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Trichinella-induced immunomodulation: Another tale of helminth success 旋毛虫诱导的免疫调节:另一个蠕虫成功的故事
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00164
F. Bruschi , D.S. Ashour , A.A. Othman

Trichinella spiralis is a unique parasite in that both the adults and larvae survive in two different intracellular niches in the same host. The immune response, albeit intense, is highly modulated to ensure the survival of both the host and the parasite. It is skewed to T helper 2 and regulatory arms. Diverse cells from both the innate and adaptive compartments of immunity, including dendritic cells, T regulatory cells, and alternatively activated macrophages are thought to mediate such immunomodulation. The parasite has also an outstanding ability to evade the immune system by several elaborate processes. The molecules derived from the parasites including Trichinella, particularly the components of the excretory–secretory products, are being continually identified and explored for the potential of ameliorating the immunopathology in animal models of diverse inflammatory and autoimmune human diseases. Herein we discuss the various aspects of Trichinella-induced immunomodulation with a special reference to the practical implications of the immune system manipulation in alleviating or possibly curing human diseases.

旋毛虫是一种独特的寄生虫,成虫和幼虫在同一寄主的两个不同的细胞内生态位中生存。免疫反应,尽管强烈,是高度调节的,以确保宿主和寄生虫的生存。它偏向于t2和监管部门。来自固有免疫和适应性免疫区室的多种细胞,包括树突状细胞、T调节细胞和选择性活化的巨噬细胞被认为介导这种免疫调节。寄生虫还具有通过几个复杂的过程逃避免疫系统的杰出能力。包括旋毛虫在内的寄生虫衍生的分子,特别是其排泄-分泌产物的成分,正在不断被鉴定和探索,以改善各种炎症和自身免疫性人类疾病动物模型的免疫病理。在这里,我们讨论了旋毛虫诱导的免疫调节的各个方面,特别提到了免疫系统操作在减轻或可能治愈人类疾病中的实际意义。
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引用次数: 9
Ascaridoid nematodes infecting commercially important marine fish and squid species from Bangladesh waters in the Bay of Bengal 在孟加拉湾孟加拉水域,蛔虫线虫感染商业上重要的海鱼和鱿鱼物种
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00157
Miguel Bao , Paolo Cipriani , Lucilla Giulietti , Mohammad Ashraful Alam , Marialetizia Palomba , Simonetta Mattiucci , Arne Levsen

Parasitic ascaridoid nematodes occur in a wide range of marine organisms across the globe. Some species of the anisakid family (Ascaridoidea: Anisakidae) can cause gastrointestinal disease in humans (i. e. anisakidosis). Despite their importance as potentially hazardous parasites, the occurrence and infection characteristics of ascaridoids are still poorly known from many host species and geographical areas. This study investigated the diversity and infection levels of ascaridoid parasites in various commercial fish and squid host species off Bangladesh. Fish and squid specimens were visually inspected for nematodes using the UV-press method. Nematodes were assigned to genus level based on morphology and identified by sequence analyses of the entire ITS region and partial 28S rDNA and mtDNA cox2 genes. Third-stage larvae (L3) of Anisakis typica occurred at low prevalence (P = 10% and 8%, respectively) in the viscera of Selar crumenophthalmus and Trichiurus lepturus, while Hysterothylacium amoyense occurred in the viscera of Sardinella fimbriata (P = 1%) and the viscera and muscle of Harpadon nehereus (P = 32%) and T. lepturus (P = 76%). Lappetascaris sp. Type A L3 occurred in the mantle of the squid Uroteuthis duvaucelii (P = 11%). Anisakis and Lappetascaris species, and H. amoyense were firstly identified in the Bay of Bengal. The potentially zoonotic A. typica was only found in fish viscera. Hysterothylacium amoyense and Lappetascaris sp., both generally regarded as non-zoonotic, occurred at low prevalence in the muscle or mantle of fish or squid, respectively. Since consumption of raw or lightly processed seafood seems to be rare in Bangladesh, the risk of acquiring anisakidosis from consuming fishery products from off Bangladesh appears to be low. Due to its reddish appearance, the visual presence of H. amoyense larvae in fish flesh may represent a food quality issue.

