茂名市区和电白县鼠类广州管圆线虫感染调查

目的了解茂名市区和电白县鼠类广州管圆线虫感染情况,为该地区广州管圆线虫防治工作提供参考依据。方法在村内和野外采用鼠夹和捕鼠笼等方法捕捉鼠类,在确定种类后,取其心肺组织检查广州管圆线虫成虫。结果共检查鼠148只,阳性15只,鼠广州管圆线虫的总感染率为10.14%。褐家鼠、黄胸鼠和小家鼠的感染率分别为12.87%、4.76%和0。野栖鼠和家栖鼠感染率分别为17.14%（12/70）和3.85%（3/78）。结论茂名市区和电白县鼠类中存在广州管圆线虫的感染,褐家鼠是最主要的终末宿主。同时,野栖鼠的感染率明显高于家栖鼠。     

广州大学城广州管圆线虫疫源地调查

目的捕捉广州大学城褐云玛瑙螺（Achatina fulica）检查广州管圆线虫（Angiostrongylus cantonensis）第Ⅲ期幼虫的感染情况。方法采集大学城10所高校生活区和教学区褐云玛瑙螺，人工消化酶消化分离第Ⅲ期幼虫，计算感染率和感染度并与广州其他地区进行比较。结果除广东外语外贸大学、华南理工大学2个校园内未捕捉到褐云玛瑙螺外，其他8所高校均发现有褐云玛瑙螺；广州大学城广州管圆线虫总体感染率为11．8％（76／642），平均感染度为872条／螺，阳性螺中最高15725条，最低17条；中山大学校园内感染率和感染度比其他校园高，分别为22．0％（56／255），1007条／螺。结论广州大学城广州管圆线虫感染率低于广州其他地区。     

广东省佛山市褐云玛瑙螺感染广州管圆线虫的调查分析

为对佛山市褐云玛瑙螺感染广州管圆线虫第Ⅲ期幼虫的情况进行调查和比较,填补佛山市广州管圆线虫病的流行病学资料,于2006年9月～2007年7月对佛山市5区(禅城区、顺德区、南海区、三水区、高明区)15个点褐云玛瑙螺进行采样收集,采用匀浆法取渣镜检广州管圆线虫Ⅲ期线虫幼虫,计算感染率并与省内深圳、江门、湛江市廉江、肇庆、罗定、阳春和茂名等市进行比较.结果显示,佛山市褐云玛瑙螺感染广州管圆线虫总的感染率为40.75%,其中禅城区、顺德区、南海区、三水区、高明区的感染率分别为25.68%、19.05%、43.48%、52.75%和48.67%.结果表明,佛山市是广州管圆线虫病疫源地,五区褐云玛瑙螺广州管圆线虫感染率中三水区最高,顺德区最低.广东省内佛山市褐云玛瑙螺的感染率高仅次于江门市.     

广东省阳春市褐云玛瑙螺和福寿螺广州管圆线虫感染的调查

目的了解广东省阳春市褐云玛瑙螺和福寿螺广州管圆线虫的感染情况。方法在阳春当地采集野生的褐云玛瑙螺和福寿螺，在实验室用人工胃液消化法检查其中的广州管圆线虫幼虫并记数。用检获的幼虫感染实验小鼠。结果共检查褐云玛瑙螺350只，福寿螺465只，广州管圆线虫第3期幼虫的阳性率分别为7，71％和0．86％；感染度分别为1～312和1～4。从上述感染的实验小鼠中检获到广州管圆线虫第4期幼虫，且其血清特异性IgM均为阳性。结论阳春是广州管圆线虫的自然疫源地，该虫中间宿主以褐云玛瑙螺感染较严重。     

云南省传播广州管圆线虫病的螺类调查研究

目的了解云南省传播广州管圆线虫病的中间宿主螺类。方法野外调查采集贝类标本,分类鉴定,寄生虫检测。结果发现传播广州管圆线虫病的中间宿主螺类共12种。结论为广州管圆线虫病在云南省的防治提供科学依据。     

The spliced leader gene of Angiostrongylus cantonensis

A 5' leader sequence has been identified on mRNAs of the parasitic nematode Angiostrongylus cantonensis. A 720-bp XhoI restriction fragment containing the gene encoding the leader sequence has been cloned and sequenced. It contains a 22-nt sequence identical to that of the leader sequence of Caenorhabditis elegans, a consensus splice site and a putative Sm antigen binding site. The gene is present as a tandem repeating unit of approximately 60 copies, and unlike C. elegans it is not associated with the 5S ribosomal RNA gene. The SL-RNA is 110 nt long and the sequence and primer extension studies suggest that it is transcribed from the tandemly repeating SL gene. It is precipitable from cell-free extracts of adult nematodes by anti-Sm anti-sera, and from RNA by anti-TMG anti-sera, thus suggesting its inclusion with small nuclear ribonucleoproteins in RNA splicing.     

