Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%–97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5ʹUTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.     

Characterization of bovine phenol sulfotransferases: evidence of a major role for SULT1B1 in the liver

Abstract

1.?Cattle are an important component of the human food chain. Drugs used either legally or illegally in cattle may therefore enter the food chain and it is thus important to understand pathways of drug metabolism in this species, including sulfation catalyzed by the sulfotransferases (SULTs).

2.?In this study, we have analyzed the sulfation of 4-nitrophenol and other compounds in male and female bovine liver and characterized recombinant bovine SULT isoforms 1A1 and 1B1 expressed in Escherichia coli.

3.?We found that, in contrast to most other mammalian species, the major phenol sulfotransferase SULT1A1 is not expressed in bovine liver. Rather SULT1B1 seems to be a major form in both male and female bovine liver.

4.?We also identified kinetic differences between bovine and human SULT1A1 and, using the human SULT1A1 crystal structure, identified two amino acid positions in the active site of bovine SULT1A1 (Ile₈₉Val and Phe₂₄₇Val) that may be responsible for these differences.     

Oral vaccine formulation combining tick Subolesin with heat inactivated mycobacteria provides control of cross-species cattle tick infestations

Tick vaccines are necessary as part of a One Health approach for the control of tick infestations and tick-borne diseases. Subolesin (SUB, also known as 4D8) is a tick protective antigen that has shown efficacy in vaccine formulations for the control of ectoparasite infestations and pathogen infection/transmission. A recent proof-of-concept study reported oral vaccination combining Rhipicephalus microplus SUB with heat inactivated Mycobacterium bovis (IV) as an immunostimulant for the control of cattle tick infestations. Based on the efficacy of Rhipicephalus decoloratus SUB for the control of multiple cattle tick species in Uganda, herein we design a controlled pen trial using an oral formulation combining R. decoloratus SUB with IV for the control of R. decoloratus and Rhipicephalus appendiculatus cattle tick infestations. Vaccine efficacy (E) of SUB + IV on tick life cycle was compared with IV and SUB alone and with PBS as control. The IgG antibody titers against SUB and M. bovis P22 and the serum levels of selected protein immune biomarkers (IL-1beta, TNF-alpha, C3) were determined and analyzed as possible correlates of protection. Oral immunization with IV and SUB alone and in SUB + IV combination were effective for the control of tick infestations (E = 71–96% for R. decoloratus and 87–99% for R. appendiculatus) with highest E (higher than 95%) for SUB + IV. The results demonstrated that oral immunization with the SUB + IV formulation resulted in effective control of cattle tick infestations through the activation of multiple immune mechanisms. These results support the application of oral vaccine formulations with SUB + IV for the control of cattle infestations with Rhipicephalus species towards improving animal health.     

Rapid determination of total conjugated linoleic acid concentrations in beef by 1H NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy was employed to determine the conjugated linoleic acid (CLA) content in beef muscle tissue. NMR is an inviting and greener alternative to traditional methods such as gas chromatography (GC), which can require time-consuming sample derivatization and use of large quantities of solvents. In this work, a new rapid and reliable lipid extraction protocol was combined with simple, non-destructive ¹H NMR spectroscopic analysis to measure the total CLA content in 41 Canadian beef ribeye steaks purchased in western Canada. The total CLA concentrations (mg/100 g meat) obtained using the new extraction and analysis methods were consistent with amounts found using the traditional lipid extraction and GC analysis (correlation coefficient of 0.961).     

Vaccination of feedlot cattle with extracts and membrane fractions from two Mycoplasma bovis isolates results in strong humoral immune responses but does not protect against an experimental challenge

Mycoplasma bovis is one of the most significant contributors to the bovine respiratory syndrome (BRD) that causes major losses in feedlot and dairy farms. Current experimental vaccines against M. bovis are ineffective and in some cases seem to enhance disease. Experimental infection with M. bovis induces a predominantly Th2 response and high levels of IgG1, which is an inferior opsonin and hence lacks protective capacity. In an attempt to induce a balanced (Th1/Th2) immune response, we have used CpG ODN 2007 as an adjuvant in a trial involving vaccination of cattle with M. bovis total extracts and/or membrane fractions and subsequent intranasal inoculation with an infective dose of M. bovis prepared from two different clinical isolates. Significant IgG1 serum responses were observed against both, extracts and fractions while IgG2 responses were significant against the extracts only. Proliferation of peripheral blood mononuclear cells (PBMC) after incubation with M. bovis cells was only observed in post-challenge samples of cattle vaccinated with both extracts and fractions but not in samples of cattle immunized with the membrane fractions alone. All groups showed transient weight losses and increased temperatures however, there were no significant differences in clinical parameters and survival rates between the groups.     