寄生蛔虫线虫在全球范围内广泛存在于海洋生物中。某些种类的八角虫科(蛔虫总科:八角虫科)可引起人类胃肠道疾病(即八角虫病)。尽管蛔虫作为具有潜在危险的寄生虫具有重要意义,但在许多宿主物种和地理区域,蛔虫的发生和感染特征仍然知之甚少。本研究调查了孟加拉国外海各种商业鱼类和鱿鱼宿主物种中蛔虫寄生虫的多样性和感染水平。鱼和鱿鱼标本采用紫外压片法目测检测线虫。根据线虫的形态划分属级,并对其ITS区和部分28S rDNA和mtDNA cox2基因进行序列分析。典型异尖线虫的第三期幼虫(L3)在眼鳞鱼和瘦毛鱼的内脏中发生率较低(P = 10%和8%),而amothyacium amothyense在细鳍沙丁鱼的内脏(P = 1%)、内氏哈帕顿鱼的内脏和肌肉中(P = 32%)和瘦毛鱼(P = 76%)中发生。Lappetascaris sp. Type A L3出现在duvaucelii乌贼的地幔中(P = 11%)。Anisakis、Lappetascaris和H. amoyense在孟加拉湾首次被发现。潜在的人畜共患型典型拟虫仅在鱼内脏中发现。一般认为非人畜共患的amayense和Lappetascaris sp.分别在鱼类和鱿鱼的肌肉和外套膜中发病率较低。由于在孟加拉国很少食用生的或轻度加工的海产品,因此食用孟加拉国外海的渔业产品而患茴香病的风险似乎很低。由于其略带红色的外观,在鱼肉中出现的amayense幼虫可能代表食品质量问题。
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引用次数: 1
Horizontal gene transfer provides insights into the deep evolutionary history and biology of Trichinella 水平基因转移提供了深入了解旋毛虫的进化历史和生物学
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00155
Dante Zarlenga , Peter Thompson , Makedonka Mitreva , Bruce A. Rosa , Eric Hoberg

Evolution involves temporal changes in the characteristics of a species that are subsequently propagated or rejected through natural selection. In the case of parasites, host switching also plays a prominent role in the evolutionary process. These changes are rooted in genetic variation and gene flow where genes may be deleted, mutated (sequence), duplicated, rearranged and/or translocated and then transmitted through vertical gene transfer. However, the introduction of new genes is not driven only by Mendelian inheritance and mutation but also by the introduction of DNA from outside a lineage in the form of horizontal gene transfer between donor and recipient organisms. Once introduced and integrated into the biology of the recipient, vertical inheritance then perpetuates the newly acquired genetic factor, where further functionality may involve co-option of what has become a pre-existing physiological capacity. Upon sequencing the Trichinella spiralis (Clade I) genome, a cyanate hydratase (cyanase) gene was identified that is common among bacteria, fungi, and plants, but rarely observed among other eukaryotes. The sequence of the Trichinella cyanase gene clusters with those derived from the Kingdom Plantae in contrast to the genes found in some Clade III and IV nematodes that cluster with cyanases of bacterial origin. Phylogenetic analyses suggest that the Trichinella cyanase was acquired during the Devonian period and independently from those of other nematodes. These data may help inform us of the deep evolutionary history and ecological connectivity of early ancestors within the lineage of contemporary Trichinella. Further, in many extant organisms, cyanate detoxification has been largely superseded by energy requirements for metabolism. Thus, deciphering the function of Trichinella cyanase may provide new avenues for treatment and control.

