Pub Date : 2020-04-01DOI: 10.29261/pakvetj/2019.114
Wan Xinwu, Hengyu Xi, Su JiZuo, Meng-jun Cheng, Wang Gang, H. Dali, Ruopeng Cai, Zijing Wang, Guan Yuan, Changjiang Sun, F. Xin, L. Lei, Sadeeq-ur-Rahman, Jianbao Dong, Han Wenyu, J. Gu
Pub Date : 2020-04-01DOI: 10.29261/pakvetj/2019.117
J. Hussen, T. Shawaf, Abdullah I. A. Al-Mubarak, N. Al-Humam, F. Almathen, H. Schuberth
To Cite This Article: Hussen J, Shawaf T, Al-Mubarak AIA, Al Humam NA, Almathen F and Schuberth HJ, 2020. Leukocytes immunophenotype and phagocytosis activity in pregnant and nonpregnant dromedary she camels. Pak Vet J, 40(2): 239-243. http://dx.doi.org/10.29261/pakvetj/2019.117 INTRODUCTION Embryonic loss is one of the major factors responsible for high economic losses in food animals. The maintenance of pregnancy is associated with modulations in different immune mechanisms, which ensure protection against pathogens and at the same time prevent immunological destruction of the conceptus (Aluvihare et al., 2004; Somerset et al., 2004). The pregnancy-associated changes occur not only in the local environment of the uterus but extend also to the peripheral immune system (Ott and Gifford, 2010; Kamat et al., 2016). For different species like human (Spadaro et al., 2019), cows (Leung et al., 2000; Oliveira et al., 2012), mares (Bazzano et al., 2014; Piccione et al., 2015) and sows (Zhang et al., 2017), local and systemic immunomodulatory effects of pregnancy have been widely studied. According to studies in human and rodents, both lymphoid and myeloid immune cells including NK cells, T cells, B cells, -T lymphocytes, neutrophils, monocytes, macrophages and dendritic cells play significant role in maintaining pregnancy (Lash et al., 2010; Nagamatsu and Schust, 2010; Groebner et al., 2011). In dairy cows, the presence of a conceptus resulted in increased numbers of peripheral blood myeloid cells with enhanced expression of chemotactic factors, which attract these cells to the uterus (Kamat et al., 2016). Little is known about the impact of pregnancy on the immune system of dromedary she camels. The aim of the current study was, therefore, the comparative analysis of immunophenotype of blood leukocytes and phagocytosis activity of neutrophils in pregnant and nonpregnant dromedary she camels. The results of the current study would lead to a better understanding of immunology of pregnancy and the identification of the immunologic factors associated with higher pregnancy rates in she camels. RESEARCH ARTICLE Pak Vet J, 2020, 40(2): 239-243. 240 MATERIALS AND METHODS Animals and blood sampling: In this study, 18 pregnant and 21 non-pregnant dromedary she camels (Camelus dromedarius), aged 10-14 years and maintained at the Camel Research Center, King Faisal University, Al-Ahsa, Saudi Arabia were used. The pregnant she camels were at their mid gestation (between 5 and 10 month based on sonographic examination and insemination history). Blood samples (5 ml blood from each she camel) were collected from she camels during the period between January and May 2019 by jugular venepuncture in EDTA containing vacutainer tubes (BD, Germany). All experimental procedures and management conditions used in this study were approved by the Ethics Committee at King Faisal University, Saudi Arabia (Permission number DSR 1811001). Microscopic counting of leukocytes: Whole
