological material (e.g., blood) directly onto filter paper for subsequent use in laboratory assays such as polymerase chain reaction (PCR), has become a common practice. Dried cells or fluid on the paper can be readily rehydrated and retrieved into a standard volume of an appropriate elution buffer but introduces a dilution factor to the sample. The use of a common cutting instrument for excising a standard-sized piece of paper that contains the material also introduces the potential for transferring biological material from one sample to subsequent samples, causing false-positive results by PCR. In the present study, filter-paper-collected blood that contained beak and feather disease virus was used to determine if viral DNA could be transferred between samples by a hole punch used to excise sequential filter papers. It was determined that false-positive results could be obtained at least 13 times after a positive sample. Subsequently, the efficacy of 4 methods of hole punch disinfection, flaming, VirkonS, bleach, and a bleach-ethanol combination, was assessed. The only effective and practical method to destroy DNA was a method where the hole punch was agitated in commercial bleach, rinsed in water, the water was displaced with 100% ethanol and air-dried. This method was simple, cheap, and relatively rapid, and allowed for the use of a single hole punch for a series of samples, without carryover contamination and consequent false-positive results.     

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

Comparison of filter-paper-eluted whole blood with serum in fowl cholera serology using the enzyme-linked immunosorbent assay

Two methods for collecting blood for measuring antibody activity of Pasteurella multocida were compared. Whole blood was collected on filter-paper strips, dried for 48 hr at room temperature, and then stored in sealed plastic bags at 4 C. Blood was also collected in the usual manner with a needle and syringe, and serum was harvested and stored at -20 C until tested. Eluates of whole blood, obtained by overnight elution of two 4.8-mm discs in 200 microliters of buffered saline at 4 C, were compared with conventionally harvested serum for antibody activity by enzyme-linked immunosorbent assay (ELISA). Paired samples, taken from the same bird at the same time, showed no significant difference (P less than 0.05) in antibody activity as measured by absorbance when the disc-elution process itself was considered to be a 1:20 dilution. It was concluded that eluates of blood, derived from whole blood dried on filter-paper strips, may be used as an alternative to sera in ELISA for measuring P. multocida antibody activity.     

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.     

Digestion of poultry manure by Musca domestica

Larvae of the house fly (Musca domestica) grew and developed normally in fresh poultry manure where the proper environmental conditions were maintained. A temperature of 27 °C and a moisture content ranging from 60 to 75% in fresh poultry manure was determined to be optimum for larval development. A moisture content of 80% or greater created anaerobic problems for larvae.

Optimum production of house fly pupae was obtained by inoculating 0.50 to 0.75 g of fly eggs per kg of fresh poultry manure. Five to eleven days were required for the eggs to be converted to pupae depending on the environmental conditions. After digestion, the poultry manure, was reduced to about half and was granular in texture. The digested residue readily dried, had less odour and contained approximately 15% protein.     

Horse infestation with the larva of the deer warble fly, Hypoderma diana Brauer, 1985 (Diptera, Hypodermatidae)

In South Bohemia a case was discovered of a yearling colt attacked by the larva of the IIIrd instar of the deer warble fly Hypoderma diana Brauer. The dead, almost mature larva of the fly was squeezed out of a subcutaneous lump above the shoulder in the first decade of April, 1985. The case is evaluated from the point of view of the possibility of the transition of specific parasites--warble flies--to another host. The attacking of a non-specific kind can occasionally occur only when there is a large number of the parasites and both kinds of host. At present the degree of attacking of deer by subcutaneous warble flies is high and therefore under favourable circumstances even domestic animals can be attacked by this type of warble fly. The above case is the first to be ascertained of a horse being attacked by a deer warble fly.     

Sensitivity of different Trypanosoma vivax specific primers for the diagnosis of livestock trypanosomosis using different DNA extraction methods

There are several T. vivax specific primers developed for PCR diagnosis. Most of these primers were validated under different DNA extraction methods and study designs leading to heterogeneity of results. The objective of the present study was to validate PCR as a diagnostic test for T. vivax trypanosomosis by means of determining the test sensitivity of different published specific primers with different sample preparations. Four different DNA extraction methods were used to test the sensitivity of PCR with four different primer sets. DNA was extracted directly from whole blood samples, blood dried on filter papers or blood dried on FTA cards. The results showed that the sensitivity of PCR with each primer set was highly dependant of the sample preparation and DNA extraction method. The highest sensitivities for all the primers tested were determined using DNA extracted from whole blood samples, while the lowest sensitivities were obtained when DNA was extracted from filter paper preparations. To conclude, the obtained results are discussed and a protocol for diagnosis and surveillance for T. vivax trypanosomosis is recommended.     