进化涉及物种特征的时间变化,这些特征随后通过自然选择繁殖或被拒绝。以寄生虫为例,宿主转换在进化过程中也起着重要作用。这些变化根植于基因变异和基因流动,基因可能被删除、突变(序列)、复制、重排和/或易位,然后通过垂直基因转移传播。然而,新基因的引入不仅仅是由孟德尔遗传和突变驱动的,还可以通过供体和受体生物之间水平基因转移的形式从外部谱系引入DNA。一旦引入并整合到接受者的生物学中,垂直遗传就会使新获得的遗传因素永久化,其中进一步的功能可能涉及对已经成为预先存在的生理能力的共同选择。在对旋毛虫(进化支I)基因组测序后,发现了一种在细菌、真菌和植物中常见的氰酸水合酶(氰化酶)基因,但在其他真核生物中很少观察到。旋毛虫的氰化酶基因序列与来自植物界的基因集群形成对比,而在一些进化枝III和IV线虫中发现的基因与来自细菌的氰化酶集群形成对比。系统发育分析表明,旋毛虫氰化酶是在泥盆纪获得的,独立于其他线虫。这些数据可能有助于我们了解当代旋毛虫谱系中早期祖先的深层进化历史和生态连通性。此外,在许多现存的生物体中,氰酸盐解毒在很大程度上已被代谢的能量需求所取代。因此,破译旋毛虫氰化酶的功能可能为治疗和控制提供新的途径。
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引用次数: 0
Serological testing for Trichinella infection in animals and man: Current status and opportunities for advancements 动物和人类旋毛虫感染的血清学检测:现状和发展机遇
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00165
María Ángeles Gómez-Morales, Simona Cherchi, Alessandra Ludovisi

Serological tests are widely used for the detection of Trichinella spp. infections in animals and humans. Despite some limitations, (such as low sensitivity in the early period after infection) ELISA and western blot testing have demonstrated good performance when excretory/secretory products from muscle larvae are used as antigens in agreement with the International Commission on Trichinellosis. Over recent decades, considerable progress has been made in the characterization of Trichinella-derived molecules in the hope of improving diagnosis, mainly during the early days post infection. Despite these efforts, validated tests using characterized antigens for early diagnosis are still not available. However, combining currently available sero-diagnostic tools with clinical and epidemiological data provides valuable information on Trichinella infections in humans and animals as shown in this review.

血清学试验广泛用于检测动物和人类中的旋毛虫感染。尽管存在一些局限性(如感染后早期敏感性较低),但与国际旋毛虫病委员会一致,ELISA和western blot检测在将肌肉幼虫的排泄/分泌产物用作抗原时表现良好。近几十年来,在旋毛虫衍生分子的表征方面取得了相当大的进展,希望改善诊断,主要是在感染后的早期。尽管做出了这些努力,但仍然没有使用特征抗原进行早期诊断的有效测试。然而,将目前可用的血清诊断工具与临床和流行病学数据相结合,可以提供有关人类和动物旋毛虫感染的宝贵信息。
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引用次数: 3
Congenital toxoplasmosis: Should we still care about screening? 先天性弓形虫病:我们还应该关注筛查吗?
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00162
Eskild Petersen , Valeria Meroni , Daniel V. Vasconcelos-Santos , Laurent Mandelbrot , Francois Peyron

Prenatal systematic screening for congenital toxoplasmosis has been performed in Austria and France since 1975 and neonatal screening for congenital toxoplasmosis has been part of the New England Newborn screening program since 1986.

In this narrative review we review the data leading up to the systematic screening programs in Austria and France, highlighting the main finding of the European Union funded research in the 1990s and early 2000s. Different descriptive studies of the effect of pre- or postnatal treatment are discussed. Toxoplasma gondii has different genetic lineages with different pathogenicity in humans. This means that results in areas with a low pathogenic lineage cannot be extrapolated to an area with highly pathogenic lineages. The importance of meat as a source of infection is discussed in the light of an increased prevalence of T.gondii in organic livestock production .

自1975年以来,奥地利和法国已开始进行先天性弓形虫病的产前系统筛查,自1986年以来,新生儿先天性弓形虫病筛查已成为新英格兰新生儿筛查计划的一部分。在这篇叙述性综述中,我们回顾了导致奥地利和法国系统筛查项目的数据,重点介绍了欧盟在20世纪90年代和21世纪初资助的研究的主要发现。对产前或产后治疗效果的不同描述性研究进行了讨论。刚地弓形虫具有不同的遗传谱系,对人类具有不同的致病性。这意味着低致病性谱系地区的结果不能外推到高致病性谱系地区。鉴于弓形虫在有机牲畜生产中的流行率增加,本文讨论了肉类作为感染源的重要性。
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引用次数: 5
Dispersal of taeniid eggs: Experimental faecal contamination of forest environment followed by DNA detection in wild berries 带绦虫卵的传播:森林环境的粪便污染实验,随后在野生浆果中进行DNA检测
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00152
Sanna Malkamäki , Antti Oksanen , Anu Näreaho , Antti Sukura

