Feasibility of using total purines as a marker for ruminal bacteria

A procedure for measuring total purine content of mixed ruminal bacteria was adapted for use in the determination of purines in pure cultures of ruminal bacteria. Recovery of adenine and guanine, alone or in mixture, was quite variable. The problem was traced to solubility of the silver salt of adenine in the acid wash solution. When the precipitating solution was used as the wash, recovery of the purines was over 97%. Recovery of a 1:1 mixture of adenine and guanine added to yeast RNA was 100.6+/-3.2%. Purine, protein, and bacterial concentrations were determined for 10 pure cultures of ruminal bacteria: Butyrivibrio fibrisolvens, D16f, H10b, and H17c; Fibrobacter succinogenes B21a; Lachnospira multiparus D25e; Lactobacillus lactis ARD26e; Prevotella ruminicola H15a; Ruminococcus albus 7; Ruminococcus flavefaciens B34b; and Streptococcus bovis ARD5d. The CV for the most-probable-number (MPN) assay (bacterial concentrations), purine analysis, and protein analysis were 55.86, 5.25 and 6.52%, respectively. Considerable variation was found among bacterial species and strains when purine and protein concentrations were compared as the amount per individual cell. More consistent values were obtained when these components were expressed on a dry matter basis. Purine:protein ratios for the 10 pure cultures ranged from .023 to .1299, with a mean value of .0883. For samples of mixed bacteria separated from ruminal fluid, this ratio was found to average .0306, which is approximately one-third of the value for the pure cultures. The value determined for the mixed bacterial sample is similar to previously reported values. Based on the ratio obtained with the pure cultures, the microbial protein flow out of the rumen has probably been overestimated in most previous reports. Limited studies suggest that the samples of mixed ruminal bacteria used as a standard are probably contaminated with feed particles containing protein, which results in lower purine:protein ratios.     

Use of hexadimethrine bromide as a heparin-neutralizing agent in canine plasma

Hexadimethrine bromide was evaluated as a heparin-neutralizing agent in a simple modification of the activated partial thromboplastin time test in canine plasma. Addition of various amounts of heparin in vitro to canine plasma indicated that heparin could be neutralized by adding 0.5 micrograms of hexadimethrine bromide 15 s before CaCl2 was added to the reaction mixture of the activated partial thromboplastin time test. In 8 dogs given (subcutaneous injection) 500 USP units of sodium heparin/kg, marked individual variations in clotting time prolongations were observed over the 12-hour period of study. The hexadimethrine bromide modification effectively neutralized the heparin-related clotting time prolongations to values that were not significantly different from base-line (preheparin) activated partial thromboplastin time values. The modification seems to be useful in confirming the presence of heparin and in monitoring heparin therapy in dogs.     

我国狍子源鞭虫的分子鉴定与进化分析

为鉴定我国黑龙江省分离的狍子源鞭虫的种类及确定其在鞭虫中的分类地位,本研究首先对鞭虫进行形态学特征观察,并利用PCR方法扩增该鞭虫r DNA ITS序列,经测序分析结果表明,r DNA ITS序列全长为1 371 bp,其中ITS1、5.8S及ITS2大小分别为800 bp、163 bp及408 bp,与之前报道的狍子源鞭虫同源性达99.8%。将其与NCBI数据库中相应鞭虫ITS序列进行比对分析,并将r DNA ITS序列作为基因标记构建鞭虫属系统发生树,结果显示本研究所分离的狍子源鞭虫与其他不同宿主的无色鞭虫均处于同一个分支,因此可鉴定本研究分离的我国狍子源鞭虫为无色鞭虫。本研究为国内首次对狍子源鞭虫进行分子鉴定及系统进化分析,为草食动物的鞭虫病分子鉴别诊断和鞭虫系统进化分析研究提供依据。     

Modification of a colorimetric analysis for lignin and its use in studying the inhibitory effects of lignin on forage digestion by ruminal microorganisms

The colorimetric acetyl bromide soluble lignin (ABSL) procedure was modified to use for analyzing intact alfalfa and its cell wall fractions for both lignin and total phenolic substances. A purified lignin extracted from alfalfa (native lignin) was used as a standard. Soluble phenolic compounds present in alfalfa did not inhibit cellulose digestion in vitro, because cell wall fractions had the same or slightly lower cellulose digestibility values than did the intact forage (intact forage = 46.5%; Morrison's cell walls = 46.4%; NDF = 42.6%; ADF = 48.7%). Disappearance of ABSL from the solid digesta was very high for intact alfalfa (48.5%), presumably reflecting either solubilization or utilization of the phenolics. However, very little ABSL was detected in the liquid fraction, suggesting that the soluble phenolic substances possibly were metabolized or modified by ruminal microorganisms. On the other hand, little, if any, of the ABSL present in the cell wall fractions disappeared after 48 h of fermentation. These data emphasize the resistance of core lignin to microbial degradation in short-term anaerobic fermentations.     

Antigenic and genetic analyses of H5 influenza viruses isolated from ducks in Asia.