引用本文：Hussen J，Shawaf T，Al Mubarak AIA，Al Humam NA，Almathen F和Schuberth HJ，2020。妊娠和非妊娠单峰骆驼的白细胞免疫表型和吞噬活性。Pak Vet J，40（2）：239-243。http://dx.doi.org/10.29261/pakvetj/2019.117引言胚胎损失是造成食用动物高经济损失的主要因素之一。妊娠期的维持与不同免疫机制的调节有关，这些机制确保了对病原体的保护，同时防止了对妊娠期的免疫破坏（Aluvihare等人，2004；Somerset等人，2004年）。妊娠相关的变化不仅发生在子宫的局部环境中，还延伸到外周免疫系统（Ott和Gifford，2010；Kamat等人，2016）。对于不同的物种，如人类（Spadaro et al.，2019）、奶牛（Leung et al.，2000；Oliveira et al.，2012）、母马（Bazzano et al.，2014；Piccione et al.，2015）和母猪（Zhang et al.，2017），妊娠的局部和系统免疫调节作用已被广泛研究。根据对人类和啮齿类动物的研究， T细胞、B细胞，-T淋巴细胞、中性粒细胞、单核细胞、巨噬细胞和树突状细胞在维持妊娠中发挥着重要作用（Lash等人，2010；Nagamatsu和Schust，2010；Groebner等人，2011年）。在奶牛中，妊娠期的存在导致外周血髓细胞数量增加，趋化因子表达增强，从而将这些细胞吸引到子宫中（Kamat等人，2016）。怀孕对单峰骆驼免疫系统的影响知之甚少。因此，本研究的目的是比较分析妊娠和非妊娠单峰骆驼血白细胞的免疫表型和中性粒细胞的吞噬活性。目前的研究结果将有助于更好地了解妊娠免疫学，并确定与母骆驼妊娠率较高相关的免疫因素。研究文章Pak Vet J，2020，40（2）：239-243。240材料和方法动物和血液取样：在本研究中，使用了18头怀孕和21头未怀孕的单峰母骆驼（Camelus dromdarius），年龄10-14岁，饲养在沙特阿拉伯阿尔阿赫萨费萨尔国王大学骆驼研究中心。怀孕的母骆驼处于妊娠中期（根据超声波检查和受精史，在5到10个月之间）。在2019年1月至5月期间，通过在含有EDTA的真空管（BD，德国）中进行颈静脉穿刺，从母骆驼身上采集血样（每头母骆驼5毫升血液）。本研究中使用的所有实验程序和管理条件均经沙特阿拉伯费萨尔国王大学伦理委员会批准（许可号DSR 1811001）。白细胞的显微镜计数：全血在PBS中以1:4稀释，然后与Türk溶液混合（最终稀释1:20；Merck Millipore），将10μl混合物倒入血细胞仪（Neubauer细胞计数器）上，在显微镜下计数。白细胞（蓝色）在细胞计数器的四个大正方形中计数，并计算白细胞总数（Camacho-Fernandez等人，2018）。单克隆抗体：如表1所示，本研究中使用了9种市售单克隆抗体（mAb）。血液白细胞分离：全血白细胞的分离是在红细胞低渗裂解后进行的（Hussen等人，2017）。简言之，将血液悬浮在蒸馏水中20秒，并加入双倍浓缩的PBS以恢复张力。重复这一过程，直到形成透明的白色白细胞颗粒表明完全的红细胞溶解。最后将分离的细胞以5x10细胞/ml悬浮在膜免疫荧光（MIF）缓冲液（含有牛血清白蛋白（5g/L）和NaN3（0.1g/L）的PBS）中。通过染料排除法（碘化丙啶；2μg/ml，Calbiochem，德国）对分离细胞的平均生存能力进行流式细胞术评估，结果始终>95%。免疫荧光和流式细胞术：在含有牛血清白蛋白（5 g/L）和NaN3（0.1 g/L）的PBS中分离的白细胞（5x10个细胞/ml）在96孔圆底微量滴定板（1x10/孔；20分钟；4°C）中用CD4、WC1、MHCII和CD14特异性单克隆抗体以三种组合进行标记，包括CD4/WC1/CD14，CD14/MHCII和CD14/CD11a/CD18（Eger等人，2015；Hussen等人，2018）。在与未标记的初级抗体孵育后，洗涤细胞两次，并与用不同荧光染料标记的小鼠次级抗体IgG1、IgM和IgG2a（BD）孵育。洗涤细胞后，将直接标记的单克隆抗体添加到CD14、CD11a、CD11b和CD18中。 最后，洗涤细胞并通过流式细胞术（FACSCalibur，Becton Dickinson Biosciences）进行分析。对于每次测量，采集100000个事件，并用FlowJo（FlowJo LLC）分析数据（图1）。吞噬作用测定：用异硫氰酸荧光素（FITC，Sigma-Aldrich，St.Louis，Missouri，USA）标记热杀死的金黄色葡萄球菌（S.aureus）细菌（Pansorbin，Calbiochem，Merck，Nottingham，UK）。将FITC缀合和热灭活的金黄色葡萄球菌悬浮在Roswell Park Memorial Institute（RPMI）培养基中，并调节至2x10个细菌/ml。将分离的骆驼白细胞接种在96孔板中（1x10/孔），并在37℃、5%CO2下与标记细菌（50个细菌/细胞）孵育40分钟。洗涤后，通过流式细胞术（FACS Calibur，Becton Dickinson Biosciences，San Jose，California，USA）分析细胞。吞噬细胞阳性细胞定义为绿色荧光细胞在总细胞中的百分比。吞噬能力（作为每个细胞摄入细菌数量的指标）定义为门控吞噬阳性中性粒细胞的平均绿色荧光强度。表1：抗体列表抗原抗体克隆标记源同种型CD4 GC50A1未标记的WSU小鼠IgM WC1 BAQ128A未标记的WSU小鼠IgG1 CD14 TÜK4 PerCP Biorad mIgG2a MHCII TH81A5 WSU mIgG2aCD11a G43-25B PE BD mIgG2aCD18 6.7 FITC BD mIgG1 mIgG2a多克隆PE Invitrogen gIgG1多克隆FITC InvitrogengIgGmIgM多克隆APC Invitrogen-gIggIg:免疫球蛋白；m： 鼠标；MHC-II：主要组织相容性复合体II类，g：山羊，WSU：华盛顿州立大学。图1：用于鉴定母驼外周血中主要白细胞群体的门控策略。在SSC/FSC点图中，根据骆驼粒细胞和单核细胞（PBMC）的正向和侧向散射特征对其进行门控。在粒细胞上设置门后，根据FL1和FL2荧光通道中嗜酸性粒细胞和中性粒细胞的不同自身荧光强度来鉴定它们。在PBMC门中，单核细胞和淋巴细胞根据其不同的CD14染色进行鉴定。S S C FSC F L 1 FL2 C D 1 4 SSC嗜酸性粒细胞-中性粒细胞-单核细胞淋巴细胞图1 S S C FC F L 1 FL 2 C D 4 SSC嗜中性粒细胞
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Pub Date : 2020-04-01DOI: 10.29261/PAKVETJ/2019.014
Kim Sh, Choi Ws, Seung Bj, Choi Sh, Lim Hy, Bae Mk, J. Sur
{"title":"Statistics of Canine Skin Tumors in Korea during 2005-2018","authors":"Kim Sh, Choi Ws, Seung Bj, Choi Sh, Lim Hy, Bae Mk, J. Sur","doi":"10.29261/PAKVETJ/2019.014","DOIUrl":"https://doi.org/10.29261/PAKVETJ/2019.014","url":null,"abstract":"","PeriodicalId":19845,"journal":{"name":"Pakistan Veterinary Journal","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69865912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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