To understand Taeniidae epidemiology, the principles of egg-dispersion dynamics under natural conditions must be known. In this study, non-zoonotic Taenia laticollis was used as a model parasite for the family Taeniidae (including Echinococcus spp.). An experiment to investigate dispersion from contaminated faeces to the surroundings was performed both with bilberries (Vaccinium myrtillus) and lingonberries (Vaccinium vitis-idaea), both of which are commercially harvested wild berries in Finland. For this experiment, 30 g of fox faeces was inoculated with 30,000 T. laticollis eggs for the bilberry experiment and 100,000 eggs for the lingonberry experiment. The faecal material was placed in the middle of good berry growth areas in four locations for bilberries and eight locations for lingonberries. After 41–42 days, berries at different distances (0–15 m) from the original contamination spot were collected and delivered to our laboratory. DNA was extracted from washed and sieved material and analysed using T. laticollis-specific semi-quantitative SYBR Green real-time polymerase chain reaction (qPCR). Taenia laticollis-specific DNA was recovered from 67% (8/12) of bilberry samples but not reliably from any of the lingonberry samples 0% (0/24), although the exposure dose was higher for those. The qPCR results suggest that under natural conditions, taeniid egg dispersion from the contamination spot is demonstrated but attachment is berry specific. The surface of bilberries may be more adhesive for taeniid eggs than the waxier and harder pericarp of the lingonberries or there might be a difference in the dispersal mechanism caused by different biotopes.

为了了解带绦虫的流行病学,必须了解自然条件下卵分散动力学的原理。本研究以非人畜共患的带绦虫(Taenia laticollis)作为带绦虫科(包括棘球绦虫)的模式寄生虫。研究人员利用越橘(Vaccinium myrtillus)和越橘(Vaccinium vitis-idaea)进行了一项调查受污染粪便向周围环境扩散的实验,这两种浆果都是芬兰商业收获的野生浆果。本实验用30 g狐狸粪便接种3万枚血蜱卵进行越橘实验,10万枚血蜱卵进行越橘实验。粪便被放置在四个越橘生长区域和八个越橘生长区域的中间。在41-42天后,收集离原始污染点不同距离(0-15 m)的浆果并送到我们的实验室。从洗涤和筛选的材料中提取DNA,并使用T. laticollis特异性半定量SYBR Green实时聚合酶链反应(qPCR)进行分析。从67%(8/12)的越桔样品中恢复了带绦虫特异性DNA,但从任何0%(0/24)的越橘样品中都不可靠,尽管这些样品的暴露剂量更高。qPCR结果表明,在自然条件下,带绦虫卵从污染点分散,但附着是浆果特异性的。与越橘果皮蜡质较硬相比,越橘果皮表面对带卵的粘附性更强,或者不同的生物群落对带卵的传播机制存在差异。
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引用次数: 0
Validation of a multilocus genotyping scheme for subtyping Cryptosporidium parvum for epidemiological purposes 用于流行病学目的的小隐孢子虫亚型多位点基因分型方案的验证
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00151
Guy Robinson , Gregorio Pérez-Cordón , Clare Hamilton , Frank Katzer , Lisa Connelly , Claire L. Alexander , Rachel M. Chalmers