The hemagglutinin (HA) of six H5 influenza virus strains isolated from ducks in Japan and China in 1976 to 1996 were analyzed antigenically and genetically. Antigenic analysis using a panel of monoclonal antibodies revealed that the HA of H5 influenza viruses isolated from ducks are antigenically closely related to each other. Phylogenetic analysis indicates that the isolates from ducks in Hokkaido were derived from an ancestor common with the highly pathogenic isolates from chickens and humans in Hong Kong in 1997.     

Regional differences in porcine adipocytes isolated from skeletal muscle and adipose tissues as identified by a proteomic approach

The content and distribution of body lipids are of special interest for production efficiency and meat quality in the farm animal industry. Triglycerides represent the most variable fraction of tissue lipids, and are mainly stored in adipocytes. Although several studies have reported regional differences in the expression of genes and their products in adipocytes from various species, the characteristics of i.m. adipocytes remain poorly described. To evaluate adipocyte features according to muscle and other fat locations, adipocyte proteins were isolated from trapezius skeletal muscle, and intermuscular, s.c., or perirenal adipose tissues from 6 female pigs (80 d of age). Protein extracts were labeled and analyzed by 2-dimensional, fluorescent, differential gel electrophoresis. The comparisons revealed that 149 spots were always differentially expressed (P < 0.05, ratio exceeding |2|-fold difference) between i.m. adipocytes and the fat cells derived from the 3 other adipose locations. The proteins that were downregulated in i.m. fat cells belonged to various metabolic pathways, such as lipogenesis (cytosolic malate dehydrogenase and isocitrate dehydrogenase, P < 0.01), glycolysis (enolases and aldolase, P 相似文献   

Feasibility of serological bulk milk testing as a method for BVD surveillance

A commercial ELISA detecting antibodies against bovine viral diarrhoea Virus (BVDV) was analysed for its applicability for bulk-milk screening. Detection limits were analysed using native and concentrated milk samples (milk treated with rennet and ammonium sulfate precipitated) from 10 cows whose sera showed different reactivity levels in the ELISA and from two cows which gave birth to persistently infected calves during the last year. Further this and a second commercial ELISA were used to screen 591 randomly selected bulk-milk samples. To clarify discrepancies thirty-nine herds were included in a follow-up study. A second bulk-milk sample and serum samples from 10 young cattle of 6 to 28 month of age per herd were analysed for antibodies against BVDV. The results of this second testing and the detection of viremic animals in 4 herds confirmed the results from initial bulk-milk testing with both tests. The analysed test is suitable for bulk-milk testing although its application is limited by vaccination.     

Feeding value of whole raw soya beans as a protein supplement for beef cattle consuming low‐quality forages

Experiments (Exp) I and II were conducted to compare raw whole soya beans (WSB), roasted (rWSB) or other protein sources as supplements of low‐quality forages fed ad libitum to beef cattle, upon DM intake (DMI), ruminal and blood parameters, and animal performance. Exp I: treatments for wheat straw fed to four ruminally cannulated steers were (i) Control‐WS: no supplement; (ii) WSB‐WS: whole soya beans; (iii) rWSB‐WS: roasted WSB; and (iv) SBM‐WS: soybean meal–wheat midds mixture; all fed at 1.4 kg DM/day. Exp II: 12 steers grazed deferred grain sorghum (DS) receiving these treatments: (i) Control‐DS: no supplement; (ii) WSB‐DS: 1.26 kg DM/day whole soya beans; and (iii) SFM‐DS: 1.35 kg DM/day of sunflower meal. In Exp I, WS DMI resulted 47, 52 and 41% greater for WSB‐WS, rWSB‐WS and SBM‐WS, respectively, than Control‐WS (p < .05). In Exp II, the DMI of DS was unaffected by supplementation; a substitution of DS by supplement was found for WSB‐DS (p < .05); however, total diet and digestible DMI increased with supplementation (p < .05). Rumen pH in Exp I remained unaffected by supplementation, but N‐NH₃ as well as blood urea‐N in Exp II increased (p < .05). In Exp II, average daily weight gains improved similarly with both supplements compared with Control‐DS. Additionally, feed‐to‐gain ratio decreased (p < .05), being lower for WSB‐DS (8.3) vs. SFM‐DS (9.9). Roasting effects of WSB as a supplement for low‐quality forages were not detected, and all protein sources increased total diet DMI and forage utilization. Only moderate cattle weight gains could be expected for unsupplemented DS.     

Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks

Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 x 10(9) and 3.2 x 10(11) colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P < or = 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the beta2-toxin.     