The economics of using artificially dried green crops in milk production

From comprehensive experiments in Denmark, it is found that traditional roughage can be substituted by artifically dried grass (cobs) without any technical changes in the milk produced, but with a reduced labour requirement. With respect to maintaining a high feed utilization wafers are preferable to cobs and cobs to pellets.The future of artificially dried crops depends greatly on the ability of the industry to secure a high and uniform quality of these products. If this is achieved, the dried feeds can substitute a great part of the grain mix (concentrates) without any significant reduction of a typical basic ration of roughage for milk cows. Dried crops can be strongly competitive in certain circumstances, depending on its relative to grain mix.As an alternative for traditional conservation methods of grassland crops, artificial drying offers technical advantages, but it is not economically competitive when energy (oil etc.) prices are high. The economic importance of dried green crops can be increased through development of the organization and technique in the drying industry to ensure better plant material, a more efficient drying temperature and more efficient further processing in the plant. An open, but very important question is: what will the price of energy for the drying plant be in the future?     

A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.     

Maturation ability after transfer of freeze‐dried germinal vesicles from porcine oocytes

The purpose of this study was to examine whether freeze‐dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze‐drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze‐dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase‐II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze‐dried GVs with intact nuclear membrane and DNA stored at ?20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze‐dried GV stored for 15 or 30 days at ?20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze‐dried GVs.     

Detection of coronavirus in calf faeces with a haemadsorption-elution-haemagglutination assay (HEHA)

A simple and rapid procedure has been developed for the detection of bovine coronaviruses in faecal specimens. The method consists of adsorption of the virus onto mouse erythrocytes at 4°C, removal of unadsorbed material and elution of adsorbed viral material at 37°C. The eluate is then used in a haemagglutination test. Specificity of the reaction is checked by a blocking assay. No non-specific reactions have been observed. The sensitivity of the test appeared to be better than that of the electron microscope, at least when crude faecal extracts are used. By ultracentrifugation of the eluates the sensitivity of the assay can be further improved.     

Detection and Identification of Mycoplasma conjunctivae in Infectious Keratoconjunctivitis by PCR. Based on the16S. rRNA. Gene

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 × 10⁵ by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

褐马鸡非损伤性取样、微量取样DNA提取的初步研究

褐马鸡是中国特有的国家Ⅰ级保护濒危鸟类,为了解决野生褐马鸡分子生物学研究中存在的取样局限性,采用非伤害性取样和非损伤性取样,对褐马鸡微量血液、皮、脚垫、指甲、羽毛、陈旧标本的皮以及粪便的DNA提取进行了初步研究。结果表明,从褐马鸡微量血液（10μL）中可以提取到浓度和纯度高的基因组DNA（41.85μg）;从褐马鸡的皮、脚垫、指甲、羽毛中可以提取出基因组DNA并扩增出目标片断;陈旧标本的皮能够提取到基因组DNA。该研究将拓宽褐马鸡分子生物学研究的采样范围,并可以提高野外非损伤性取样在保护遗传学中的应用。     

Molecular characterization of Brazilian isolates of orf virus

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.     

HPLC-MS/MS法检测猪肉中苯并菲啶类生物碱的研究

利用高效液相色谱串联三重四极杆质谱技术建立了猪肉中痕量苯并菲啶类生物碱定量检测方法。将猪肉样品匀浆经1％盐酸一甲醇（10：90,V／V）超声提取后,离心分离取上清液经脱脂后氮气吹干浓缩,甲醇复溶。采用C18色谱柱（2．1mm×150mm,2．7μm）分离,流动相由乙腈与0．2％甲酸组成,梯度洗脱,柱温35℃,流速0.3mL／min;采用液相色谱-电喷雾离子源质谱检测。血根碱和白屈菜红碱在0．5～50ng／mL浓度范围内与峰面积具有良好的线性关系．线性相关系数分别为R2=0．9996和R2=0．9995．平均加标回收率分别为91．50％和91．30％,重复性RSD为11．68％和9．90％。精密度RSD为2．58％和3．89％。测定饲喂博落回散的猪的肌肉中血根碱和白屈菜红碱的残留量分别为4．76ng／g和2．09ng／g。该方法灵敏度高,重复性好,适合于猪肉中苯并菲啶类残留的定量检测。     