Subtyping Cryptosporidium parvum for outbreak investigations or epidemiological surveillance usually relies on DNA sequence analysis of a gene coding for a 60 KDa glycoprotein (gp60). Although gp60 can be useful for allelic discrimination and to help investigate sources and routes of transmission, the presence of common subtypes and recombination during the parasite's sexual life-cycle demand a multilocus-based method for more discriminatory genotyping. While whole genome sequencing would provide the ultimate approach, it is a time consuming and expensive option for faecal parasites such as Cryptosporidium that occur at low density and are difficult to propagate routinely. In this study, we selected and evaluated a panel of previously identified variable-number tandem-repeat (VNTR) markers, to establish a multilocus genotyping scheme based on fragment sizing, appropriate for inter-laboratory surveillance and outbreak investigations. Seven VNTR markers were validated in vitro and demonstrated typeability of 0.85 and discriminatory power of 0.99. The discriminatory power was much greater than the currently used gp60 sequencing (0.74), which identified 26 subtypes, compared to 100 different MLVA profiles within the same sample set. The assay was robust, with repeatable results and reproducibility across three laboratories demonstrating the scheme was suitable for inter-laboratory comparison of C. parvum subtypes. As the majority of genotypes (79%) were unique among epidemiologically unrelated samples, there was efficiency to infer linkage, and epidemiological concordance was observed in historical outbreaks. We propose that the multilocus variable number of tandem repeats analysis scheme is suitable to assist outbreak investigations.

为疫情调查或流行病学监测分型细小隐孢子虫通常依赖于编码60 KDa糖蛋白(gp60)的基因的DNA序列分析。虽然gp60可以用于等位基因的区分,并有助于调查传播来源和途径,但在寄生虫的性生命周期中,常见亚型和重组的存在需要一种基于多位点的方法来进行更具歧视的基因分型。虽然全基因组测序将提供最终的方法,但对于像隐孢子虫这样的粪便寄生虫来说,这是一个耗时且昂贵的选择,因为隐孢子虫的密度很低,而且很难常规繁殖。在这项研究中,我们选择并评估了一组先前确定的可变数量串联重复(VNTR)标记,以建立基于片段大小的多位点基因分型方案,适用于实验室间监测和疫情调查。体外验证了7个VNTR标记,其分型能力为0.85,区分力为0.99。与同一样本集中的100种不同的MLVA谱相比,目前使用的gp60测序(0.74)鉴定出26种亚型,其鉴别能力远远大于当前使用的gp60测序(0.74)。该试验具有稳健性,在三个实验室中具有可重复的结果和再现性,表明该方案适用于小孢子虫亚型的实验室间比较。由于大多数基因型(79%)在流行病学上不相关的样本中是独特的,因此可以有效地推断联系,并且在历史暴发中观察到流行病学一致性。我们建议多位点可变重复序列分析方案适合于协助疫情调查。
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引用次数: 8
Is Cryptosporidium a hijacker able to drive cancer cell proliferation? 隐孢子虫是一种能够驱动癌细胞增殖的劫机者吗?
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00153
Gabriela Certad

The pathophysiological mechanisms of Cryptosporidium infection are multifactorial and not completely understood. Some advances achieved recently revealed that the infection by Cryptosporidium parvum induces cytoskeleton remodeling and actin reorganization through the implication of several intracellular signals involving, for example, PI3K, Src, Cdc42 and GTPases. It has also been reported that the infection by C. parvum leads to the activation of NF-κβ, known to induce anti-apoptotic mechanisms and to transmit oncogenic signals to epithelial cells. Despite the growing evidence about the hijacking of cellular pathways, potentially being involved in cancer onset, this information has rarely been linked to the tumorigenic potential of the parasite. However, several evidences support an association between Cryptosporidium infection and the development of digestive neoplasia. To explore the dynamics of Cryptosporidium infection, an animal model of cryptosporidiosis using corticoid dexamethasone-treated adult SCID (severe combined immunodeficiency) mice, orally infected with C. parvum or Cryptosporidium muris oocysts was implemented. C. parvum-infected animals developed digestive adenocarcinoma. When mechanisms involved in this neoplastic process were explored, the pivotal role of the Wnt pathway together with the alteration of the cytoskeleton was confirmed. Recently, a microarray assay allowed the detection of cancer-promoting genes and pathways highly up regulated in the group of C. parvum infected animals when compared to non-infected controls. Moreover, different human cases/control studies reported significant higher prevalence of Cryptosporidium infection among patients with recently diagnosed colon cancer before any treatment when compared to the control group (patients without colon neoplasia but with persistent digestive symptoms). These results suggest that Cryptosporidium is a potential oncogenic agent involved in cancer development beyond the usual suspects. If Cryptosporidium is able to hijack signal transduction, then is very likely that this contributes to transformation of its host cell. More research in the field is required in order to identify mechanisms and molecular factors involved in this process and to develop effective treatment interventions.