The complete mitochondrial genome of the rodent intra-arterial nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis

The two rodent intra-arterial nematodes, Angiostrongylus cantonensis and Angiostrongylus costaricensis, can cause human ill-health. The present study aimed to characterize and compare the mitochondrial (mt) genomes of these two species, and clarify their phylogenetic relationship and the position in the phylum Nematoda. The complete mt genomes of A. cantonensis and A. costaricensis are 13,497 and 13,585 bp in length, respectively. Hence, they are the smallest in the class of Chromadorea characterized thus far. Like many nematode species in the class of Chromadorea, they encode 12 proteins, 22 transfer RNAs, and two ribosomal RNAs. All genes are located on the same strand. Nucleotide identity of the two mt genomes is 81.6%, ranging from 77.7% to 87.1% in individual gene pairs. Our mt genome-wide analysis identified three major gene arrangement patterns (II-1, II-2, and II-3) from 48 nematode mt genomes. Both patterns II-1 and II-2 are distinct from pattern II-3, which covers the Spirurida, supporting a closer relationship between Ascaridida and Strongylida rather than Spirurida. Thymine (T) was highly concentrated on coding strands in Chromadorea, but balanced between the two strands in Enoplea, probably due to the gene arrangement pattern. Interestingly, the gene arrangement pattern of mt genomes and phylogenetic analysis based on concatenated amino acids indicated a closer relationship between the order Ascaridida and Rhabditida rather than Spirurida as indicated in previous studies. These discrepancies call for new research, reassessing the position of the order of Ascaridida in the phylogenetic tree. Once consolidated, the findings are important for population genetic studies and target identification.     

Matrix metalloproteinases activity demonstrated in the infective stage of the nematodes, Angiostrongylus cantonensis

Ingestion of the larval nematode Angiostrongylus cantonensis can cause the human eosinophilic meningitis known as angiostrongyliasis. Analysis of the extracts and excretory-secretory (ES) products of A. cantonensis larvae and adult stages on gelatin substrate zymography demonstrated the presence of distinct gelatinolytic enzymes. In worm extracts, inhibitor studies showed that the metalloproteinases revealed in L₁ (23 kDa), L₃ (66, 42 and 30 kDa), young adult worm (72 and 94 kDa) and adult worm (72 and 94 kDa). In ES products, the L₁ revealed one low (42 kDa) and two high (105 and 94 kDa) molecular weight proteolytic bands that degraded gelatin in substrate gels. The L₃ revealed three low (66, 50, and 30 kDa) and one high (105 kDa) molecular weight proteolytic bands. Inhibitor studies confirmed that the 105 and 94 proteolytic bands of the L₁, and the 50 and 30 kDa proteolytic bands of the L₃ classification were metalloproteinases. These metalloproteinases secreted in the infective larvae may be associated with the parasite dissemination or pathogenesis.     

Effects of milbemycin D on adult Angiostrongylus cantonensis in rats

Effects of milbemycin D against adult Angiostrongylus cantonensis in rats were examined. The first-stage larval counts in rat faeces (larvae per gram of faeces per female worm recovered, LPG/female) were most conspicuously reduced in the group treated with nine consecutive weekly doses of 5.0 mg/kg. The effect was more marked in the group treated with five or ten successive daily doses of 5.0 mg/kg than the group treated with a single dose of 25.0 or 5.0 mg/kg. Host lung-body weight ratio and number of recovered worms were reduced significantly only in the group treated with five or ten successive daily doses of 5.0 mg/kg. These results suggest that the action of milbemycin D on the reproductive system of the worms might be differentiated from its killing action. The in vitro motility of females recovered from rats medicated with nine consecutive weekly doses of 5.0 mg/kg was inhibited, and almost all females and males were semitransparent and colourless. Results obtained from sectioned worms showed little content in their digestive tracts and uteri. In addition, there were few eggs and first-stage larvae in the lung tissues of treated rats. These suggest that milbemycin D affects the reproductive functions of the worms through an indirect mode of action including paralysis and inhibition in food intake and energy and/or synthetic metabolism.Studies of chemotherapy of parasitic helminths XXIX     

Polyclonal B cell activation during the course of Angiostrongylus cantonensis infection

The numbers of IgM antibody forming cells against various heterologous antigens: SRBC, HRBC, TNP and FITC, were found to be elevated in the spleens of rats after infection with Angiostrongylus cantonensis. In these rats, the number of splenic B cells and total IgG and IgM levels in serum also increased spontaneously about 2- to 3-fold of uninfected rats during the course of the infection. These results suggested that A. cantonensis infection may activate B cells polyclonally in the spleens of the infected rats. While, it was clearly observed that the antibody response to SRBC injection was significantly depressed in these rats, compared to those of uninfected rats. The relation between the polyclonal B cell activation and the immunodepression was discussed.     

