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PRRSV surveillance, elimination and immunity in boars and boar semen.

机译：公猪和公猪精液的PRRSV监测，消除和免疫力。

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摘要

Porcine reproductive and respiratory syndrome (PRRS) is caused by a single stranded positive sense RNA virus. The virus is widespread and economically devastating to the swine industry. Transmission through insemination of tainted semen, biosecurity breaches, and pig-to-pig contact have led to the need for better understanding of superior diagnostics and elimination techniques.;The first objective of this study was to evaluate the feasibility of using PCR to detect PRRSV in pooled samples through the estimation of the sensitivity of RT-PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twenty-nine boars were inoculated with a low virulent PRRSV isolate. Serum, blood swab, and semen samples were collected every 2 to 3 days for two weeks. The data showed that serum was the best sample to detect PRRSV during acute infection, with the blood swab performed in a close second. PRRSV was not detected in semen samples in most cases. Pooling samples in pools of 3 and 5 resulted in a decrease in sensitivity, particularly within the first 3 days after infection. Sensitivity was reduced by 6% and 8% when serum or blood swab samples were run in pools of 5, respectively.;The second objective of this study was to use a previously developed density gradient centrifugation technique to purify boar semen of PRRSV. However, semen was purified by using a "scaled up" method from a 15 ml tube to a 50 ml tube for this study, allowing for a more expedient and useful method. A majority of the samples were purified from PRRSV using this method but further refinements or other methods will be necessary to ensure PRRSV is eliminated consistently from all PRRSV positive semen samples.;The final objective of this study was to measure seminal cytokine levels in PRRSV infected (n=8) and non-infected (n=4) boars in an attempt to determine the local immunological factors that influence the duration and viral load of PRRSV shedding in the semen of boars. A sandwich ELISA was performed using swine specific MAbs to detect pro-inflammatory (IL-1beta, IL-8, IL-6, TNF alpha (R&D Systems Inc., Mpls., MN)), Th2 (IL-10 (R&D)) and Th1 (IL-12 (R&D), IFN-gamma (Biosource, Carlsbad, CA)) cytokines. Due to the observance of inhibitors in boar seminal plasma, a 100 kDa Amicon filter (Millipore, Corp., Billerica, MA) was used to remove high molecular weight proteins, glycoproteins and lipids from the sample prior to performing the enzyme immunoassay. Both PRRSV infected and non-infected boars had levels of IL-12 ranging from 626--1442 pg/ml, IFN-gamma levels ranged from 177--597 pg/ml, IL-1beta levels ranged from 28--942 pg/ml and IL-8 levels ranged from 127--564 pg/ml. Other cytokine levels appear to be negligible or below the level of detection of the assays. IL-12 levels were significantly higher in the seminal plasma of PRRSV-infected boars when contrasting cytokine seminal plasma levels with the non-infected group (P=0.0424). INF-gamma seminal cytokine levels were statistically higher in the seminal plasma when contrasting the concentrations with the serum cytokine concentrations in the infected group (P0.001).;In summary, the first objective of this study validated the blood swab sample as a specimen for detection of PRRSV with PCR; although, serum is still the "gold standard". The first objective also demonstrated that pooling of samples for PCR will decrease the sensitivity of the assay. The "PRRSV elimination from semen" objective demonstrated that more improvement needs to be completed on the "scaled-up technique" to obtain consistent PRRSV-free semen. The immunity objective was successful in measuring seminal cytokine levels in boars. IL-12 is known to promote the production of INF-gamma which may correlate to the significant findings of the seminal cytokine concentrations of these two cytokines in this study. Further studies with a virulent strain of PRRSV that elicits a strong immune response may lead to more insights with the immunological studies.
机译：猪生殖和呼吸综合症（PRRS）是由单链阳性正义RNA病毒引起的。该病毒对养猪业来说是广泛传播的，并且在经济上具有毁灭性。通过精液污染，生物安全性破坏以及猪与猪之间的接触进行传播，导致需要更好地了解卓越的诊断和消除技术。本研究的第一个目标是评估使用PCR检测PRRSV的可行性通过评估RT-PCR对分别运行的不同生物样品（3个库和5个库）的敏感性，对29个公猪接种了低毒PRRSV分离株。每两到三天收集一次血清，血液拭子和精液样本，持续两周。数据显示，血清是在急性感染期间检测PRRSV的最佳样品，而血液拭子在紧随其后的时间内进行。在大多数情况下，在精液样本中未检测到PRRSV。在3和5的池中合并样品会导致敏感性降低，尤其是在感染后的前3天内。当将血清或血液拭子样本放入5个池中运行时，灵敏度分别降低了6％和8％。该研究的第二个目标是使用先前开发的密度梯度离心技术纯化PRRSV的公猪精液。但是，通过使用“放大”方法从15 ml试管到50 ml试管中纯化精液，以进行更方便，更有用的方法。使用该方法从PRRSV中纯化了大多数样品，但仍需要进一步改进或采取其他方法以确保从所有PRRSV阳性精液样品中始终消除PRRSV .;本研究的最终目标是测量PRRSV感染者的精浆细胞因子水平（n = 8）和未感染（n = 4）的公猪试图确定影响公猪精液中PRRSV脱落持续时间和病毒载量的局部免疫学因素。使用猪特异性MAb进行夹心ELISA，以检测促炎因子（IL-1beta，IL-8，IL-6，TNFα（R＆D Systems Inc.，Mpls。，MN）），Th2（IL-10（R＆D） ）和Th1（IL-12（R＆D），IFN-γ（Biosource，Carlsbad，CA））细胞因子。由于在公猪精浆中观察到抑制剂，因此在进行酶免疫测定之前，使用100 kDa Amicon过滤器（Millipore，Corp。，Billerica，MA）从样品中去除高分子量蛋白质，糖蛋白和脂质。 PRRSV感染和未感染的公猪的IL-12水平为626--1442 pg / ml，IFN-γ水平为177--597 pg / ml，IL-1beta水平为28--942 pg / ml ml和IL-8水平范围为127--564 pg / ml。其他细胞因子水平似乎可以忽略不计或低于测定的检测水平。将细胞因子精浆血浆水平与未感染组进行对比时，PRRSV感染公猪的精浆中IL-12水平显着升高（P = 0.0424）。与感染组的血清细胞因子浓度相比，精浆中的INF-γ精液细胞因子水平在统计学上较高（P <0.001）。总之，本研究的第一个目标是验证血拭子样本是否为标本用于通过PCR检测PRRSV；虽然，血清仍然是“黄金标准”。第一个目标还表明，将样品汇总用于PCR将降低测定的灵敏度。 “从精液中消除PRRSV”的目标表明，需要进一步改进“放大技术”才能获得一致的不含PRRSV的精液。免疫目标成功地测量了公猪精液中的细胞因子水平。已知IL-12会促进INF-γ的产生，这可能与本研究中这两种细胞因子的精浆细胞因子浓度的重要发现有关。对PRRSV强毒株引起强烈免疫反应的进一步研究可能会为免疫学研究带来更多见识。

著录项

  • 作者

    Clement, Travis.;

  • 作者单位

    South Dakota State University.;

  • 授予单位 South Dakota State University.;
  • 学科 Biology Animal Physiology.;Biology Veterinary Science.;Biology Zoology.
  • 学位 M.S.
  • 年度 2008
  • 页码 123 p.
  • 总页数 123
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生理学;动物学;动物学;
  • 关键词

  • 入库时间 2022-08-17 11:39:01

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