隐孢子虫感染的病理生理机制是多因素的,尚不完全清楚。最近取得的一些进展表明,细小隐孢子虫感染通过涉及PI3K、Src、Cdc42和gtpase等细胞内信号诱导细胞骨架重塑和肌动蛋白重组。也有报道称,小孢子虫的感染会导致NF-κβ的激活,已知NF-κβ可诱导抗凋亡机制并向上皮细胞传递致癌信号。尽管越来越多的证据表明劫持细胞通路可能与癌症发病有关,但这些信息很少与寄生虫的致瘤潜能联系起来。然而,一些证据支持隐孢子虫感染与消化道肿瘤发展之间的联系。为了探讨隐孢子虫感染的动力学,采用皮质类固醇地塞米松治疗的成年SCID(严重联合免疫缺陷)小鼠,口服感染小隐孢子虫或隐孢子虫卵囊,建立隐孢子虫病动物模型。感染小弧菌的动物发生了消化腺癌。当研究这一肿瘤过程的机制时,Wnt通路和细胞骨架的改变的关键作用得到了证实。最近,与未感染的对照组相比,微阵列分析可以检测到在细小梭菌感染动物组中高度上调的促癌基因和途径。此外,不同的人类病例/对照研究报告,与对照组(没有结肠瘤变但有持续消化系统症状的患者)相比,在接受任何治疗前,最近诊断为结肠癌的患者中隐孢子虫感染的患病率明显更高。这些结果表明隐孢子虫是一种潜在的致癌因子,参与癌症的发展超出了通常的怀疑。如果隐孢子虫能够劫持信号转导,那么这很可能有助于其宿主细胞的转化。需要在该领域进行更多的研究,以确定参与这一过程的机制和分子因素,并制定有效的治疗干预措施。
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引用次数: 4
Editorial Board Page 编委会页面
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/S2405-6766(22)00025-7
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引用次数: 0
A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium 醇基卵囊灭活法用于隐孢子虫检测方法的建立
IF 2.5 Q1 Immunology and Microbiology Pub Date : 2022-06-01 DOI: 10.1016/j.fawpar.2022.e00163
Biniam Hagos, Robert E. Molestina

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

隐孢子虫是专性的细胞内寄生虫,可在儿童和免疫功能低下的成人中引起危及生命的腹泻。在摄入可在水和食物中污染、持续存在并抵抗消毒的厚壁卵囊后,通过粪-口途径传播。次氯酸钠、过氧化物、臭氧、甲醛和氨是隐孢子虫卵囊的适宜消毒剂。这些化学物质的有效浓度可能是有毒的,不适合下游研究使用无活力的卵囊。卵囊灭活方法,如紫外线、加热和乙醇或甲醇处理,通常更容易用于常规实验室使用,但它们在隐孢子虫检测开发中的适用性有限。本研究的目的是评价隐孢子虫卵囊的灭活方法,以便于在实验室中应用,并测试整个灭活卵囊在定量PCR (qPCR)中的实用性。实验是在加热(75 °C/10 min)或随时间增加乙醇和甲醇浓度的条件下进行的。采用碘化丙啶(PI)染色法、体外清除法和感染Hct-8细胞系的方法来评价处理的效果。将卵囊暴露于50%乙醇或甲醇中24 h,孢子子的脱落并未受到损害,尽管观察到明显的PI掺入。在体外实验中,这些化学物质的浓度≥70%才能完全抑制Hct-8细胞的清除和感染。通过光镜和免疫荧光观察,在4 °C下保存30 天的灭活卵囊保留了囊壁的完整性和抗原性。此外,直接应用于cop基因qPCR检测的非活卵囊为加标水样中隐孢子虫的鉴定和定量提供了有用的参考试剂。总之,我们已经建立了一种实用的方法来灭活在实验室的小C.卵囊,适用于开发针对寄生虫的检测或诊断分析。
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Food and Waterborne Parasitology
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