MicroRNA expression profile in the third- and fourth-stage larvae of Angiostrongylus cantonensis

The pathogenesis of angiostrongyliasis, resulting from the third-stage and the fourth-stage Angiostrongylus cantonensis larvae invasion of the human central nervous system, remains elusive. MicroRNAs are important regulators of gene expression and involved in many biological processes. The aim of this study was to determine and characterize miRNAs of third (L3) and fourth (L4) larvae of A. cantonensis by Solex deep sequencing. A total of 629 conserved miRNAs (526 and 376 miRNAs in L3 and L4 larvae of A. cantonensis, respectively) and three novel candidate miRNA from L3 and L4 larva of A. cantonensis were identified with bioinformatic analysis. There were 163 miRNAs upregulated and 54 miRNAs downregulated (fold changes ≥5.0) in the L4 of A. cantonensis compared with that of L3 of A. cantonensis. Interestingly, Gene Ontology “biological process” classifications revealed that 26 miRNAs of significantly differential expression are associated with the immune system, which implies that these miRNAs might participate in the pathogenesis of angiostrongyliasis by regulating genes involved in immune response pathways. Furthermore, the differential expression patterns of 26 conserved miRNAs between L3 and L4 of A. cantonensis were verified. The results of real-time PCR and Northern blot showed that the aca-miR-124 and aca-miR-146a-5p have a low level expression in L3 larvae but high level expression in L4 larvae. Transfection of aca-miR-124 mimics alone significantly downregulated the mRNA expression of IL-6 and IL-1β and TNF-a in the N9 cells, compared to the combination transfection of aca-miR-124 mimics and inhibitor (P?<?0.05), suggesting that miR-124 of A. cantonensis have an important role in the suppression of microglia activation. In conclusion, the study presents a general picture of the expression of small RNAs in L3 and L4 of A. cantonensis and highlights conserved miRNAs differentially expressed between L3 and L4 larvae. Our data revealed that miRNAs of parasite may mediate important roles in A. cantonensis immune evasion and aca-miR-146a-5p can serve as a potential biomarker to diagnose angiostrongyliasis.     

阿苯达唑治疗广州管圆线虫感染的免疫学效应研究

动态观察小鼠人工感染广州管圆线虫并接受阿苯达唑治疗后外周血嗜酸性粒细胞数量、循环抗原CAg、IL-5水平变化,探讨阿苯达唑治疗广州管圆线虫感染的免疫学效应.将160只BALB/c小鼠随机分组成4组:实验组、感染未给药对照Ⅰ组、未感染给药对照Ⅱ组、健康对照Ⅲ组.动态计数各组小鼠外周血嗜酸性粒细胞,采用单抗金标层析法和酶联...     

A transcriptomic study on the pepsin-activated infective larvae of Angiostrongylus cantonensis

Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death.     

广州管圆线虫GAMMA—BBH基因的克隆表达鉴定和荧光定量检测

目的亚克隆和表达广州管圆线虫γ-丁基甜菜碱羟化酶（γ-butyrobetaine hydroxylase，GAMMA-BBH）基因．对该蛋白属性进行鉴定，定量检测不同虫龄该目的基因表达量。方法PCR扩增GAMMA—BBH cDNA基因．产物经Nco Ⅰ和HindⅢ限制性内切酶酶切后连接至原核表达载体重组为pET—BBH，在B121（DE3）中用IPTG诱导表达．SDS—PAGE、Westernblot和酶活性测定鉴定表达产物，用荧光定量PCR方法检测广州管圆线虫不同虫龄GAMMA—BBH的表达量。结果PCR和核苷酸序列测定结果表明pET—BBH构建正确并能在IPTG诱导下表达，SDS—PAGE和Western blot鉴定表明表达产物分子量与预期分子量（Mr=48500）一致，酶学测定显示表达产物有GAMMA—BBH的酶活性，荧光定量PCR方法检测结果显示GAMMA—BBH在虫体不同虫龄期表达量不一致。结论目的基因能正确编码并表达具有酶活性的GAMMA—BBH，该酶的表达量与虫龄和寄生部位可能有关。     

Angiostrongylus cantonensis: Immunoblot analysis of the antigens recognized by rats

Following infection of rats with Angiostrongylus cantonensis, occurrence of anti-parasite antibody in the serum was determined with special reference to the antigens recognized by host IgG antibodies, using SDS-PAGE combined with an immunoblotting technique. Three saline extracts of digestive organ, reproductive organ and body wall, isolated from adult female A. cantonensis, were used as crude antigenic solutions. Then 7 to 49 days after infection, IgG antibodies directed predominantly against a single antigen, referred to as Ac-1 antigen, were detected. After 91 days or more, infected rats formed antibodies not only against the Ac-1 antigen, but also against a wide variety of other components with molecular weights in the range of 26000–220000 dalton. By using an antiserum against Ac-1 antigen as a probe, it was shown that the molecular weight and subunit structure, as well as the immunoelectrophoretic mobility, varied according to the organ from which the antigenic extract was prepared. The Ac-1 protein in the extract of the reproductive organ, one of the major sources of the Ac-1 antigen, showed the same electrophoretic mobility as -globulin. It has a molecular weight in the range of 100 000–200 000 dalton under both non-reducing and reducing conditions. Immunohistochemical studies, using the same antiserum and sectioned adult female worms, found Ac-1 antigen in the cytoplasm of oocytes at different stages of development, and in the lateral cord.