Estimation of undegradable intake protein in forages using neutral detergent insoluble nitrogen at a single in situ incubation time point

Two experiments were conducted to evaluate the use of neutral detergent insoluble nitrogen (NDIN) at a single in situ incubation time point to estimate the undegradable intake protein (UIP) in forages as well as to compare rates of NDIN degradation. Forage samples in Exp. 1 comprised diet samples collected from range and meadow pastures monthly from May through September. In Exp. 2, clipped samples of alfalfa, birdsfoot trefoil, kura clover, and smooth bromegrass, and diet samples of the mixed legume-grass and smooth bromegrass were evaluated. Forage samples were incubated in situ for their mean retention time (MRT) estimated from IVDMD plus a 10-h passage lag to yield the total MRT (TMRT). Samples were also incubated for 0 h, 10 h, 75% TMRT, and 96 h. Undegradable intake protein was measured at 75% TMRT and TMRT, and calculated using fractional rates of degradation and passage with a 10-h passage lag. Rates of ruminal NDIN degradation were calculated using the slope of the regression of the natural logarithm of the potentially degradable NDIN remaining (96-h undegradable fraction subtracted) against time. The estimated UIP values obtained using 75% TMRT were highly correlated with those obtained using fractional rates of degradation and passage plus accounting for a 10-h passage lag in Exp. 1 (R2 = 0.95) and Exp. 2 (R2 = 0.98). Rates of NDIN degradation of range and meadow samples in Exp. 1 were slower (P < 0.05) from 0 to 10 h in May and June compared with rates from 10 h to 75% TMRT, but rates of degradation were not different (P = 0.34 to 0.71) for the rest of the collection periods. Rates of degradation were not different from 0 to 10 h and 10 h to 75% TMRT in Exp. 2 for diet (P = 0.82) or clipped samples (P = 0.86). The UIP of the forages in these experiments was accurately estimated using NDIN at a single in situ incubation time point equivalent to 75% of the TMRT, and rates of protein degradation can be obtained at this time point when 0- and 96-h incubations are included.     

Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid

An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.     

Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013

Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.     

Pathogenicity for chickens of a reovirus isolated from turkeys

A viral agent that was isolated from livers of commercial turkey poults that died at approximately two weeks of age was characterized as a reovirus. Experimental infection of day-old chickens with this reovirus isolate resulted in the development of tenosynovitis, hepatitis, and myocarditis. In vitro neutralization of the turkey reovirus isolate by antiserum against chicken reovirus correlated with in vivo protection of maternally immune chickens from day-old oral challenge with the turkey reovirus isolate.     

Evaluation of fecal samples from mares as a source of Rhodococcus equi for their foals by use of quantitative bacteriologic culture and colony immunoblot analyses

OBJECTIVE: To determine whether mares are a clinically important source of Rhodococcus equi for their foals. SAMPLE POPULATION: 171 mares and 171 foals from a farm in Kentucky (evaluated during 2004 and 2005). PROCEDURES: At 4 time points (2 before and 2 after parturition), the total concentration of R equi and concentration of virulent R equi were determined in fecal specimens from mares by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively. These concentrations for mares of foals that developed R equi-associated pneumonia and for mares with unaffected foals were compared. Data for each year were analyzed separately. RESULTS: R equi-associated pneumonia developed in 53 of 171 (31%) foals. Fecal shedding of virulent R equi was detected in at least 1 time point for every mare; bacteriologic culture results were positive for 62 of 171 (36%) mares at all time points. However, compared with dams of unaffected foals, fecal concentrations of total or virulent R equi in dams of foals with R equi-associated pneumonia were not significantly different. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that dams of foals with R equi-associated pneumonia did not shed more R equi in feces than dams of unaffected foals; therefore, R equi infection in foals was not associated with comparatively greater fecal shedding by their dams. However, detection of virulent R equi in the feces of all mares during at least 1 time point suggests that mares can be an important source of R equi for the surrounding environment.     

Retrospective identification as influenza virus of a hemagglutinating agent isolated from turkeys in Israel in 1973

A hemagglutinating (HA) agent was isolated in Israel in 1973 from an outbreak of a respiratory disease on a turkey farm. The HA agent was preserved in a viable form until 1981 and was then identified retrospectively as influenza virus A/turkey/Ramon, Israel/73(H5N2). This finding helps to reconstruct the ecological picture of influenza virus strains circulated in avian reservoir in Israel during the last decade.     

A comparison of N-butylscopolammonium bromide and butorphanol tartrate for analgesia using a balloon model of abdominal pain in ponies.

The analgesic effect of N-butylscopolammonium bromide (0.3 mg/kg) using a balloon-induced model of colic in ponies was evaluated and compared with butorphanol tartrate (0.1 mg/kg). Eight adult ponies were used and each received both treatments during the two different trials. The order in which the treatment was received was randomly assigned. At the start of each trial, moderate abdominal pain was induced by inflation of a balloon placed in the lumen of the caecum. The ponies were evaluated every 5 minutes, and a cumulative pain score (CPS) was assigned. Two baseline measurements were recorded, followed by the administration of one of the two treatments. Assessments were continued for 60 minutes, or until moderate abdominal pain returned. Three ponies out of 8 responded to treatment with butorphanol tartrate, while 6 out of 8 ponies responded to N-butylscopolammonium bromide. There were no statistical differences in the CPS or duration of drug action between treatments.