Establishing conditions for the storage and elution of rabies virus RNA using FTA? cards

The Flinders Technology Associates filter paper cards (FTA^® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA^® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA^® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA^® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA^® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA^® cards.     

Real-time fluorescence resonance energy transfer PCR with melting curve analysis for the detection of Opisthorchis viverrini in fish intermediate hosts

A real-time fluorescence resonance energy transfer (FRET) PCR combined with a melting curve analysis was developed for the detection of Opisthorchis viverrini in its fish intermediate host, cyprinoid fishes. Real-time FRET PCR is based on a fluorescence melting curve analysis of a hybrid between an amplicon generated from a family of repeated DNA elements, the pOV-A6 specific probe sequence (Genbank Accession No. S80278), a 162bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled probes. The real-time FRET PCR could detect as little as a single metacercaria artificially inoculated in 30 fish samples. The O. viverrini infected fishes were distinguished from non-infected fishes and from the genomic DNA of other parasites by their melting temperature. Sensitivity and specificity of this method were both 100% in the laboratory setting and it outperformed the microscopic method on field-collected samples as well. Melting curve analysis is a rapid, accurate, and sensitive alternative for the specific detection of O. viverrini infected fishes. It allows a high throughput and can be performed on small samples. The assay has not only great potential for epidemiological surveys of fish intermediate hosts but it could also be adapted as screening tool for a range of foodborne parasites in freshwater fishes.     

Determination of the number of somatic cells in milk using the rapid diphenylamine DNA filter method

The rapid reaction of the diphenylamine agent with DNA was used for the determination of the counts of somatic cells in cow's milk, using the DNA filter method. The method is based on the filtration of a warmed (65-70 degrees C) mixture of milk with Triton X-100 through the Synpor nitrocellulose membrane filter, pore size 2 to 5 microns, and subsequent DNA determination of the collected somatic cells by the colour reaction of diphenylamine. A 2ml quantity of distilled water and 4 ml of diphenylamine reagent were added to the membrane filters with somatic cells. The mixture is warmed in water bath at 90 to 100 degrees C for 20 min., then it is cooled, centrifuged (3500 X g, 15 min.), and the optical density is measured at 595 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content to the counts of cells. The average variation coefficient of the determination was 5.9% and the coefficient of correlation between the diphenylamine DNA filter method and the direct microscopy of the somatic cells on membrane filters was r = 0.997. Using the diphenylamine DNA filter method, the counts of somatic cells can also be determined from milk samples stored in frozen condition or from the filters with collected cells kept at the temperature of 4 degrees C (10 days) or 25 degrees C (3 days). Milk stabilized with formaldehyde can also be used for the determination if stored at 4 degrees C.     

Tsetse flies as vectors of trypanosomes

Glossina spp. can be naturally infected with trypanosomes belonging to the subgenera Duttonella, Nannomonas and Trypanozoon; rates of infection vary but are, in general, highest with those trypanosomes which have the simplest cycle of development in the insect and lowest in those with the most complicated cycle of development. Differences in rates of infection have mainly been accounted for in terms of such factors as the maintenance temperature of puparia and adults, the age of the fly at the time of the infective feed and, perhaps most important, the type of host animals on which the flies feed. Differences in infectibility occur between species of Glossina and may occur between different individuals of a single species. The nature of the mechanism involved is unknown; there is no evidence that trypanosomes have any pathogenic effect on Glossina. Cyclical transmission of a strain of Trypanosoma brucei through Glossina seems to have little effect on the antigenic characters of the strain, the tsetse fly acting only as a carrier of the strain which either remains unaltered throughout the period required for cyclical development and for the rest of the life of the fly or reverts to a so-called basic antigenic type.