安全放牧控制洲滩地区耕牛血吸虫病流行的效果

本文报道了和县陈桥洲建立安全牧场、实施放牧规划控制耕牛血吸虫病流行的效果。结果耕牛感染率两年后由建场前的26.1％下降为2.0％,下降了92.5％。滩地钉螺感染率也显著下降,野粪的污染已明显减轻。初步显示实施安全放牧不失为控制洲滩地区耕牛血吸虫病流行的有效措施之一。     

心脏瓣膜置换术患者围术期细胞因子浓度变化以及脑苷肌肽的影响

目的探讨体外循环（CPB）下心脏瓣膜置换术患者围术期血清细胞因子浓度的变化以及脑苷肌肽对其影响。方法40例CPB下心脏瓣膜置换术患者，随机分为脑苷肌肽组（A组）和对照组（B组），每组20例。A组于麻醉诱导后静脉注射脑苷肌肽20ml（每毫升含神经节苷脂50pg、多肽3．2mg），B组给等容积生理盐水。分别于给药前（T1）、CPB结束（12）及结束后2h（T3）、6h（T4）、24h（T5）各时间点采集颈内静脉球部血，测定细胞因子白介素-6（IL-6）、白介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）浓度。结果两组IL-6、IL-8和TNF-α浓度在CPB后均显著升高，组间比较显示B组IL-6和IL-8浓度在T3、T4和髑时间点均显著高于A组（P〈0．05），B组TNF-α浓度在12、T3、T4和T5时间点均显著高于A组（P〈0．05）。结论脑苷肌肽可以减少体外循环心脏瓣膜置换手术中炎性细胞因子IL-6、IL-8和TNF-α的释放。     

脑苷肌肽联合丁苯酞治疗老年脑梗死的临床疗效观察

目的观察脑苷肌肽联合丁苯酞治疗老年脑梗死的临床疗效。方法选取2011年3月—2014年5月佛山市三水区人民医院收治的老年脑梗死患者106例,随机分为对照组和治疗组,每组53例。对照组静脉滴注丁苯酞氯化钠注射液100 m L/次,2次/d。治疗组静脉滴注脑苷肌肽注射液20 m L,加入5%葡萄糖注射液300 m L,1次/d,其他治疗同对照组。两组均连续治疗14 d。比较两组的临床疗效,同时比较两组治疗前后的NIHSS、m RS评分和Barthel指数(BI)变化。结果对照组和治疗组总有效率分别为73.58%、90.57%,两组比较差异有统计学意义(P0.05)。两组患者治疗后NIHSS评分、m RS评分均显著降低,BI显著升高,同组治疗前后差异有统计学意义(P0.05);治疗后治疗组这些观察指标改善程度优于对照组,两组比较差异有统计学意义(P0.05)。结论脑苷肌肽联合丁苯酞治疗老年脑梗死患者的临床疗效显著,能够提高患者的生活质量,改善患者神经细胞的功能,值得临床推广应用。     

Gene gun DNA immunization of cattle induces humoral and CD4 T-cell-mediated immune responses against the Theileria parva polymorphic immunodominant molecule

Theileria parva kills over one million cattle annually in sub-Saharan Africa. Parasite genetic complexity, cellular response immunodominance, and bovine MHC diversity have precluded traditional vaccine development. One potential solution is gene gun (GG) immunization, which enables simultaneous administration of one or more DNA-encoded antigens. Although promising in murine, porcine, and human vaccination trials, bovine GG immunization studies are limited. We utilized the model T. parva antigen, polymorphic immunodominant molecule (PIM) to test bovine GG immunization. GG immunization using a mammalian codon optimized PIM sequence elicited significant anti-PIM antibody and cell-mediated responses in 7/8 steers, but there was no difference between immunized and control animals following T. parva challenge. The results suggest immunization with PIM, as delivered here, is insufficient to protect cattle from T. parva. Nonetheless, the robust immune responses elicited against this model antigen suggest GG immunization is a promising vaccine platform for T. parva and other bovine pathogens.     

Protein,fat, moisture and cooking yields from a U.S. study of retail beef cuts

Nutrient data from the U.S. Department of Agriculture (USDA) are an important resource for U.S. and international databases. To ensure that data for retail beef cuts in USDA's National Nutrient Database for Standard Reference (SR) are current, a comprehensive, nationwide, multi-phase study was conducted. Samples were collected and analyzed in three phases based on primal category. Using a statistically based sampling plan, 72 beef carcasses per phase were obtained with nationally representative quality and yield grades, genders and genetic types. Retail cuts were fabricated, cooked and dissected to obtain component weights. Nutrient values were determined by validated laboratories using quality assurance procedures. Full nutrient profiles were made available in SR (http://www.ars.usda.gov/nutrientdata). Results for 16 beef retail cuts were compared for cooking yield and protein, fat and moisture concentrations. For example, cooked fat levels differed among three roasted cuts and among three grilled cuts from chuck, rib and loin (p < 0.01). Cooking yield for roasted ribeye (76%) was lower (p < 0.001) than for grilled ribeye (83%) or for chuck eye grilled (80%) or roasted (84%). This study demonstrates the importance of maintaining data for a variety of retail beef cuts due to their unique properties and different cooking methods.